Professional Documents
Culture Documents
Combined Protocols
Combined Protocols
Contents
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1.
1.1.
1.2.
1.3.
Introduction
Technology for routine three dimensional (3D) cell culture
Choosing the right AlvetexScaffold format
Handling AlvetexScaffold
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10
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10
Note: Please See Section 10: A Review of Imaging Techniques Compatible with Three
Dimensional Culture of Cells Grown in AlvetexScaffold)
HPM/18/09/2012
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5.2. Processing AlvetexScaffold 3D Cultures for PFA Fixation and Paraffin Wax
Embedding for Immunofluorescence
5.3. Processing AlvetexScaffold 3D Cultures for Bouins Fixation and Paraffin Wax
Embedding for Haematoxylin and Eosin Staining
5.4. Processing AlvetexScaffold 3D Cultures for Resin Embedding and Toluidine Blue
Staining for Light Microscopy
5.5. Processing AlvetexScaffold 3D Cultures for Scanning Electron Microscopy (SEM)
5.6. Processing AlvetexScaffold 3D Cultures for Cryosectioning
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7.
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1.
2.
3.
4.
Fox et al (2010). Validation of reference gene stability for APAP hepatotoxicity studies
in different in vitro systems and identification of novel potential toxicity biomarkers.
In Vitro Toxicology, 24 (7), 1962-1970.
5.
6.
Bokhari et al (2007). Novel cell culture device enabling three-dimensional cell growth
and improved cell function. Biochem. Biophys. Res. Comm.,354, 1095-1100.
7.
8.
9.
General Information
Product Descriptions:
Description
Catalogue Number
Contains:
AlvetexScaffold
12 Well Plate Starter Pack
AVP002-2
2 X 12 Well Plates
(AVP002)
AlvetexScaffold
Well Insert Holder
Starter Pack
AVP015-2
AlvetexScaffold
6 Well Insert Starter Pack
AVP004-12
12 X 6 Well Inserts
(AVP004)
AlvetexScaffold
12 Well Insert Starter Pack
AVP005-12
12 X 12 Well Inserts
(AVP005)
Intended Use
AlvetexScaffold is intended for in vitro research only.
CAUTION: Not intended for human or animal diagnostic or therapeutic uses.
Protocols and Instructions
To view or download the AlvetexScaffold protocol that best suits your chosen cell type
and experimental design simply visit: www.reinnervate.com/alvetex/protocols and
choose from the following categories:
Preparation and use of AlvetexScaffold Products
Example Protocols for the Growth of Specific Cell Types
Protocols for Analytical Techniques
Protocols for Specialised Applications
Storage & Shelf Life
Store at room temperature. The product is sterile and is expected to be stable indefinitely
whilst the blister pack and peel away lid are intact.
Precautions
All handling of AlvetexScaffold products should be performed wearing gloves according
to standard aseptic methods required for cell culture in a Class I/II cabinet. All products
have been terminally sterilised by gamma irradiation and remain sterile until opened.
Contact Information for placing orders
For all other enquiries: NETPark Incubator, Thomas Wright Way, Sedgefield, Co Durham,
TS21 3FD, United Kingdom t: +44 (0)1740 625266 f: +44 (0)1740 625251
e.mail: info@reinnervate.com
Contact Information for technical assistance
Technical Services: techsupport@reinnervate.com
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Protocol Booklet
Introduction
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The product has been terminally sterilised by gamma irradiation and remains sterile until
opened. AlvetexScaffold requires an ethanol (EtOH) wash prior to use to render it
hydrophilic. The material is compatible with a broad range of standard molecular, cellular and
histological techniques.
In deciding which AlvetexScaffold format to use, the following factors should be considered
in combination:
Cell type and duration of culture,
The desired depth of cell penetration in the 3D cell culture,
The type of assay to be performed or application to be used for.
The rate of cell growth in AlvetexScaffold is affected by cell metabolism, proliferation rate,
motility and cell size. It is therefore important to choose the AlvetexScaffold format that will
achieve optimal growth for the chosen cell type.
For example, in Figure 1. two different cell types were grown for the same time period using
AlvetexScaffold 12-well plates (AVP002), resulting in two cultures with different characteristics:
Figure 1. 3T3 (A.) and HepG2 (B.) cultures grown on AlvetexScaffold in 12-well plate format
for 7 days.
3T3 cells are small, highly proliferative and invasive, and therefore readily penetrated through
the entire scaffold. In contrast, HepG2 cells are slower growing cells that have a high tendency
for cell-cell attachment and consequently penetrated only the top 30% of the AlvetexScaffold
membrane. In both cases 3D cell culture was achieved. Micrographs taken at 20x magnification.
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AlvetexScaffold formats have been specifically designed to enable optimal 3D cell culture in
both short and long term experiments. The 12-well plate format is ideal for short term cell
cultures where the medium is replaced every 1 to 2 days. The AlvetexScaffold membrane sits
at the bottom of each well and the cells are exposed to the medium only from above. This is
desirable for studies where easy access is required to the cells as they predominantly reside in
the top portion of the scaffold (e.g. for transfection studies). Alternatively a shorter experiment
in well inserts is also suitable, as the cells will not have had time to penetrate the scaffold.
The well inserts are capable of supporting cell growth for up to 3 weeks for assays and
applications where higher cell numbers are desirable (Table 1.). The design of the well insert
allows for greater cell penetration into the scaffold and higher cell yields because
AlvetexScaffold is suspended in the medium such that cells receive nutrients from above
and below. Therefore, they sustain optimal growth for longer and achieve greater
differentiation creating a cultured tissue that more closely resembles the growth of cells in
the body. Well inserts can be used to create co-cultures either in plates or in the well insert
holder in the Petri dish.
Application / AlvetexScaffold format
AVP002
(12-well plate)
AVP004-3
(6-well insert)
AVP005-3
(12-well insert)
+++
+++
+++
N/A
++
++
++
++
++
++
N/A
++
N/A
+++
+
+
+
+++
+++
+++
N/A
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
N/A
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
Table 1. Suggested guidelines for the use of AlvetexScaffold formats for cell applications and assays. + + + most suitable,
+ + suitable, + least suitable, N/A= not applicable.
Ranking is based on AlvetexScaffold disc format suitability, the likely cell yields and therefore signal generation, and whether
exogenously added chemicals/cells can be contained to only one side of the membrane.
*The growth of cells cannot be followed by traditional light microscopy as in 2D, but as with ex-vivo tissues,
3D structures have to be evaluated using histology or confocal microscopy. Alternatively cell
proliferation can be monitored using a viability assay such as the MTT.
Figure 2. HaCaT cells grown on AlvetexScaffold in 12 well insert (AVP005-3) in a 6-well plate
(A) and (B) in well insert holder in deep Petri dish (AVP015). Note significantly more proliferation
and cell invasion in the cultures grown in the well insert holder system, where more nutrients
were available. Micrographs taken at 20x magnification.
Thus, AlvetexScaffold products provide cell biologists with a broad range of choice and great
flexibility when designing their cell culture experiments.
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The well insert holder in a deep Petri dish (AVP015) is designed to hold well inserts in a large
volume Petri dish reducing the need for frequent media changes. This format should be used
for prolonged cell growth of highly proliferative and demanding cell types (Figure 2.). The
well inserts can be positioned at three different levels in the insert holder to allow for cultures
to be raised to the air liquid interface (for air liquid interface differentiation) and subsequent
permeability / barrier testing (Table 1.). 3D co-cultures can also be set-up in one or two of the
well inserts within the same Petri dish.
Handling AlvetexScaffold
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Figure 1. AlvetexScaffold discs can easily be removed from the 12-well plate using flat ended
forceps (Fisher Scientific, MNK-155-M).
Handling AlvetexScaffold
Figure 2. Removal of AlvetexScaffold disc from well-insert. Unclip the base of the well
insert all the way around (A), and pull down to release the disc (B). Alternatively the disc can
be cut out by holding the well insert upright (C) or upside down (D).
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In well insert formats, the AlvetexScaffold membrane can be removed using forceps (Figure
2. A & B) or can be cut out using a scalpel with a size 11 blade (C & D).
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Open the 24-well plate carefully to ensure that the clips holding the
membranes are not displaced.
Carefully aspirate the EtOH solution and immediately wash the membrane in
~1-1.5 ml of appropriate medium for ~1 min.
Carefully aspirate medium wash and replace with final wash medium (use
same type as for cell seeding). The AlvetexScaffold is now ready: aspirate medium
just before application of cells. If preparation of cell suspension is delayed, incubate
plate with medium at 37 C, 5 % CO2 until further use.
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can be pre-coated with standard cell culture reagents such as collagen, fibronectin,
laminin, poly-D/L-lysine, poly-L-ornithine and Matrigel to encourage cell adhesion,
differentiation and optimise function. Perform this step after the EtOH treatment and
appropriate buffer wash steps instead of medium. For specific coating protocols
please refer to www.reinnervate.com/workflow.
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For most applications initial cell seeding densities of 0.25-1.0x106 cells in 5075 l per disc are recommended. Seeding in a low volume enables cells to attach
predominantly to the AlvetexScaffold and avoids cell loss on other surfaces.
When inoculating, aspirate washing medium thoroughly from the plate and
carefully dispense cells on the middle of the membranes. Replace lid and incubate in
a humidified incubator at 37 C with 5 % CO2 for 30 minutes to 3 hours to facilitate
cell attachment.
After this time gently flood the wells with medium by dispensing up to 1.5 ml of
medium per well.
With 3D cell culture there will be many more cells growing per unit volume of
medium. Therefore, users must refresh media more frequently; ideally once a day,
however this will also depend on the population doubling rate and nutrient demands
of the cell type cultured.
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Similarly to 2D culture, if using serum-free medium, consider the use of coating agents
to enhance cell attachment.
Prior to cell seeding, AlvetexScaffold can also be pre-coated with standard cell culture
reagents such as collagen, fibronectin, laminin, poly-D/L-lysine, poly-L-ornithine and
MatrigelTM to encourage cell adhesion, differentiation and optimise function. Perform
this step after the EtOH treatment and appropriate buffer wash steps instead of medium.
Optimisation of seeding and 3D cell culture using the AlvetexScaffold 12-well format
3D cell culture is different to conventional 2D cell culture and as such requires optimisation
according to cell type:
For most applications initial cell seeding densities of 0.5-2.0x106 cells in 100-150 l per
disc are recommended. Seeding in a low volume enables cells to attach predominantly
to the AlvetexScaffold disc and avoids cell loss on other surfaces.
When inoculating, aspirate washing medium thoroughly from the plate and carefully
dispense cells on the middle of the discs. Replace lid and incubate in a humidified
incubator at 37 oC with 5% CO2 for 30 minutes to 3 hours to facilitate cell attachment.
After this time gently flood the wells with medium by dispensing up to 4 ml of medium
per well.
With 3D cell culture there will be many more cells growing per unit volume of medium.
Therefore, users must refresh media more frequently; ideally once a day, however this
will also depend on the population doubling rate and nutrient demands of the cell type
cultured.
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Aspirate and replace with final wash medium (use same type of medium as for cell
seeding). The scaffold is now ready for cell seeding: aspirate medium just before
application of cells. If preparation of cell suspension is delayed, incubate plate with
medium at 37 C with 5% CO2 until further use.
Similarly to 2D culture, if using serum-free medium, consider the use of coating agents to
enhance cell attachment. Prior to cell seeding, AlvetexScaffold can also be pre-coated with
standard cell culture reagents such as collagen, fibronectin, laminin, poly-D/L-lysine, poly-Lornithine and MatrigelTM to encourage cell adhesion, differentiation and optimise function.
Perform this step after the EtOH treatment followed by an appropriate buffer wash step instead
of medium.
Optimisation of seeding and 3D cell culture using the AlvetexScaffold 6-well and
12-well insert formats
3D cell culture is different to conventional 2D and as such requires optimisation according to
cell type, assay being performed and insert configuration used (Figure 2):
i. Media from
below only:
for cells grown in 3D
at air-liquid interface
iii. Media
interconnected:
for routine 3D growth
of cells with high
metabolic activity/
proliferation rate
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Well insert and holder type
Feeding volumes
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Below only(i)
Above and
Above and below
below separately(ii) interconnected(iii)
7 1 ml/well
10 0.5 ml/well
7 1 ml/well
10 0.5 ml/well
Table 1. Feeding volumes for the different well insert configurations (i-iii, see also Figure 2.)
In 3D cell culture there will be more cells per unit volume of medium. Therefore, users must
refresh medium more frequently typically every 21 days, however this will also depend on the
population doubling rate, nutrient demands of the cell type cultured and the volume of medium
used as described above.
If any signs of cell attachment and growth are evident on the bottom of the plate, transfer the
well inserts into a new plate, re-feed and then incubate as usual.
Figure 1. AVP005-3 (left) and AVP004-3 (right) in well insert holder system in deep Petri dishes.
AlvetexScaffold well insert formats and their use in the well insert holder
The deep Petri dish enables users to grow their 3D cultures in larger volumes of media
compared to an ordinary multiwell plate. Up to 95 ml of media can be used in the deep Petri
dish and is therefore capable of sustaining long term 3D culture experiments (3-4 weeks) and
reducing the frequency of medium exchanges. If required, a magnetic stirrer bar can be
placed in the bottom of the dish to circulate media and facilitate exchange.
The well insert can be positioned at three different levels in the insert holder: high, medium
and low (Figure 2).
High
Medium
Low
Figure 2. Well insert settings within the well insert holder (AVP015).
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A single well insert holder capable of housing up to three well inserts (6- or 12-well inserts,
Figure 1.) in a deep Petri dish is supplied in a pack. The Petri dish itself is not tissue culture
treated. The whole product has been terminally sterilised by gamma irradiation and remains
sterile until opened.
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This feature allows cultures to be raised to the air liquid interface by moving the insert to a
different level within the same holder.
Positioning the well inserts at different levels may be used to conserve expensive media or
allow for increasing media volumes for demanding cell types over the course of a long term
experiment.
Additionally, cultures can be fed from below the well insert only (i.), media from above and
below separately (ii.) and media interconnected (iii.) through the windows at the side of the
well inserts (Figure 3):
i.
ii.
iii.
Low
20ml 1ml
40ml 3ml
70ml 5ml
Medium
34ml 2ml
50ml 3ml
80ml 3ml
High
48ml 2ml
70ml 5ml
92ml 3ml
12
Low
20ml 1ml
40ml 3ml
72ml 5ml
Medium
34ml 2ml
52ml 3ml
82ml 3ml
High
48ml 2ml
70ml 5ml
92ml 3ml
The well insert holder system also allows for the 3D co-culture of more than one cell type
by seeding different cells in one or two of the well inserts within the same Petri dish. 3D cocultures can also be set up within the same well insert. Alternatively, a support cell line can
be cultured at the base of the Petri dish in 2D and another in 3D in the well inserts.
As the Petri dishes are untreated, coat with poly-L-lysine, or similar, to facilitate cell
attachment. Ensure that suitable media is chosen that will simultaneously support the growth
of both cell types cultured. The well insert holder will fit into deep Petri dishes with
approximate dimensions of > 86 mm (internal diameter) x > 25 mm (height).
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Feeding Volume
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HaCaT cells
HepG2 cells
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12-well inserts (AVP005-3) in well insert holder in deep Petri dish (AVP015)
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with poly-D or Llysine in order to facilitate cell attachment and migration within the scaffold.
Poly-L-lysine is a synthetic amino acid that has been widely used as a coating agent in tissue
culture to enhance cell attachment to untreated plastic and glass. When Poly-L-lysine is applied,
it renders the material positively charged and increases the electrostatic attraction between the
surface and the negatively charged cells thus improving adhesion. The use of Poly-D-lysine is
favoured with cells displaying high proteolytic activity as it is less prone to breakdown than the
naturally-occurring L-enantiomer. Otherwise, Poly-D-Lysine and Poly-L-Lysine are equivalent for
these purposes. Refer to manufacturers instructions when choosing poly-lysine formats and
dilutions required for the desired cell type.
Note also that the available molecular weight (MW) ranges of poly-lysine vary. The lower the
MW, the less viscous the solution, while the higher the MW, the more binding sites per
molecule. In this protocol 3T3 fibroblasts were grown on poly-L-lysine (Sigma-Aldrich, P4707)
coated AlvetexScaffold in 12-well plate (AVP002) format for 7 days. The MW of poly-L-lysine
ranged from 70-150 kDa, which remained easy to handle while providing sufficient binding
sites for cell attachment.
Method:
1. Prepare AlvetexScaffold for coating by first treating with 70% ethanol followed by two
PBS washes as described in the relevant product information leaflet. Leave
AlvetexScaffold in the second PBS wash until ready to apply the poly-L-lysine solution.
2. Aspirate the second PBS wash and add 500 l of poly-D or L-lysine per well. Replace plate
lids and leave to stand for 1 hour at room temperature.
3. Tilt the 12-well plate and gently aspirate any excess fluid from the edge of the wells. If
using well insert AlvetexScaffold formats, remove excess fluid from AlvetexScaffold by
gently tapping the plate or Petri dish on the worktop. Check that no residual fluid is hanging
from the base of the well inserts. Aspirate to remove any residual coating agent from the
bottom of the wells.
4. Prepare cells for seeding in the appropriate culture media and seed directly on the wet polyL-lysine coated AlvetexScaffold membrane in the volumes relevant for the
AlvetexScaffold product format. Allow the cells to settle for 30-90 minutes in an incubator
(5 % CO2, 37 C) before flooding with media.
03
Results:
Pre-coating of AlvetexScaffold discs with poly-L-lysine resulted in enhanced infiltration of
3T3 cells into the scaffold compared with control cultures in uncoated AlvetexScaffold. Cells
were seen to reside deep within the scaffold and in greater abundance after 7 days of growth
in treated discs. These findings indicate that pre-treatment of AlvetexScaffold with well
established tissue culture coating agents is able to enhance the attachment and growth of
appropriate cell types into the 3D structure.
Uncoated AlvetexScaffold control
100 m
50 m
50 m
Figure 1. Brightfield micrographs showing the structure of 3T3 cells cultured on non-coated
and poly-L-lysine coated AlvetexScaffold discs in 12 well plate format
(AVP002) after 7 days culture
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with a thin layer of
collagen I in order to facilitate and enhance cell attachment and migration within the scaffold.
Example data shown herein was obtained using this protocol to grow HepG2 hepatocytes on
collagen I coated AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in 6-well plate format.
Method:
1. Prepare AlvetexScaffold for coating by first treating with 70% ethanol followed by two
PBS washes as described in the relevant product information leaflet. Leave
AlvetexScaffold in the second PBS wash until ready to apply the collagen solution.
2. Dilute rat tail collagen I (BD Biosciences, 354236) to a concentration of 0.8 mg/ml using
cell culture grade water. Handle the reagents on ice, using pre-chilled pipette tips to
perform the dilution and subsequent application onto AlvetexScaffold.
3. Aspirate the second PBS wash from AlvetexScaffold disc and carefully pipette 500 l of
the diluted collagen solution onto each disc. Replace plate lids and leave to stand for 1
hour at room temperature.
4. Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the base of
the well inserts. Aspirate to remove any residual coating agent from the bottom of the
wells. If using AlvetexScaffold in 12-well plate format, tilt the plate and gently aspirate any
excess fluid from the edge of the wells.
5. Prepare cells for seeding in the appropriate culture media and seed directly on the wet
collagen coated AlvetexScaffold membrane in the volumes relevant for AlvetexScaffold
product format. Allow the cells to settle for 30-90 minutes in an incubator (5 % CO2, 37
C) before flooding with media.
05
Results:
Pre-coating of AlvetexScaffold discs with collagen I resulted in enhanced infiltration of HepG2
cells into the scaffold compared with control cultures in untreated AlvetexScaffold. Cells were
seen to reside deep within the scaffold after 7 days of growth in treated discs, while cells
grown in untreated AlvetexScaffold occupied only the upper half of the scaffold. These findings
indicate that pre-treatment of AlvetexScaffold with extracellular matrix products is able to
enhance the attachment and growth of appropriate cell types into the 3D structure.
Uncoated AlvetexScaffold control
3 Day
200 m
7 Day Culture
50 m
200 m
50 m
7 Day Culture
50 m
200 m
50 m
Figure 1. Brightfield micrographs at low (10x) and high (40x) magnification showing HepG2
cells cultured for up to 7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well
insert (AVP004-3) in 6-well plate format. Cells were fixed, sectioned and counterstained with
haematoxylin and eosin.
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with MatrigelTM to
facilitate and enhance attachment and differentiation of both normal and transformed anchorage
dependent cells. Example data shown herein was obtained using this protocol to grow HepG2
hepatocytes in AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in 6-well plate format.
Method:
1. Prepare AlvetexScaffold for coating by first treating with 70% ethanol followed by two
PBS washes as described in the relevant product information leaflet. Leave
AlvetexScaffold in the second PBS wash until ready to apply the MatrigelTM solution.
2. Dilute Matrigel (BD Biosciences, 356234; pre-thawed overnight on ice) to a concentration
of 0.8 mg/ml (1 in 10 dilution) using appropriate cell culture media (e.g. MEM for the
example below). Handle the reagents on ice, using pre-chilled pipette tips to perform the
dilution and subsequent application onto AlvetexScaffold.
3. Aspirate the second PBS wash from AlvetexScaffold disc and carefully pipette 350 l of
the diluted Matrigel solution onto each disc. Replace plate lids and leave to stand for 12 hours at room temperature.
4. Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the base of
the well inserts. Aspirate to remove any residual coating agent from the bottom of the
wells. If using AlvetexScaffold in 12-well plate format, tilt the plate and gently aspirate any
excess fluid from the edge of the wells.
5. Prepare cells for seeding in the appropriate culture media and seed directly on the wet
Matrigel coated AlvetexScaffold membrane in the volumes relevant to the
AlvetexScaffold product format. Allow the cells to settle for 30-90 minutes in an incubator
(5 % CO2, 37 C) before flooding with media.
Results:
Pre-coating of AlvetexScaffold discs with Matrigel resulted in enhanced infiltration of cells
into the scaffold compared with control cultures in untreated AlvetexScaffold. Cells were seen
to occupy the entire depth of the scaffold after 7 days of growth in Matrigel-coated discs,
while cells grown in untreated AlvetexScaffold occupied only the upper half of the scaffold.
These findings indicate that pre-treatment of AlvetexScaffold with extracellular matrix products
is able to enhance the growth of appropriate cell types into the 3D structure.
Uncoated AlvetexScaffold control
3 Day
200 m
7 Day Culture
50 m
200 m
50 m
Matrigel-coated AlvetexScaffold
3 Day
200 m
7 Day Culture
50 m
200 m
50 m
Figure 1. Brightfield micrographs at low (10x) and high (40x) magnification showing HepG2
cells cultured for up to 7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well
insert (AVP004-3) in well insert holders in 6-well plate format. Cells were fixed, sectioned and
counterstained with haematoxylin and eosin.
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with fibronectin
in order to facilitate and enhance cell attachment and migration within the scaffold.
Example data shown herein was obtained using this protocol to grow HepG2 hepatocytes
on fibronectin coated AlvetexScaffold for 7 days in 6-well inserts (AVP004) in a 6-well
plate format.
Method
1.
2.
3.
Aspirate the second PBS wash from AlvetexScaffold disc and carefully pipette 300 l
of the diluted fibronectin solution onto each disc. Replace plate lids and leave to stand
for 1 hour at room temperature.
4.
Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the
base of the well inserts. Aspirate to remove any residual coating agent from the
bottom of the wells. If using AlvetexScaffold in 12-well plate format, tilt the plate and
gently aspirate any excess fluid from the edge of the wells.
5.
Prepare cells for seeding in the appropriate culture media and seed directly on the wet
fibronectin coated AlvetexScaffold membrane. Seed cells in volumes relevant for the
specific AlvetexScaffold format being used (see product information booklet for
volume details). Allow the cells to settle for 30-90 minutes in an incubator (5 % CO2,
37 C) before flooding with media.
Figure 1. Brightfield micrographs at low (10x) and high (40x) magnification showing HepG2 cells cultured
for up to 7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well insert (AVP004) in 6-well
plate format. Cells were fixed, sectioned and counterstained with Haematoxylin and Eosin.
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Results
Pre-coating of AlvetexScaffold discs with fibronectin resulted in enhanced infiltration of
HepG2 cells into the scaffold compared with control cultures in untreated AlvetexScaffold.
Cells were seen to have reached deeper within the scaffold after 7 days of growth in treated
discs, while cells grown in untreated AlvetexScaffold occupied only the upper part of the
scaffold. These findings indicate that pre-treatment of AlvetexScaffold with extracellular
matrix products is able to enhance the attachment and growth of appropriate cell types
into the 3D structure.
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Introduction:
Extra-cellular matrix (ECM) coating of in vitro culture surfaces is commonly used to enhance
cell-substrate adhesion, encourage cell-matrix signalling and to protect shear-sensitive cells
in perfusion devices. The following protocol outlines how to coat the top surface of
AlvetexScaffold membranes with a thin gel layer of collagen I. This method can be used to
promote adhesion and culture of a monolayer of epithelial cells at the surface of
AlvetexScaffold, either as a monoculture for diffusion assays, or as co-culture for tissue
engineering (e.g. a surface epithelial compartment layered on top of a fibroblastic
compartment grown inside AlvetexScaffold). The data shown exemplify the application of
this protocol to grow colorectal adenocarcinoma CaCo-2 cells on collagen I coated
AlvetexScaffold for 4 days in 6-well inserts (AVP004).
Method:
1.
2.
Note: Ensure all reagents are maintained on ice and use pre-chilled pipette tips
to perform the dilution and subsequent application onto AlvetexScaffold.
Dilute rat tail collagen I (BD Biosciences, 354236) to a concentration of 2 mg/ml using
cell culture grade water and adjust the pH with NaOH according to manufacturers
instructions.
Note: Manufacturers instructions for BD Biosciences rat tail collagen (cat # 354236) can be
found at: www.bdbiosciences.com/external_files/dl/doc/manuals/live/web_enabled/354236
_pug.pdf
3.
Aspirate the second PBS wash from AlvetexScaffold disc. Do not allow the scaffold
to dry out. Immediately and carefully pipette an appropriate volume of the diluted
collagen solution onto each disc. Depending on the desired thickness of the collagen
layer, use 100-400 L of collagen solution per disc for the 12-well plate format
(AVP002) and the 6-well insert format (AVP004) or 75-200 L of collagen solution for
the 24-well plate (AVP006) and 12-well insert format (AVP005).
4.
5.
Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the
HPM/18/07/2012
6.
7.
Prepare cells for seeding in the appropriate culture media and seed directly on the
wet collagen-layered AlvetexScaffold membrane, in the volumes relevant for the
AlvetexScaffold product format used. See the Quick Start protocol available from
www.reinnervate.com/alvetex/workflow for details.
11
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base of the well inserts. Aspirate to remove any residual gelling agent from the
bottom of the wells. If using AlvetexScaffold in 24- or 12-well plate format, tilt the
plate and gently aspirate any excess fluid from the edge of the wells.
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CaCo-2 cells (ATCC, HTB-37TM) were routinely maintained in T-75 flasks. CaCo-2 complete
medium consisted of: high-glucose DMEM media (Lonza, BE12-614F) supplemented with 20
% v/v FBS, 0.1 mM non-essential amino acids, 10 mM/L HEPES (pH 7.4), 2 mM L-glutamine
and 100 U/ml Penicillin/Streptomycin. AlvetexScaffold 6-well inserts (AVP004) in 6-well
plates, were layered with 200 l of 2 mg/ml collagen I as described above.
5 ml medium was added to the bottom of each well, i.e on the outside of the
AlvetexScaffold insert, taking care not to trap air bubbles underneath the insert. Cells were
seeded on top of each disc at a density of 0.5 x 106 cells in 500 l medium suspension and
were left to attach overnight in an incubator (5 % CO2, 37 C). The following day, medium
was carefully added to each sample up to 10 ml per well. Culture medium was changed
every two days. After 4 days and 10 days culture, samples were fixed, embedded, sectioned
and stained for hematoxylin & eosin or toluidine blue, according to established protocols
(see www.reinnervate.com/alvetex/workflow).
Results:
Culture of CaCo-2 cells on collagen I coated AlvetexScaffold resulted in an even cell
monolayer of epithelial morphology. Apical structures characteristic of differentiated intestinal
epithelium (microvilli), were evident after 10 days culture (Figure 4).
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Method:
1.
Perform a PBS wash prior to running this assay if cells in AlvetexScaffold were cultured
in phenol-red containing medium. If using the AlvetexScaffold 12-well plate format,
gently remove the discs using flat-ended forceps, gently dip in PBS and place into a new
12-well plate. If using well inserts, dip in PBS and then unclip the base of the insert to
release the disc.
2.
Place each disc into a fresh 12-well plate and set up one well without AlvetexScaffold
as the negative control.
3.
Prepare 1 mg/ml MTT reagent (Sigma, M5655) solution in Phenol Red-free DMEM
(Lonza, BE12-917F) and filter sterilise.
4.
Add 1ml of MTT reagent to each well and cover with aluminium foil as it is light sensitive.
5.
6.
7.
Aspirate off the MTT solution from each disc and replace by pipetting 1 ml of acidified
isopropanol onto each disc plus the negative control.
8.
Cover plate in aluminium foil and place on rotating platform for 10 minutes at 100 rpm
to ensure blue formazan product is fully solubilised.
9.
Pipette 20 l of sample containing the formazan dye into a 96-well plate and add 180 l
of isopropanol to each well (This represents a 1 in 10 dilution of the formed product.
Different dilutions may be required depending on the viable cell numbers expected.)
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Introduction
This chromogenic assay involves the conversion of a yellow, water soluble compound, MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) to a purple, insoluble
formazan. This reaction only takes place when mitochondrial reductase enzymes are active,
and therefore the conversion can be directly related to the number of viable (living) cells. The
MTT reagent alone results in very low background absorbance values in the absence of cells
on AlvetexScaffold. It is recommended that for each cell type the linear relationship between
cell number and signal produced should be first established in order to investigate the limits
of the assay.
This is an example protocol, which may require further optimisation depending on the rate
of cell growth of a particular cell type on AlvetexScaffold.
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Introduction:
Cell culture in AlvetexScaffold allows the formation of multilayered, high-density cell
populations which approximate the complexity and structure of in vivo tissues. When viewing
an unstained, unsectioned AlvetexScaffold 3D culture under a standard brightfield
microscope, the combined density and thickness of the scaffold and the 3D culture within it
prevent the clear visualisation of individual cells. However, common visible dyes can be
successfully used to confirm cell attachment or to check confluency.
05
8. After removing the last PBS wash, add 150 L of PBS to each culture and
proceed with visualisation.
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Example results
1.0 Preparation of CHO-K1 Cultures on AlvetexScaffold
CHO-K1 cells (ATCC, CCL-61) were seeded on AlvetexScaffold 12-well plate format
(AVP002) at a density of 0 cells, 0.5 million cells and 2 million cells per scaffold, in a
seeding volume of 150 L with a settling time of 30 minutes before adding 4 ml Kaighns
modified nutrient mixture consisting of F-12K (Gibco, 21127022) supplemented with 10 %
(v/v) foetal calf serum, 100 g/ml penicillin and 10 g/ml streptomycin. (See also Example
protocols for the culture of the CHO-K1 cell line on AlvetexScaffold on
www.reinnervate.com/alvetex/protocols). Cells were visualised using the Neutral Red
staining technique after 1 day.
CHO-K1 cells were also seeded on AlvetexScaffold 6-well plate format (AVP004) at a density
of 0.5 million cells per scaffold as above and were maintained by complete media exchange
every two days for 6 days. Cells were visualised using the Neutral Red staining technique
after 2, 4 and 6 days.
Figure 1. Macroscopic appearance of cultures on AlvetexScaffold. A. Neutral Red staining of CHO-K1 cells
after 24 hours. B. Methylene Blue staining of HepG2 cells after 24 hours. Note the increase in staining
intensity with higher cell numbers.
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HepG2 cells (ATCC, HB-8065) were seeded on AlvetexScaffold 12-well plate format
(AVP002) at a density of 0 cells, 0.5 million cells and 2 million cells per scaffold, in a
seeding volume of 150 L and with a settling time of 30 minutes before adding 4 ml
of EMEM cell culture medium (Gibco, 21090055) supplemented with 10 % (v/v) foetal
calf serum, 2 mM L-glutamine, 100 g/ml Penicillin and 10 g/ml Streptomycin. (See also
Example protocols for the culture of the HepG2 cell line on AlvetexScaffold on
www.reinnervate.com/alvetex/protocols). Cells were visualised using the Methylene Blue
staining technique after 1 day.
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Reinnervate recommends the use of Neutral Red staining for the visualisation of individual
cells within the scaffold over Methylene Blue. Immediately after staining with Neutral Red,
the microscopic appearance of the cultures was observed using a Leica ICC50HD
microscope using LAS EZ software.
Neutral Red solution can be used as a very quick and simple staining technique to follow culture
growth and cell survival within AlvetexScaffold over a time course. Note as cell density
increased with time, the staining intensity also increased both macroscopically and
microscopically (Figures 3 and 4). Although Neutral Red can be compatible with live cell
experiments as it is non-toxic at low concentrations, it is recommended that the user should
check the effect of the dye on culture growth and cell survival first. Reinnervate recommends
setting up extra scaffolds for dye analysis alongside with the experimental setup.
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Figure 3. Neutral red staining of CHO-K1 cells cultured in AlvetexScaffold 6-well inserts (AVP004) in 6 wellplate format for up to 6 days. Cells were seeded at an initial seeding density of 0.5 x106 cells/well. Note the
intensity of the staining and the cell surface coverage increase with greater culture period. Scalebar 200 m.
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Figure 4. Close up view of Fig 3. Day 2 cells viewed by 20x objective. Note that this staining method clearly
enables the visualisation of individual cell nuclear morphologies. Scale bar 200 microns.
Below are example results for live cell labelling of 3D cultures in AlvetexScaffold. It is
recommended that for each staining agent used, the original manufacturers instructions be
followed. Imaging of the specimens will also need to be optimised according to the
specifications of the microscope used.
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Introduction
Live cell imaging allows real time monitoring of cell shape and form during cell differentiation,
proliferation and migration. AlvetexScaffold offers a unique ability to study cell growth in 3D
by providing an environment through which individual cells can navigate for extended periods
of time and where confluent cell populations routinely multilayer.
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Transfection reagent: e.g. linear, 25 kDa polyethylenimine (PEI) (Park Scientific Ltd.,
Catalogue No: 23966-2), diluted to a stock concentration of 0.9mg/ml in
dimethylsulfoxide (DMSO) (Sigma-Aldrich, Catalogue No: D-8779), followed by
filtration through a 0.2m mesh, aliquotted and stored at -20C.
Vector DNA: e.g. gWIZ GFP mammalian expression vector (Amsbio, Catalogue No:
P040400), provided at working concentration (1g/l) and stored at -20oC.
DiI Labelling:
CellTrackerTM CM-DiI (Invitrogen, Catalogue No: C-7000), diluted to a stock concentration
of 2mg/ml in dimethylformamide (DMF) (Sigma-Aldrich, Catalogue No: D-4551) and stored
at -20C.
Hoechst Counterstaining:
Hoechst 33342 (Invitrogen, Catalogue No: H-3570), as supplied at the stock concentration of
10mg/ml and stored at 4oC.
Equipment and Materials Required for Imaging:
Confocal fluorescent microscope with live cell imaging capacity, i.e. time-lapse acquisition,
temperature-controlled stage and CO2 supply. HEPES-buffered cell culture media can be
used in cases where a CO2 supply is unavailable. In the example results below, a Zeiss LSM
510 was used.
Methods
1.0. DiI Labelling
Note: Hydrophobic dyes (eg. DiI, calcein) can be used to label cells grown on
AlvetexScaffold , but the dye must be added prior to cell seeding and the cells thoroughly
washed after staining to minimise binding of residual dye to AlvetexScaffold. As some
hydrophobic dyes will partition between dividing cells, the time in culture before imaging
must also be kept in mind to prevent excessive dye dilution within a growing cell population.
1.1. Expand populations of cells in culture medium as monolayer cultures in
conventional 2D plastic-ware according to standard procedures.
1.2. See further information at www.reinnervate.com/alvetex/protocols on how
to choose, handle and prepare the AlvetexScaffold for cell culture.
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GFP Labelling:
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Note: Although this example protocol describes the transfection of live cells in suspension
before seeding into AlvetexScaffold, adherent 3D AlvetexScaffold cultures can also be
transfected and imaged.
2.1. Expand populations of cells in culture medium as monolayer cultures in
conventional 2D plastic-ware according to standard procedures.
2.2. See further information at www.reinnervate.com/alvetex/protocols on how to
choose, handle and prepare the AlvetexScaffold for cell culture.
2.3. In a sterile 1.5ml tube, mix 2l of vector DNA with 11.11l of PEI solution, i.e.
equivalent to a DNA:PEI ratio of 1:5 (w/w), per well. Leave for 15 minutes at room
temperature.
2.4. Meanwhile, prepare a single-cell suspension of cells in media using Trypsin-EDTA
to remove cells from 2D plastic-ware. Count cells and prepare seeding suspensions
in medium to allow for a seeding density of 1x106 cells in 0.5ml, per well.
2.5. Add 0.5ml of cell suspension to the DNA:PEI solution and mix gently. This is
equivalent to 2g DNA per million cells.
2.6. Seed cells on AlvetexScaffold according to the format used and leave the cultures
in a 37C, 5% CO2 cell culture incubator. Replace the medium with appropriate
fresh cell culture medium the following day and return the cultures to the 37C,
5% CO2 cell culture incubator until the desired imaging timepoint. Protect
from light.
Example Results
CHO-K1 cells (ATCC, CCL-61) were maintained in F-12K medium (Kaighns modification,
Gibco, 21127022) supplemented with 10% (v/v) foetal calf serum, 100g/ml penicillin and
10g/ml streptomycin (for full methods refer to Example protocol for the culture of CHO-K1
cells on AlvetexScaffold located at www.reinnervate.com/alvetex/protocols). For imaging,
cultures were switched to Hams F-10 nutrient mixture buffered with HEPES (Gibco,
22390025), supplemented with 10% (v/v) foetal calf serum, 100g/ml penicillin and 10g/ml
streptomycin.
Below are captured images of live cells over a time-course: labelled with CellTracker
(Figure 2.) and cells transfected with GFP (Figure 3.).
Figure 2: CHO-K1 cells were stained with CellTrackerTM CM-DiI (1/1000 dilution) immediately
prior to seeding onto AlvetexScaffold 6-well inserts. After five days, cultures were imaged
on a ZEISS LSM 510 confocal microscope. Cells were stained with Hoechst 33342 the
evening prior to imaging. Note the dividing cell (arrows).
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For all the example labelling protocols described above (sections 1.0 and 2.0), cells can be
counterstained with Hoechst 33342 to check for labelling efficiency. Shortly prior to imaging,
add Hoechst 33342 at a dilution of 1/1000 of the supplied stock solution. Leave for 30
minutes at room temperature, then replace the medium with fresh cell culture medium and
proceed with imaging. Protect from light until imaging.
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Figure 3: CHO-K1 cells were grown on AlvetexScaffold 6-well inserts. After five days,
cultures were transfected with G-Wiz GFP construct and left to recover for a further 24 hours
before imaging on a ZEISS LSM 510 confocal microscope. Pictures shown are flattened
Z-stacks (6 sections, total height 18m). Note the development of a cytoplasmic extension
during the 3:30 hour recording period (arrows).
2.0. Method
2.1. If using the AlvetexScaffold 12-well plate format (AVP002, 22 mm disc), gently remove the
clip and lift the 3D culture using flat-ended metal forceps. If using well inserts (AVP004-3, 22
mm disc or AVP005-3, 15 mm disc) unclip the base of the insert to release the 3D culture.
2.2. Place each 3D culture into a fresh well
Note: To improve the efficiency of the subsequent washing, fixing and blocking steps of this
protocol, each 3D culture should be homogenously exposed to a suitably large volume of
solution. Therefore it is recommended that at this stage 3D cultures grown on 22 mm discs
be placed into 6-well plates and 3D cultures grown on 15 mm discs be placed into 12-well
plates. The plates maybe gently rocked to facilitate diffusion.
2.3. Wash the 3D culture in PBS, carefully aspirate and repeat the PBS wash twice
Note: Thorough washing of 3D cultures requires an excess volume of PBS. Therefore it is
recommended to use at least 5 ml of solution for 3D cultures placed into 6-well plates and at
least 3 ml of solution for 3D cultures placed into 12-well plates for each washing step.
2.4. Fix the 3D culture using either a fresh 4% paraformaldehyde solution (in PBS) for 10 min
at room temperature or an ice-cold methanol/acetone (1:1 v/v) solution for 20 min in the fridge.
Note: The choice of the fixative solution should be in accordance with the manufacturers
instructions for the antibodies used. As above, use excess volume of fixing solution. Use a
glass vessel if fixing with methanol/acetone mixture.
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1.0. Introduction
Immunofluorescence uses the recognition of cellular targets by fluorescent dyes or antigenspecific antibodies coupled to fluorophores. Depending on the antibody or dye used, proteins,
lipids and DNA can be visualised within individual cells and tissues. For 2D cell cultures, 3D cell
culture sections or tissue sections, conventional microscopy is generally sufficient to visualise
samples processed by immunofluorescence. For thicker specimens or when sectioning is not
desired, confocal microscopy is to be preferred. Confocal microscopy relies on the combination
of point illumination and a pinhole to eliminate most of the out-of-focus light signal and allows
for reconstruction of 3D volumes, making it ideal to image cultures grown in full-thickness
AlvetexScaffold.
Below is an example protocol. It is recommended that for each antibody or dye used, the
original manufacturers instructions for fixing, blocking and incubating methods be followed.
Imaging of the specimens will also need to be optimised according to the specifications of the
confocal microscope used.
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2.12. At the end of the primary antibody incubation period, return the 3D culture facing
upwards to a new plate and wash the 3D culture three times in the PBS/BSA wash solution.
(see 2.3 for volumes required).
2.13. Place the 3D culture facing upwards onto a new, dry glass slide. Circle with a PAP or
DAKO pen as previously (step 2.9.).
2.14. Pipette the secondary antibody solution onto the 3D culture inside a humidified chamber.
Protect from light and leave for 1h at room temperature (or as required by the manufacturers
instructions). In the meantime, label microscope slides.
2.15. At the end of the secondary antibody incubation period, return the 3D culture facing
upwards to a new plate and wash the 3D culture three times in the PBS/BSA washing solution
(see step 2.12.).
2.16. Put a drop of mounting medium on a microscope slide. One drop will be required for each
3D culture. Place the 3D culture facing upwards on the microscope slide and add one further
drop of mounting medium on top of the 3D culture. Cover the 3D culture with a coverglass
while taking care not to create air bubbles, then seal with nail varnish or tape. If using nail
varnish, leave to dry for 10-30 min at room temperature.
2.17. The sample is now ready for imaging by microscopy. If imaging is to be done at a later
date, store the prepared microscope slides in the dark at 40C.
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2.11. Leave the 3D culture in the primary antibody solution for the desired time at the required
temperature. This is usually 1-2 hours at room temperature or overnight at 2-8oC. In the
meantime, dilute the secondary antibodies or dyes in the PBS/BSA wash solution at the
working concentrations as recommended by the manufacturer.
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3.0 Special Considerations for the Imaging of Cell Cultures Grown in AlvetexScaffold
by Confocal Microscopy
The material used to manufacture AlvetexScaffold will produce minimal autofluorescence and
should not result in background signal when using confocal microscopy. Efficient washing and
blocking steps will prevent any significant binding of secondary antibodies to AlvetexScaffold.
However, lipophilic dyes, such as Nile Red (see below), will bind strongly to AlvetexScaffold.
This feature can be used to conveniently visualise the scaffold within the cell culture.
AlvetexScaffold discs are supplied at a thickness of 200 m, which might exceed the Z-stack
capacity of some models of confocal microscopes. The exact depth of sample that can be
successfully imaged will vary according to the density of the cell culture and the fluorophore
used, so that depths of more than 100 m can be imaged from a cell-free AlvetexScaffold disc,
while depths of between 50 and 100 m can be expected from a cell-seeded AlvetexScaffold
disc. As high-density cell cultures grown in AlvetexScaffold approximate the complexity and
structure of in vivo tissues, fluorophores specifically developed for in vivo deep imaging can be
used to improve performance if needed.
4.0. Example pictures
4.1. AlvetexScaffold stained with Nile Red
A working solution was prepared by diluting stock solution of Nile Red (Sigma, N3013) 1 in
1000. The stock solution was initially made up in methanol at 1 mg/ml, kept in a fridge and
protected from light. AlvetexScaffold was incubated 10 minutes at room temperature with
the working solution in the dark, washed three times in PBS and mounted.
Note: Nile Red staining is best carried out after the secondary antibody stage following the
PBS wash. Adhere strictly to incubation times and concentrations as higher concentrations of
the working solution will produce strong staining in the red channel and a signal in the green
channel.
Figure 2. HepG2 cells grown for 3 days in AlvetexScaffold 12-well plate format (AVP002).
Cells were stained with Hoechst 33342 (blue), cytokeratin 8 (green) and Nile Red (red).
Pictures were taken on a Zeiss LSM 510 confocal microscope. Note the background signal
from AlvetexScaffold in the blue and green channels is very low. Scale bar 10 m.
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Introduction:
The alamarBlue assay is based on the biological reduction of resazurin to the fluorescent
molecule, resorufin. Resazurin is nontoxic and will readily penetrate into viable cells
whereupon it is metabolised. The read out options for the assay are absorbance or
fluorescence, the latter being the more sensitive. The resazurin substrate is blue in colour and
virtually non-fluorescent, resulting in very low background values in the fluorescent read out.
The amount of resorufin product increases with time and deviation from linearity can be
anticipated at later time points. Therefore, it is recommended that for each cell type the linear
relationship between both cell number and time, and signal produced should be first
established in order to investigate the limits of the assay.
Methods:
These are example protocols which may require further optimisation depending on the rate
of cell growth of a particular cell type on AlvetexScaffold. The protocols describe cell viability
analysis of upcyte hepatocytes (Medicyte, www.medicyte.com) using the alamarBlue
Assay (Invitrogen, DAL1010, 1025, 1100). Upcyte cells were thawed, immediately seeded
onto the AlvetexScaffolds and medium added after a 3 hour attachment period. At various
time-points, the growth and viability of the upcyte hepatocytes were monitored as
described below.
HPM/01/5/2012
1.
2.
Remove the AlvetexScaffold disc from the culture vessel and dismantle inserts
where applicable. For 22 mm AlvetexScaffold discs, transfer to the wells of a
clean 12-well plate. For 15 mm AlvetexScaffold discs, transfer to the wells of a
clean 24-well plate.
3.
4.
5.
Transfer 100 l aliquots of each sample to replicate wells of a black 96-well plate.
6.
2.
Remove the AlvetexScaffold insert from the culture vessel and transfer to a
clean plate. For 22 mm AlvetexScaffold inserts, transfer to a clean 6-well plate.
For 15 mm AlvetexScaffold inserts, transfer to a clean 12-well plate.
3.
4.
5.
Transfer 100 l aliquots of each sample to replicate wells of a black 96-well plate.
6.
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1.
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Method:
1.
To create a cell number standard curve, harvest cells from 2D cultures using an
appropriate detachment method.
2.
Count cells and re-suspend appropriate cell number ranges in 1 ml lysis buffer
(10 mM Tris pH 8, 1 mM EDTA and 0.2 % (v/v) Triton X-100). Note: Always work
RNase and DNase free where possible.
3.
Vortex samples for 10 seconds every five minutes for half an hour, keeping on ice
throughout. Note: At this point samples can be stored at -80 C until the assay is
ready to be performed.
4.
5.
6.
7.
Add 100 l of PicoGreen reagent, mix and incubate at room temperature for
5 minutes wrapped in aluminum foil.
8.
9.
For cells cultured in AlvetexScaffold, wash in PBS and remove disc from culture
format used using forceps and place in a 1.5 ml tube
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Introduction:
The following protocol outlines a method for determining cell numbers within
AlvetexScaffold using the PicoGreen Assay. PicoGreen is an ultrasensitive ( 50 cells)
fluorescent nucleic acid dye which provides linear results over multiple orders of magnitude
with a single dye concentration. It is therefore the dye of choice over Hoescht and DAPI for
this type of quantitative assay. Example data was obtained using this protocol with HepG2
hepatocytes cultured in AlvetexScaffold for 7 days.
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Results:
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Conclusions:
The PicoGreen assay can be used to evaluate the growth characteristics of HepG2 cells
cultured on 22 mm AlvetexScaffold discs in 6-well insert format. HepG2 cells show an initial
lag phase for 24 hours before moving into log phase growth, with a population doubling time
of just over 48 hours (from gradient evaluation, 56 hours). After 4 days of culture HepG2
cell growth slows with a plateau region showing no increase in cell population between 4 and
7 days.
This method is a useful approach to determine cell number within a 3D culture.
Methods:
These are example protocols which may require further optimisation depending on the rate
of cell growth of a particular cell type on AlvetexScaffold. The protocols describe cell viability
analysis of upcyte hepatocytes (Medicyte) using the CellTiter 96 AQueous Non-Radioactive
Cell Proliferation Assay (Promega). Upcyte cells were thawed, immediately seeded onto
the AlvetexScaffolds and medium added after a 3 hour attachment period. At various timepoints, the growth and viability of the upcyte hepatocytes were monitored as described below.
HPM/01/5/2012
1.
2.
Dilute the MTS/PMS solution 1:5 in appropriate diluent (e.g. PBS, or phenol red-free
culture medium).
3.
Remove the AlvetexScaffold disc from the culture vessel and dismantle
inserts where applicable. For 22 mm AlvetexScaffold discs, transfer to the wells
of a clean 12-well plate. For 15 mm AlvetexScaffold discs, transfer to the wells
of a clean 24-well plate.
4.
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Introduction:
This chromogenic assay involves the biological reduction by viable cells of the tetrazolium
compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (or MTS). The MTS assay reagent is composed of MTS and the electron coupling
agent phenazine methosulfate (PMS). The formazan product of MTS reduction is soluble in
tissue culture medium. This reaction only takes place when mitochondrial reductase enzymes
are active, and therefore the conversion can be directly related to the viability of cells in
culture. The MTS reagent alone results in very low background absorbance values in the
absence of cells. It is recommended that for each cell type the linear relationship between
cell number and signal produced should be first established in order to investigate the limits
of the assay.
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5.
6.
Transfer 100 l aliquots of each sample to replicate wells of a clear 96-well plate.
7.
2.
Dilute the MTS/PMS solution 1:5 in appropriate diluent (e.g. PBS, or phenol red-free
culture medium).
3.
Remove the AlvetexScaffold insert from the culture vessel and transfer to a
clean plate. For 22 mm AlvetexScaffold inserts, transfer to a clean 6-well plate.
For 15 mm AlvetexScaffold inserts, transfer to a clean 12-well plate.
4.
5.
6.
Transfer 100 l aliquots of each sample to replicate wells of a clear 96-well plate.
7.
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Fixation methods
Fixation is performed in order to preserve the tissue or culture from the putrefaction
(destruction by micro-organisms), autolysis (self-digestion by lysosomal enzymes) and
abnormal metabolism of the isolated tissues deprived of oxygen and nutrients. Fixation of 3D
cultures in AlvetexScaffold can be achieved by chemical fixation. The use of uncontrolled
high heat by flame is not recommended.
Fixation is usually the first stage in a multistep process to prepare a sample for microscopy
or other analysis. So, when choosing a fixative, the final purpose of the section must be
considered and matched by the requirements for the analytical technique. Some fixatives
are suitable for general structure analysis, others for evaluating cytological detail and some
for histochemistry. Survival of tissue antigens for immunochemical staining depends on the
type and concentration of fixative, on fixation time, and on the size of the tissue specimen
to be fixed. Given the thin nature of AlvetexScaffold, fixation of cells is rapid, uniform and
efficient, preserving the 3D culture in a life-like condition.
02
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Introduction
These histology protocols contain a series of detailed methods that will allow the user to
examine the morphology of their cultured cells subsequent to 3D growth. These are not in
any way exhaustive and there is scope to include additional methods as the user deems
appropriate. Users should also perform their own risk assessments as safety evaluations
contained in these protocols are for information only.
03
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Paraformaldehyde (4 % PFA):
Suitable for paraffin embedding and sectioning. It is the fixative of choice for immunocytochemical analysis. Samples can also be stained for general histology but the degree
of fixation is less vigorous than Bouins so the quality of the morphology obtained will
be less. This fixative allows for subsequent immuno-detection of certain antigens and
should therefore be used when the objective is to study morphology and protein
expression simultaneously. (See Figure 2.)
Method:
1. Working in a fume cupboard wearing appropriate PPE heat up 1 litre
of Phosphate Buffered Saline (PBS) to 60 C on a hotplate stirrer.
2. Add 40 g of paraformaldehyde (PFA) powder to the warm PBS. Use a magnetic
stirrer and hotplate to dissolve PFA.
3. Slowly add drops of 1 M sodium hydroxide solution until the solution is clear.
4. Filter the fixative and adjust pH to 7.3-7.4.
5. Allow fixative to cool to 53 C before use.
6. Aliquots can be stored at 20 C with a shelf life of 6 months.
Safety precautions:
CARCINOGENIC, IRRITANT, CORROSIVE, and TOXIC.
Karnovskys fixative:
Is a mixture of paraformaldehyde and glutaraldehyde. It is suitable for use when preparing
samples for light microscopy in resin embedding and sectioning, and for electron
microscopy. This fixative should always be prepared fresh. (See Figures 3 and 4
respectively).
Method:
Karnovskys fixative is 2 % paraformaldehyde, 2.5 % glutaraldehyde in 0.1 M
phosphate buffer pH 7.4.
1.
Prepare 8 % PFA:
2.
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04
05
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06
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07
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Introduction
Immunofluorescence uses the recognition of cellular targets by fluorescent dyes or antigenspecific antibodies coupled to fluorophores. Depending on the antibody or dye used,
proteins, lipids and DNA can be visualised within individual cells and tissues. AlvetexScaffold
can easily be processed like a standard tissue sample, allowing established
immunofluorescence methods to be followed with excellent results. An example protocol is
outlined below.
3.2. If using well inserts in Petri dishes, remove inserts from holder and place in
a conventional 6-well plate. To fix, add in 5 ml 4 % PFA fixative at 4 C. Fix
specimens at 4 C for a minimum of 12 hours but no longer than 24 hours.
3.3. Aspirate off the fixative, wash 3 times using 5 ml PBS to thoroughly remove
excess fixative, discarding the waste liquid.
3.4. If using inserts, remove AlvetexScaffold from the well insert either by
separating the two parts of the device or using a scalpel to cut out the scaffold.
At this time samples can be transferred to a tissue processing cassette to
minimize direct handling and damage to the 3D culture.
3.5. Aspirate off the PBS and add 5 ml of 30 % ethanol. Leave to equilibrate for
at least 15 minutes. Aspirate off the ethanol and discard.
3.6. Repeat with 50 %, 70 %, 80 %, 90 % and then 95 % ethanol. A gradual
dehydration of the sample will result in less tissue shrinkage. Material can
be stored in 95 % ethanol prior to paraffin embedding.
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3.1. Aspirate off the medium and carefully wash the 3D culture in PBS twice.
08
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5.8. Incubate with primary antibody diluted in blocking buffer (this will be
specific to the antigen chosen and will be used at a pre-determined
concentration) overnight at 4 C in a humidified chamber, or as stated in
the datasheet for the specific antibody used.
10
Example results
Figure 1. below shows an example of a 3D AlvetexScaffold culture fixed with 4 % PFA,
embedded in paraffin wax, sectioned and probed with Ki67 antibody using
immunocytochemistry and standard fluorescence microscopy.
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Protocol Booklet
5.10. Incubate with secondary antibody diluted in blocking buffer (this will be
specific to the antigen chosen and will be used at a pre-determined
concentration) for 2 hours in the dark at room temperature.
11
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Introduction
Following fixation a variety of different cytological stains can be used to visualise cell components
in detail. Here we describe the use of Haematoxylin and Eosin, a general structural stain, one of
the most commonly used stains throughout histology. The hemalum component of haematoxylin
stains cell nuclei blue. Counterstaining of samples with an alcoholic eosin Y solution stains other
eosinophilic structures in various shades of red, pink and orange. Most of the cell cytoplasm is
eosinophilic and therefore stains pink. AlvetexScaffold can easily be processed like a standard
tissue sample, allowing established histology methods to be followed with excellent results.
An example protocol is outlined below.
Method
1.0 Materials Required
Bouins fixative (for full recipe see Histology Series Part 1. Choosing
the Right Fixative to Preserve 3D Cell Cultures at
www.reinnervate.com/alvetex/protocols)
Dehydration ethanols (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, 100 %)
Histoclear (Note: the use of Histoclear is recommended over the use of Xylene).
Paraffin wax
Mayers Haematoxylin solution
Eosin solution (2 g eosin in 1000 ml 95 % alcohol)
Alkaline alcohol
DPX mountant
Basic equipment: spade forceps to handle 3D cultures, scalpel (to trim/cut
AlvetexScaffold cultures if required), disposable pipettes, convection oven,
embedding moulds, microtome.
2.5. Aspirate off the PBS and add 5 ml of 30 % ethanol. Leave to equilibrate for
at least 15 minutes. Aspirate off the ethanol and discard.
2.6. Repeat with 50 %, 70 %, 80 %, 90 % and then 95 % ethanol. A gradual
dehydration of the sample will result in less tissue shrinkage. Material can
be stored in 95 % ethanol prior to paraffin embedding.
3.0 Paraffin Embedding and Sectioning
3.1. Fully
dehydrate
3.0 Paraffin
Embedding
andspecimens
Sectioningstored in 95 % ethanol by replacing with 100 %
absolute ethanol for at least 30 minutes.
3.1. Fully dehydrate specimens stored in 95 % ethanol by replacing with 100 %
ethanol
for at least
30 minutes.
3.2. absolute
Aspirate off
the ethanol
and replace
with Histoclear for at least 30 minutes.
Histoclear
will dissolve
certainwith
types
of plastic,
it is
best
3.2. (Note:
Aspirate
off the ethanol
and replace
Histoclear
fortherefore
at least 30
minutes.
to process samples in glass tissue processing cassettes).
(Note: Histoclear will dissolve certain types of plastic, therefore it is best
to process
in glass
3.3. Replace
thesamples
Histoclear
with atissue
50:50processing
solution of cassettes).
Histoclear and molten paraffin
wax (60 C) mix and incubate in a convection oven at 60 C for 30 minutes.
3.3. Replace the Histoclear with a 50:50 solution of Histoclear and molten paraffin
wax Replace
(60 C) mix
incubate in amix
convection
at 60 wax
C forand
30incubate
minutes.at
3.4.
the and
Histoclear:wax
with 100 oven
% molten
60 C for a further 60 minutes.
3.4. Replace the Histoclear:wax mix with 100 % molten wax and incubate at
C forthe
a further
60tominutes.
3.5. 60
Transfer
scaffold
plastic embedding moulds and orientate into the
required embedding position, with plane of section in mind. Embed in
3.5. Transfer the scaffold to plastic embedding moulds and orientate into the
molten wax.
required embedding position, with plane of section in mind. Embed in
wax.
3.6. molten
Allow wax
to cool and set at room temperature for 1-2 hours or overnight.
3.6. Once
Allow the
waxwax
to cool
set at room
temperature
for 1-2 hours
or from
overnight.
3.7.
has and
hardened,
remove
the wax embedded
block
the
plastic mould. The sample is now ready for sectioning on a suitable
3.7. Once the wax has hardened, remove the wax embedded block from the
microtome (e.g. Leica RM2125).
plastic mould. The sample is now ready for sectioning on a suitable
(e.g.
Leica RM2125).
3.8. microtome
Following the
microtome
manufacturers instructions throughout, align the
correctly
with the microtome
blade instructions
and proceedthroughout,
to cut 10 m
3.8. block
Following
the microtome
manufacturers
align the
sections of the sample block.
block correctly with the microtome blade and proceed to cut 10 m
of the sample
block.
3.9. sections
Transfer sections
to a slide
water bath (40 C), floating them on the surface
of the water to enable them to flatten out.
3.9. Transfer sections to a slide water bath (40 C), floating them on the surface
the water
to enable
themtotoslides
flattenbyout.
3.10.ofTransfer
selected
sections
flotation. Superfrost Plus slides
(Thermo, 4951PLUS4) are recommended.
3.10. Transfer selected sections to slides by flotation. Superfrost Plus slides
(Thermo,
areleave
recommended.
3.11. Place
on a4951PLUS4)
slide drier and
overnight. The sections
should now be readyfor staining.
3.11. Place on a slide drier and leave overnight. The sections
should now be readyfor staining.
12
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2.4. If using inserts, remove AlvetexScaffold from the well insert either by
separating the two parts of the device or using a scalpel to cut out the
scaffold. At this time samples can be transferred to a tissue processing
cassette to minimize direct handling and damage to the 3D culture.
13
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5 minutes
2 minutes
1 minute
4.5. 70 % alcohol
1 minute
1 minute
5 minutes
30 seconds
30 seconds
30 seconds
4.11. 95 % alcohol
30 seconds
1 minute
10 seconds
4.14. 95 % alcohol
10 seconds
15 seconds
30 seconds
3 minutes
4.18. Histoclear
3 minutes
4.19. Retain slides in Histoclear until ready to coverslip to prevent drying out.
14
Example results
Figure 1. shows an example of paraffin embedded 3D AlvetexScaffold culture, sectioned
and stained with Hematoxylin and Eosin.
Figure 1. Human keratinocyte
cell line (HaCaT) grown in
AlvetexScaffold (7 days air
exposure). The subsequent
culture was fixed in Bouins
fixative and processed for
paraffin wax embedding and
sectioning. The sectioned
materials (10 m) were stained
with Haematoxylin and Eosin
for morphological analysis.
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Protocol Booklet
5.1. Mount in DPX by spotting DPX around the section and carefully cover with
a coverslip ensuring no air bubbles are present. Allow to air dry for at least
one hour.
15
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Introduction
Following fixation, a variety of cytological stains can be used allowing different cell
components to be visualised in detail. When structural integrity of sample architecture is
paramount, samples are embedded in resin. Resin embedding allows ultrathin sections of
samples to be obtained with no loss of structural detail.
Here we describe the use of toluidine blue as a stain of sectioned material for light
microscopy. Toluidine blue is a general histology stain that, in basic solutions, binds to nucleic
acids and all proteins. It is also commonly used for staining thin sections (0.5-1 m)
of resin embedded tissues to aid with the orientation and visualisation of samples
subsequently prepared for electron microscopy. While everything in the sample stains
with the dye, structural details remain highly prominent due to the thinness of the sections.
AlvetexScaffold can easily be processed like a standard tissue sample, allowing
established histology methods to be followed with excellent results. An example protocol is
outlined below.
Method
1.0 Materials Required
Karnovskys fixative (2 % paraformaldehyde; 2.5 % glutaraldehyde in
0.1 M phosphate buffer pH 7.4. (for full recipe see Histology Series Part 1.
Choosing the Right Fixative to Preserve 3D Cell Cultures at
www.reinnervate.com/alvetex/protocols)
0.1 M phosphate buffer (for full recipe see Histology Series Part 1.
Choosing the Right Fixative to Preserve 3D Cell Cultures at
www.reinnervate.com/alvetex/protocols)
Dehydration ethanols (30 %, 50 %, 75 %, 95 %, 100 %)
1 % buffered osmium tetroxide (1 % osmium tetroxide solution
(Agar scientific Ltd), 0.1 M phosphate buffer (pH 7.4). (for full recipe detail
see Histology Series Part 1. Choosing the Right Fixative to Preserve 3D Cell
Cultures at www.reinnervate.com/alvetex/protocols)
L R White Resin (Agar Scientific Ltd)
1 % toluidine blue in 1 % sodium borate
DPX
Basic equipment: spade forceps to handle 3D cultures, scalpel (to trim/cut
AlvetexScaffold cultures if required), disposable pipettes, embedding
moulds, wire loop
2.2. Remove well inserts from cradle and carefully cut small samples (2-3 mm
square) from the scaffold disc.
2.3. Immerse samples in Karnovskys fixative at 4 C for 90 minutes. The ratio
of fixative volume to specimen size should be approximately 20:1.
2.4. After fixation, aspirate the fixative into a waste container for disposal.
2.5. Wash the specimen twice for 2 minutes in 3 ml 0.1 M phosphate to remove
excess fixative, discarding waste liquid.
2.6. Add 20x the specimen volume of 30 % ethanol. Leave to equilibrate for
5 minutes preferably on a rotating wheel. Pour off the ethanol and discard.
Repeat twice.
2.7. Repeat step 2.6 with 50 %, 75 %, 95 % and then absolute ethanol.
The sample can be stored in absolute ethanol prior to resin embedding.
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2.1. Aspirate off the medium and carefully wash the 3D culture twice with PBS.
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Example Results
Figure 1 shows an example of a resin embedded 3D AlvetexScaffold culture, sectioned
and stained with Toluidine Blue.
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Introduction
Scanning electron microscopy (SEM) is becoming a popular method for visualisation of
cultures grown in 3D. SEM is a form of electron microscopy where images are obtained by
scanning samples using a high-energy beam of electrons. As the samples are scanned the
electrons interact with atoms located on the sample surface, and it is these interactions that
are detected, and processed, leading to high-resolution images depicting the samples
topography and composition.
For conventional imaging, SEM samples must be electrically conductive and completely dry
(due to the specimen chamber being at high vacuum).To achieve this, samples usually
undergo chemical fixation to preserve and stabilise their structure. Standard SEM fixing
solutions are Karnovskys fixative followed by a second fixing in osmium tetroxide, which
increases the bulk conductivity of the sample. Samples are completely dried using a critical
point dryer, and finally, fixed samples are sputter coated with gold to further enhance
their conductivity.
AlvetexScaffold can easily be processed like a standard tissue sample, allowing established
methods to be followed with excellent results. An example protocol is outlined below.
Imaging of the specimens will need to be optimised according to the specifications of the
scanning electron microscope used.
Method
1.0 Materials required
Karnovskys fixative (2 % paraformaldehyde; 2.5 % glutaraldehyde in
0.1 M phosphate buffer pH 7.4. (for full recipe detail see Histology
Series Part 1. Choosing the Right Fixative to Preserve 3D Cell Cultures
at www.reinnervate.com/alvetex/protocols)
Dehydration ethanols (30 %, 50 %, 75 %, 95 %, 100 %)
1 % buffered osmium tetroxide (1 % osmium tetroxide solution; 0.1 M
phosphate buffer (pH 7.4). (For full recipe details see Histology Series
Part 1. Choosing the Right Fixative to Preserve 3D Cell Cultures at
www.reinnervate.com/alvetex/protocols)
0.1 M phosphate buffer pH 7.4
Basic equipment: spade forceps to handle 3D cultures, scalpel (to trim
AlvetexScaffold cultures if required), disposable pipettes.
Specialised equipment to prepare samples for SEM: critical point dryer,
sputter coater.
2.0. Protocol
2.2. Remove well inserts from cradle and carefully cut small samples
(2- 3 mm square) from the scaffold disc.
2.3. Immerse samples in Karnovskys fixative at 4 C for 90 minutes. The ratio
of fixative volume to specimen size should be approximately 20:1.
2.4. After fixation, aspirate the fixative into a waste container for disposal.
2.5. Wash the specimen twice for 2 minutes in 3 ml 0.1 M phosphate to
remove excess fixative, discarding waste liquid.
2.6. Post fix samples in 1 % buffered osmium tetroxide for 90 minutes at
4 C, then remove the osmium tetroxide solution.
2.7. Add 20x the specimen volume of 30 % ethanol. Leave to equilibrate
for 5 minutes preferably on a rotating wheel. Pour off the ethanol and
discard. Repeat twice.
2.8. Repeat step 2.7 with 50 %, 75 %, 95 % and then absolute ethanol.
The sample can be stored in excess absolute ethanol.
2.9. Follow the manufacturers instructions to completely dry the specimen
using a critical point dryer.
2.10. Follow the manufacturers instructions to gold plate the sample using
a sputter coater. The gold coating will increase the samples
conductivity and consequently improve image quality.
2.11. The samples are now ready for imaging using a conventional scanning
electron microscope.
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2.1. Aspirate off the medium and carefully wash the 3D culture twice with PBS.
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Example Results
Figure 1 below shows an example of a 3D AlvetexScaffold culture imaged using the
SEM technique.
A
Processing AlvetexScaffold
3D Cultures for Cryosectioning.
AlvetexScaffold 3D cell cultures can be easily processed for cryosectioning. This document
describes three separate methods and shows example data obtained from the culture and
processing of HaCaT and HepG2 cells.
Liquid nitrogen
HPM/01/5/2012
Histoclear
Alkaline alcohol
DPX mountant
22
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Introduction:
Tissue freezing and sectioning is a rapid method of generating tissue samples (cryosections)
for histological analysis, and obviates the need for wax embedding. The method is popular
for preparing samples for immunostaining since the need for antigen retrieval is also
eliminated.
Processing AlvetexScaffold
3D Cultures for Cryosectioning.
23
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Humidified chamber
Vectashield/DAPI mountant
3.0 Freezing:
3.1
Aspirate culture medium and carefully wash the 3D culture in PBS twice.
3.2
If using well inserts, remove inserts from cradle and place in a conventional
6-well plate.
3.3
Bisect the membrane using surgical scissors or a single edge razor blade.
3.4
At this point one of the following three techniques can be chosen depending
upon personal preference and experience:
Processing AlvetexScaffold
3D Cultures for Cryosectioning.
3.2 Method 2: Samples can be soaked in sucrose before freezing to remove excess
water (more suitable for cells in collagen gels)
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24
Processing AlvetexScaffold
3D Cultures for Cryosectioning.
25
4.0 Sectioning
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Protocol Booklet
4.1 At least twenty-four hours after freezing, the samples are ready for sectioning
on a suitable cryostat (e.g. Leica CM1850).
4.2 Let the sample sit in the cryostat for 20-30 minutes before sectioning to allow
the sample to adjust to the temperature.
4.3 Following the cryostat manufacturers instructions throughout, align the block
correctly with the cryostat blade and proceed to cut 5-10 m sections.
4.4 Transfer sections to microscope slides. Histobond slides are recommended
since sections adhere to these slides well.
4.5 Let the sections dry for at least 2 hours before processing. The sections should
now be ready for histological and/or immunocytochemical analysis.
5.0 Analysis
5.1 Histological analysis
Set up a series of slide baths and process sample slides using slide holders
in the following sequence of solutions, for the times indicated:
5.1.1 Rehydrate section in PBS.
10 minutes
15 minutes
5 minutes
1 minute
5 minutes
30 seconds
30 seconds
30 seconds
5.1.9 95 % alcohol
30 seconds
1 minute
10 seconds
5.1.12 95 % alcohol
10 seconds
15 seconds
30 seconds
Processing AlvetexScaffold
3D Cultures for Cryosectioning.
3 minutes
5.1.16 Histoclear
3 minutes
5.1.17 Slides remain in Histoclear until ready to coverslip to prevent drying out.
5.1.18 Mount slides by spotting DPX solution around the section and cover with
a coverslip. Allow to air dry for at least one hour.
Figure 1 shows examples of frozen 3D AlvetexScaffold cultures, cryosectioned and stained
with Haematoxylin and Eosin.
26
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Processing AlvetexScaffold
3D Cultures for Cryosectioning.
27
Example Data:
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Protocol Booklet
Processing AlvetexScaffold
3D Cultures for Cryosectioning.
28
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29
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01
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02
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Introduction
The following protocol outlines a method for the extraction of total protein from cells cultured
in AlvetexScaffold. Example data was obtained using this protocol to extract protein from
HepG2 hepatocytes cultured in AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in Well
Insert Holder in Deep Petri Dish (AVP015) format.
Method:
1. Prepare lysis buffer: 1 % v/v Igepal CA-630 (Sigma, Product No. I3021), 50 mM Tris-HCl
pH 8.0, 150 mM NaCl, 1 mM MgCl2, and Complete Mini Protease Inhibitor Cocktail (Roche
Diagnostics, 11 836 153 001).
Note: The Protease Inhibitor Cocktail is available in two forms, with or without EDTA lysate,
see manufacturer instructions for recommendation.
2. Remove AlvetexScaffold disc from culture format (e.g. well insert or 12-well multiwell
plate) and wash by gentle immersion in PBS (e.g. in a Petri dish). Transfer into a 1.5 ml tube.
3. Add 1 ml of lysis buffer and vortex for 10 seconds. Place on ice for 30 minutes with
vortexing (10 sec.) every 5 minutes.
4. Clarify lysates by centrifugation at approx 15,000 rpm for 3 minutes and place supernatant
into a fresh tube.
5. The samples can be frozen at this stage (e.g. at -80oC), for use at a later date.
6. Determine total protein content of samples using a protein determination assay (e.g. BioRad protein assay).
Example: Extraction of Total Protein from HepG2 Hepatocytes Cultured in AlvetexScaffold in 3D
Cell Culture:
HepG2 cells (ATCC, HB-8065) were routinely maintained in T-75 flasks. HepG2 complete media
consisted of: MEM media (Gibco, 21090) supplemented with 10 % v/v FBS, 2 mM L-glutamine
and 100 U/ml Penicillin/Streptomycin. Cells were seeded onto AlvetexScaffold discs in 6-well
inserts (AVP004-3) in well insert holders in deep Petri dishes (Product No. AVP015), at a density
of 1 x 106 cells in 150 l media per insert. After settling for 3 hours in an incubator (5 % CO2,
37C) complete media was carefully added (70 ml per Petri dish). Cultures were maintained for
7 days, with a single media change on day 5. After 7 days, well inserts were dismantled and
cultures were processed according to the protocol described above.
Sample 1
Sample 2
1.08 (0.01)
1.02 (0.01)
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04
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Protein extracts were also analysed by SDS-polyacrylamide gel electrophoresis and western
blotting (Figure 2). Protein extracts from two biological replicate cultures produced well-resolved
and highly reproducible SDS-PAGE profiles as determined via Coomassie Brilliant Blue R
staining. Western blotting demonstrated that the constitutive protein -actin was both intact
and equally loaded in both sample lanes.
Method:
1. Wash AlvetexScaffold culture by gentle immersion in PBS using flat-ended forceps and
transfer to a clean 12-well plate.
2. Lyse cells by adding 600 l Qiagen RNeasy kit lysis buffer RLT per well.
3. Place on a rotating platform (100 rpm) for 10 minutes at room temperature.
4. Homogenise the lysate by passage up and down through a 20-gauge needle 10 times
using a sterile plastic syringe.
5. Add an equal volume of 70 % ethanol to the homogenised lysate and pipette up and down
10 times to mix.
6. Transfer sample (up to 700 l at a time) to an RNeasy spin column in a 2 ml collection
tube. Close cap and microcentrifuge for 15 seconds at 8000 x g. Discard flow-through.
Repeat for remaining volume of lysate. Optional: On-column DNase digestion can be
performed at this point (steps 6 a-d). Alternatively proceed to step 7.
a. Add 350 l Buffer RW1 to the column. Close cap, and microcentrifuge for 15 seconds
at 8000 x g to wash the membrane. Discard flow-through.
b. Prepare DNase 1 reagent by adding 10 l DNase 1 stock solution (prepared according
to manufacturers instructions) to 70 l Buffer RDD. Mix by gentle pipetting or gentle
tube inversion.
c. Add the 80 l diluted DNase 1 reagent to the column membrane and incubate at room
temperature for 15 minutes.
d. Add 350 l Buffer RW1 to the column. Close cap and microcentrifuge for 15 seconds
at 8000 x g. Discard flow-through. Proceed to step 8.
7. Add 700 l Buffer RW1 to the column. Close cap and microcentrifuge for 15 seconds at
8000 x g, reusing the collection tube. Discard flow-through.
8. Add 500 l Buffer RPE (appropriately diluted with ethanol according to manufacturers
instructions) to the column. Close cap and microcentrifuge for 15 seconds at 8000 x g.
Discard flow-through.
05
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Introduction
The following protocol outlines the use of a commercial extraction kit (Qiagen RNeasy kit,
74104) for the isolation of total RNA from cells cultured in AlvetexScaffold. Example data was
obtained using this protocol to extract RNA from HepG2 hepatocytes cultured in
AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in Well Insert Holder in Deep Petri Dish
(AVP015) format.
06
9.
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Protocol Booklet
Add a further 500 l Buffer RPE to the column. Close cap and microcentrifuge for 2
minutes at 8000 x g. Discard flow-through.
10. Transfer column to a clean 2 ml collection tube. Close cap and microcentrifuge at full speed
for 1 minute.
11. Transfer column to a clean 1.5 ml collection tube and add 50 l RNase-free water onto the
column membrane. Close cap and leave at room temperature for 5 minutes.
Microcentrifuge for 1 minute at 8000 x g to elute RNA.
Sample 2
185.1 (0.044)
182.7 (0.470)
A260/A280
2.123 (0.012)
2.117 (0.006)
Table 1. RNA yield and purity analysis. RNA samples from duplicate HepG2 AlvetexScaffold
cultures were diluted 5-fold in 10 mM Tris-HCl, pH 7.0. Values shown represent the mean of
triplicate readings per sample (SD), taken on a NanoDrop 1000 spectrophotometer (Thermo
Scientific).
07
DNA
28 S
18 S
Figure 1. Agarose gel RNA profile from duplicate HepG2 AlvetexScaffold 3D cultures
(Samples 1 and 2). 28S and 18S rRNA bands are clearly visible confirming RNA integrity
(1% agarose gel; visualisation via ethidium bromide staining; 1 kb molecular weight ladder:
Promega, G571A).
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Sample
1
2
01
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Methods:
1.0. Standard Enzymatic Method
1.1. Unclip inserts and carefully remove the AlvetexScaffold discs using
flat-ended forceps.
1.2. Gently wash each disc in PBS, and transfer to a new 6-well plate.
1.3. 3 ml of 0.25 % Trypsin-EDTA (Sigma, T4049).
1.4. Incubate plate at 37 C, 5 % CO2 on a shaking platform set to 100 rpm,
for 15 minutes.
1.5. Transfer the resulting cell suspension to a 15 ml centrifuge tube.
1.6. Add 3 ml medium to the well containing the AlvetexScaffold membrane
and gently triturate to remove residual detached cells.
1.7. Combine the two solutions in the 15 ml centrifuge tube to neutralise
the trypsin.
1.8. Centrifuge at 1000 rpm for 5 minutes, to pellet the cells.
1.9. Resuspend the cell pellet in an appropriate volume of medium for
downstream processes.
2.0. Enzyme and Syringe Method
2.1. Unclip inserts and carefully remove the AlvetexScaffold discs using
flat-ended forceps.
2.2. Gently wash each disc in PBS, and transfer to a new 6-well plate.
2.3. 3 ml of 0.25 % Trypsin-EDTA (Sigma, T4049).
2.4. Incubate plate at 37 C, 5 % CO2 on a reciprocating shaker set to 100
strokes/min, for 15 minutes.
02
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Introduction:
The following protocol outlines a method for the retrieval of cells cultured in AlvetexScaffold.
Example data were obtained using this protocol to extract HepG2 hepatocyte cells
cultured in AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in the Well Insert Holder
in a Deep Petri Dish (AVP015) format. Two methods of cell extraction may be employed;
a standard method and a syringe-based method which produces higher average cell yields.
Both methods demonstrate that partial retrieval of cells from AlvetexScaffold is possible.
03
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Example: HepG2 cells grown for 7 days in AlvetexScaffold 6-well insert in a Petri dish format.
HepG2 cells (ATCC, HB-8065) were routinely maintained in T-75 flasks. HepG2 complete
media consisted of: MEM media (Gibco, 21090) supplemented with 10 % v/v FBS, 2 mM Lglutamine and 100 U/ml Penicillin/Streptomycin. Cells were seeded onto AlvetexScaffold
discs in 6-well inserts (AVP004-3) in Well Insert Holders in Deep Petri dishes (AVP015), at a
density of 1x 106 cells in 100 l media per insert. After settling for 1 hour in an incubator
(5 % CO2, 37 C), complete media was carefully added (70 ml per Petri dish). Cultures were
maintained for 7 days, with media changes on days 2 and 4. After 7 days, cultures were
processed according to the protocol described above.
The total number, and number of viable cells retrieved were counted using Trypan
Blue exclusion method. AlvetexScaffold discs were analysed post-retrieval by MTT assay
(Figure 1.) and histological staining to estimate cell numbers remaining in the 3D scaffold
(Figure 2.).
On the basis of residual in-scaffold MTT assay data, 12 % of cells were retrieved (compared
to untreated control discs) using the standard protocol described above. When the syringebased method was employed, cell recovery increased to 24 %. Cell viability post-retrieval
was 99 % for both methods, as determined by cell counting in conjunction with Trypan
Blue exclusion (data not shown).
04
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05
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Figure 2. Bright field micrographs showing the morphology of HepG2 cells cultured for 7 days on
AlvetexScaffold in 6-well insert within a Petri dish format (x10 and x40 objective). A: untreated HepG2 cells
and B: HepG2 cells after Trypsin-EDTA treatment for 15 minutes. Cells were fixed, embedded in paraffin
wax, sectioned (10 m) and counterstained with Haematoxylin and Eosin. The effect of Trypsin action can
clearly be seen on residual cells which lose contact with each other and adopt a rounded morphology.
01
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Specialised Applications
using AlvetexScaffold
02
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03
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04
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05
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06
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1.
Figure 1. Phase contrast micrographs of 3T3 cells grown in conventional 2D culture plates.
Images show cells at low (left) and high (right) confluency. Scale bars: 200 m.
2.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm). The
supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
5.
AlvetexScaffold 12-well plates were prepared for seeding with 70% ethanol (2 ml per
well) and media washes (twice with 3 ml of media each) as described in the product
information leaflet.
6.
125 l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
was equivalent to 1x106 cells per well.
7.
The plate was incubated 3 hours at 37 C with 5% CO2 to allow the cells to settle into
the scaffold.
8.
4 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete media exchange after every
1-2 days.
Histology
03
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Figure 2. Brightfield
micrographs showing the
structure of 3T3 cells cultured
for 7 days on 22 mm diameter
AlvetexScaffold discs presented
in 2-well plate format. Cells were
fixed, embedded in paraffin wax,
sectioned (10 m) and
counterstained with
haematoxylin and eosin.
04
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1.
Figure 1. Phase contrast micrographs of 3T3 cells grown in conventional 2D culture plates.
Images show cells at low (left) and high (right) confluency. Scale bars: 200 m.
2.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
5.
AlvetexScaffold 6-well inserts were prepared for seeding by dipping in 70% ethanol
and washing twice with 7 ml of media per well.
6.
125 l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
was equivalent to 1x106 cells per well.
7.
The plate was incubated 60 min at 37 C with 5% CO2 to allow the cells to settle into the
scaffold.
8.
10 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
Histology
05
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06
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3.
3.
4.
4.
5.
5.
6.
6.
7.
7.
8.
8.
9.
9.
1
1
2
2
Przyborski S.A. (2001). Isolation of human embryonal carcinoma stem cells by immuno-magnetic sorting.
Przyborski
Isolation of human embryonal carcinoma stem cells by immuno-magnetic sorting.
Stem
Cells,S.A.
19, (2001).
500-504.
Stem Cells, 19, 500-504.
Stewart R., Christie V. & Przyborski S.A. (2003). Manipulation of human pluripotent
Stewart R.,carcinoma
Christie V.stem
& Przyborski
S.A.development
(2003). Manipulation
of human Stem
pluripotent
embryonal
cells and the
of neural subtypes.
Cells,
embryonal carcinoma stem cells and the development of neural subtypes. Stem Cells,
07
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08
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1.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm). The
supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
5.
AlvetexScaffold 6-well inserts were prepared for seeding by dipping in 70% ethanol
and washed twice with 7 ml of media per well.
6.
125 l of the cell suspension was added to the centre of the AlvetexScaffold disc,
which was equivalent to 500,000 cells per well.
7.
The plate was incubated 30 min at 37 C with 5% CO2 to allow the cells to settle into the
scaffold.
8.
10 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
Przyborski S.A. (2001). Isolation of human embryonal carcinoma stem cells by immuno-magnetic sorting.
Stem Cells, 19, 500-504.
Stewart R., Christie V. & Przyborski S.A. (2003). Manipulation of human pluripotent embryonal carcinoma
stem cells and the development of neural subtypes. Stem Cells, 21, 248-256.
09
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Figure 2. Brightfield
micrographs showing the
structure of TERA2.cl.SP12
cells cultured for 7 days on 22
mm diameter AlvetexScaffold
discs presented in 6-well
inserts in 6-well plates.
Cells were fixed, embedded in
paraffin wax, sectioned (10 m)
and counterstained with
haematoxylin and eosin.
10
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Introduction:
HaCaT is an immortalised human adult keratinocyte cell line. This cell line has been known
to retain a capacity for normal differentiation up to multiple passages similar to normal human
epidermal keratinocytes (NHEK), and hence offers a suitable model for keratinisation studies.
However, even with their highly preserved differentiation capacity, terminal differentiation
can remain incomplete under airexposed conditions. This protocol describes a method for
differentiation of HaCaT cells under air-exposed conditions for up to 21 days.
Method:
1.
HaCaT cells were routinely maintained in T-75 flasks in 2D using complete media
(Dulbeccos Modified Eagles Medium supplemented with 10 % v/v FBS , 2 mM
L-glutamine and 100 U/ml Penicillin/Streptomycin).
HPM/01/5/2012
2.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet re-suspended in an appropriate
volume of media for dilution for cell counting by Trypan Blue.
3.
4.
11
5.
The media was aspirated and replaced with a second media wash. This was
aspirated just before application of the cells: 1 x 106 cells in 50 l were added
to the middle of each well insert.
Note: If preparation of cell suspension is delayed, incubate plate with medium
at 37 C with 5 % CO2 until further use.
6.
The plate was incubated for 30 minutes at 37 C with 5 % CO2 to allow the cells
to settle, wells were then carefully topped up with 10 ml of KGM-2 and cultured
for 3 days under submerged conditions. Culture media was fully changed after
2 days.
7.
Well inserts were then moved to Reinnervates custom-made Well insert holder
in a deep Petri dish (AVP015) at medium setting, and maintained at the air/liquid
interface (37 ml/per Petri dish) for up to 21 days to promote stratification. Culture
media was refreshed by partial media exchange (50:50) every 2 days.
8.
Cultures were examined histologically at the end of each week. 3D cultures were
processed in either Bouins fixative or 4 % paraformaldehyde, wax embedded,
sectioned (10 m) and analysed by haematoxylin and eosin staining or
immunohistochemistry. For full experimental details please refer to specific
histology protocols located at www.reinnervate.com/alvetex/workflow.
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Note: If preferred, ethanol treatment can also be done in situ in the plates by
dispensing sufficient ethanol solution to cover the well inserts and
AlvetexScaffold disc.
12
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Complete media consisted of: MEM media (Gibco 21090) supplemented with 10% v/v
FBS, 2 mM L-glutamine and 100 U/ml Penicillin/Streptomycin.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm). The
supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
5.
AlvetexScaffold 6-well inserts were prepared for seeding by dipping in 70% ethanol
and washed twice with 7 ml of media per well.
6.
125 l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
was equivalent to 2x106 cells per well.
7.
The plate was incubated 30 min at 37 C with 5% CO2 to allow the cells to settle into the
scaffold.
8.
10 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
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1.
15
16
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Figure 2. Brightfield
micrographs showing the
structure of HepG2 cells
cultured for 7 days on 22 mm
diameter AlvetexScaffold discs
presented in the 6-well plate
format. Cells were fixed,
embedded in paraffin wax,
sectioned (10 m) and
counterstained with
haematoxylin and eosin.
Complete media consisted of: MEM media (Gibco 21090) supplemented with 10 % v/v
FBS, 2 mM L-glutamine and 100 U/ml Penicillin/Streptomycin.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm). The
supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
5.
AlvetexScaffold 12-well plates were prepared for seeding with 70% ethanol (2 ml per well)
and media washes (twice with 3 ml of media each) as described in the product
information leaflet.
6.
125 l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
was equivalent to 2x106 cells per well.
7.
The plate was incubated 3 hours at 37 C with 5% CO2 to allow the cells to settle into
the scaffold.
8.
4 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete media exchange after every
1-2 days.
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1.
17
18
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Figure 2. Brightfield
micrographs showing the
structure of HepG2 cells
cultured for 7 days on 22 mm
diameter AlvetexScaffold discs
presented in the 12-well plate
format. Cells were fixed,
embedded in paraffin wax,
sectioned (10 m) and
counterstained with
haematoxylin and eosin.
Complete medium consisted of: F-12K nutrient mixture (Kaighns modification; Gibco
21127022) supplemented with 10% v/v FBS and 100g/ml Penicillin and 10g/ml
Streptomycin. Note: Medium already contains L-glutamine.
3.
4.
Cells were re-suspended at a concentration of 1x106 cells / 150l for seeding per well.
5.
AlvetexScaffold 6-well inserts fitted into 6-well plates were prepared for seeding by washing
in 70% ethanol and rinsing once with 10ml of medium per well.
6.
150l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
was equivalent to 1x106 cells per well.
7.
The plate was incubated 30 minutes at 37C with 5% CO2 to allow the cells to settle into
the scaffold.
8.
10ml of medium was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete medium exchange after every
2-3 days.
HPM/01/5/2012
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1.
19
20
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Complete medium consisted of: F-12K nutrient mixture (Kaighns modification; Gibco
21127022) supplemented with 10% v/v FBS and 100g/ml Penicillin and 10g/ml
Streptomycin. Note: Medium already contains L-glutamine.
3.
4.
Cells were re-suspended at a concentration of 1x106 cells / 150l for seeding per well.
5.
AlvetexScaffold 12-well discs were prepared for seeding by washing in 70% ethanol and
rinsing once with 4ml of medium per well.
6.
150l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
was equivalent to 1x106 cells per well.
7.
The plate was incubated 30 minutes at 37C with 5% CO2 to allow the cells to settle into
the scaffold.
8.
4ml of medium was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete medium exchange after every
1-2 days.
HPM/01/5/2012
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1.
21
22
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Methods:
Preparation for 3D cell culture on AlvetexScaffold
1. MCF-7 cells were routinely maintained in T-75 flasks.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
HPM/22/8/2012
23
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Introduction:
AlvetexScaffold is currently available in four different cell culture formats: 24-well plate
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005).
24-well and 12-well plates are suitable for shorter term cultures and for applications
where limited cell penetration into the scaffold is required. Well insert formats
generally support longer term cultures and deeper cell penetration into the scaffold.
They also provide for conveniently tailored media set ups (see Quick Start Protocol,
www.reinnervate.com/alvetex/workflow). 6-well inserts can be placed in conventional
6-well plates, while 12-well inserts can be placed in either 6-well plates or 12-well plates,
depending on media requirements. Alternatively, both insert types can be housed in the
dedicated Well Insert Holder in Deep Well Petri Dish (AVP015) to allow for increased media
volumes and prolonged cell culture. The availability of two different AlvetexScaffold sizes
enables choice on the basis of desired culture size and cell expenditure.
24
Follow one of the below methods according to your choice of AlvetexScaffold format.
4.
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Protocol Booklet
b.
c.
100 l of the cell suspension was added to the centre of the AlvetexScaffold,
which was equivalent to 1 x 106 cells per well.
d.
The plate was incubated for 60 minutes at 37 C with 5 % CO2 to allow the cells
to settle into the scaffold.
e.
10 ml of media was added to each well taking care not to dislodge cells from the
membrane.
f.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
Note: This method can be applied to the use of AlvetexScaffold in 12-well insert format,
AVP005. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
b.
c.
d.
The plate was incubated for 60 minutes at 37 C with 5 % CO2 to allow the cells
to settle into the scaffold.
e.
2 ml of media was added to each well taking care not to dislodge cells from the
membrane.
f.
Plates were re-incubated and maintained by complete media exchange every 2-3
days for the first 10 days, then every day.
Note: This method can be applied to the use of AlvetexScaffold in 12-well plate format,
AVP002. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
The cells showed good linear growth and expansion in 3D which plateaued between 14
and 21 days. We would not recommend growing cultures of MCF-7 cells beyond 14-18
days as when they reach confluency in 3D they can become overcrowded and the quality
of the culture is reduced resulting in cell death.
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25
26
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Figure 2. Brightfield micrographs showing the structure of MCF-7 cells cultured for A) 3
days, B) 7 days, C) 10 days or D) 14 days on 22 mm diameter AlvetexScaffold presented
in 6-well inserts. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and
counterstained with haematoxylin and eosin.
27
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Figure 3. Biochemical analysis of cell viability using a standard MTT assay. Data from 3
sample replicates of MCF-7 cells are shown (mean SD). Each well was sampled in
triplicate with mean value shown. Cells were cultured for up to 21 days on 22 mm
AlvetexScaffold presented in 6-well inserts.
28
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Figure 4. Brightfield micrographs showing the structure of MCF-7 cells cultured for A)
3 days, B) 7 days, C) 10 days or D) 14 days on 15 mm diameter AlvetexScaffold
presented in 24-well plates. Cells were fixed, embedded in paraffin wax, sectioned
(10 m) and counterstained with haematoxylin and eosin.
29
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Figure 5. Biochemical analysis of cell viability using a standard MTT assay. Data from
3 sample replicates of MCF-7 cells are shown (mean SD). Each well was sampled
in triplicate with mean value shown. Cells were cultured for up to 18 days on 15 mm
AlvetexScaffold presented in 24-well plate format.
30
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Introduction:
AlvetexScaffold is currently available in four different cell culture formats: 24-well plate
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005). 24-well
and 12-well plates are suitable for shorter term cultures and for applications where limited
cell penetration into the scaffold is required. Well insert formats generally support longer
term cultures and deeper cell penetration into the scaffold. They also provide for conveniently
tailored media set ups (see Quick Start Protocol, www.reinnervate.com/alvetex/workflow).
6-well inserts can be placed in conventional 6-well plates, while 12-well inserts can be placed
in either 6-well plates or 12-well plates, depending on media requirements. Alternatively,
both insert types can be housed in the dedicated Well Insert Holder in Deep Well Petri Dish
(AVP015) to allow for increased media volumes and prolonged cell culture. The availability of
two different AlvetexScaffold sizes enables choice on the basis of desired culture size and
cell expenditure.
Methods:
Preparation for 3D Cell Culture on AlvetexScaffold:
1.
Primary rat MSCs were derived from the bone marrow of 6-8 week old male Wistar
rats (Harlan) using standard, previously published, methodology [1], and used at P1
for experiments. MSCs were routinely maintained in T-75 flasks.
2.
Complete growth media consisted of: DMEM High glucose supplemented with
10 % v/v heat inactivated FBS, 1X non-essential amino acids, 2 M L-glutamine
and 100 U/ml Penicillin/Streptomycin.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
AlvetexScaffold 6-well inserts in 6-well plate format were prepared for seeding
by dipping in 70 % ethanol and washed twice with media (7 ml per well).
b.
100 l of the cell suspension was added to the centre of the AlvetexScaffold disc,
which was equivalent to 1 x 106 cells per well.
c.
The plate was incubated for at least 15 minutes at 37 C with 5 % CO2 to allow the
cells to settle into the scaffold.
d.
HPM/06/8/2012
e.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
Figure 1. Brightfield micrographs showing the structure of primary rat MSCs cultured for
14 days on 22 mm diameter AlvetexScaffold discs presented in 6-well insert in 6-well
plate format. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and
counterstained with haematoxylin and eosin. Scale bar 100 m.
Figure 2. Biochemical analysis of cell viability using a standard MTT assay. For each
timepoint, data from 3 sample replicates of primary rat MSCs are shown (n=3, mean
SEM). Cells were cultured for 4, 7 and 14 days on 22 mm AlvetexScaffold discs
presented in 6-well insert in 6-well plate format.
1.
Croft, A.P. and S.A. Przyborski, Formation of neurons by non-neural adult stem cells:
potential mechanism implicates an artifact of growth in culture. Stem Cells, 2006. 24(8):
p. 1841-51.
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Protocol Booklet
Note: This method can be applied to the use of AlvetexScaffold in 12-well insert format,
AVP005. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
31
32
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Introduction:
AlvetexScaffold is currently available in four different cell culture formats: 24-well plate
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005). 24-well
and 12-well plates are suitable for shorter term cultures and for applications where limited
cell penetration into the scaffold is required. Well insert formats generally support longer
term cultures and deeper cell penetration into the scaffold. They also provide for conveniently
tailored media set ups (see Quick Start Protocol, www.reinnervate.com/alvetex/workflow).
6-well inserts can be placed in conventional 6-well plates, while 12-well inserts can be placed
in either 6-well plates or 12-well plates, depending on media requirements. Alternatively,
both insert types can be housed in the dedicated Well Insert Holder in Deep Well Petri Dish
(AVP015) to allow for increased media volumes and prolonged cell culture. The availability of
two different AlvetexScaffold sizes enables choice on the basis of desired culture size and
cell expenditure.
Methods:
Preparation for 3D Cell Culture on AlvetexScaffold:
1.
Complete growth media consisted of: DMEM High glucose supplemented with
10 % v/v heat inactivated FBS, 1X non-essential amino acids, 2 M L-glutamine
and 100 U/ml Penicillin/Streptomycin.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
HPM/06/8/2012
b.
50 l of the cell suspension was added to the centre of the AlvetexScaffold disc,
which was equivalent to 0.5 x 106 cells per well.
c.
5 ml of media was added to each well taking care not to allow media over the
windows of the insert, i.e. independent feeding from below. The plate was incubated
overnight at 37 C with 5 % CO2 to allow the cells to settle into the scaffold.
d.
A further 5 ml of media was added to each well the following morning taking care
not to dislodge cells from AlvetexScaffold.
e.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
Note: This method can be applied to the use of AlvetexScaffold in 6-well insert format,
AVP004. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
Figure 2. Brightfield micrographs showing the structure of MG63 cells cultured for 7 days
on 15 mm diameter AlvetexScaffold discs presented in 12-well insert in 6-well plate
format. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and
counterstained with haematoxylin and eosin. Scale bar 100 m.
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Protocol Booklet
a.
33
34
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Introduction:
AlvetexScaffold is currently available in four different cell culture formats: 24-well plate
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005). 24-well
and 12-well plates are suitable for shorter term cultures and for applications where limited
cell penetration into the scaffold is required. Well insert formats generally support longer
term cultures and deeper cell penetration into the scaffold. They also provide for conveniently
tailored media set ups (see Quick Start Protocol, www.reinnervate.com/alvetex/workflow).
6-well inserts can be placed in conventional 6-well plates, while 12-well inserts can be placed
in either 6-well plates or 12-well plates, depending on media requirements. Alternatively,
both insert types can be housed in the dedicated Well Insert Holder in Deep Well Petri Dish
(AVP015) to allow for increased media volumes and prolonged cell culture. The availability of
two different AlvetexScaffold sizes enables choice on the basis of desired culture size and
cell expenditure.
Methods:
Preparation for 3D Cell Culture on AlvetexScaffold:
1.
Complete growth media consisted of: DMEM High glucose supplemented with
10 % v/v heat inactivated FBS, 2 M L-glutamine and 100 U/ml Penicillin/Streptomycin.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
5.
HPM/14/9/2012
b.
50 l of the cell suspension was added to the centre of the AlvetexScaffold disc,
which was equivalent to 0.5 x 106 cells per well.
c.
The plate was incubated for at least 1 hour at 37 C with 5 % CO2 to allow the cells
to settle into the scaffold.
d.
2 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
e.
Plates were re-incubated and maintained by complete media exchange after every
1-2 days.
Note: This method can be applied to the use of AlvetexScaffold in 12-well plate format,
AVP002. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
AlvetexScaffold 6-well inserts in 6-well plate format were prepared for seeding
by dipping in 70 % ethanol followed by media washes (twice, with 7 ml per well).
b.
Wells were filled from the outside of the insert with enough medium to allow it
to rise inside the insert and cover the substrate, but not to go over the sides of the
inserts, i.e. 7ml 1ml, as described for feeding above and below separately in the
Quick Start Protocol located at (www.reinnervate.com/alvetex/workflow).
c.
100 l of the cell suspension was added in droplets over the surface of
the AlvetexScaffold disc, which was equivalent to 1 x 106 cells per well.
d.
The plate was incubated overnight at 37 C with 5 % CO2 to allow the cells to settle
into the scaffold.
e.
Media was added to each well to a total volume of 10ml the following morning taking
care not to dislodge cells from AlvetexScaffold.
f.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
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a.
35
36
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a.
AlvetexScaffold 12-well inserts in 12-well plate format were prepared for seeding
by dipping in 70 % ethanol followed by media washes (twice with 4 ml per well).
b.
Wells were filled from the outside of the insert with enough medium to allow it
to rise inside the insert and cover the substrate, but not to go over the sides of
the inserts, i.e. 2.4ml 0.2ml, as described for feeding above and below separately
in the Quick Start Protocol (www.reinnervate.com/alvetex/workflow).
c.
50 l of the cell suspension was added in droplets over the surface of the
AlvetexScaffold disc, which was equivalent to 0.5 x 106 cells per well.
d.
The plate was incubated overnight at 37 C with 5 % CO2 to allow the cells to settle
into the scaffold.
e.
Media was added to each well to a total volume of 4ml the following morning taking
care not to dislodge cells from AlvetexScaffold.
f.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
Figure 6. Brightfield micrographs showing the structure of SW620 cells cultured for
7 days on 15 mm diameter AlvetexScaffold discs presented in the 24-well plate format.
Cells were fixed, embedded in paraffin wax, sectioned (10 m) and counterstained with
haematoxylin and eosin. Scale bar: 300 m
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Figure 7. Biochemical analysis of cell viability using a standard MTT assay. Data from
3 sample replicates of SW620 cells are shown (n=3, mean SD). Cells were cultured
for 3 days on 15 mm AlvetexScaffold discs presented in the 24-well plate format.
Figure 2. Brightfield micrographs showing the structure of SW620 cells cultured for
7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well inserts in the
6-well plate format. Cells were fixed, embedded in paraffin wax, sectioned (10 m)
and counterstained with haematoxylin and eosin. Scale bar: 300 m.
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Figure 3. Biochemical analysis of cell viability using a standard MTT assay. Data from
3 sample replicates of SW620 cells are shown (n=3, mean SD). Cells were cultured
for 3 days on 22 mm AlvetexScaffold discs presented in 6-well inserts in the 6-well
plate format.
Figure 4. Brightfield micrographs showing the structure of SW620 cells cultured for 7
days on 15 mm diameter AlvetexScaffold discs presented in 12-well insert in 12-well
plate format. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and
counterstained with haematoxylin and eosin. Scale bar: 300 m
39
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Figure 5. Biochemical analysis of cell viability using a standard MTT assay. Data from 3
sample replicates of SW620 cells are shown (n=3, mean SD). Cells were cultured for 3
days on 15 mm AlvetexScaffold discs presented in 12-well insert in 12-well plate format.
40
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Introduction:
AlvetexScaffold is currently available in four different cell culture formats: 24-well plate
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005). 24-well
and 12-well plates are suitable for shorter term cultures and for applications where limited
cell penetration into the scaffold is required. Well insert formats generally support longer
term cultures and deeper cell penetration into the scaffold. They also provide for conveniently
tailored media set ups (see Quick Start Protocol, www.reinnervate.com/alvetex/workflow).
6-well inserts can be placed in conventional 6-well plates, while 12-well inserts can be placed
in either 6-well plates or 12-well plates, depending on media requirements. Alternatively,
both insert types can be housed in the dedicated Well Insert Holder in Deep Well Petri Dish
(AVP015) to allow for increased media volumes and prolonged cell culture. The availability of
two different AlvetexScaffold sizes enables choice on the basis of desired culture size and
cell expenditure.
Methods:
Preparation for 3D Cell Culture on AlvetexScaffold:
1.
Complete growth media consisted of: DMEM High glucose supplemented with
10 % v/v heat inactivated FBS, 2 M L-glutamine and 100 U/ml Penicillin/Streptomycin.
2.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
3.
4.
HPM/14/9/2012
b.
50 l of the cell suspension was added to the centre of the AlvetexScaffold disc,
which was equivalent to 0.5 x 106 cells per well.
c.
The plate was incubated for at least 1 hour at 37 C with 5 % CO2 to allow the cells
to settle into the scaffold.
d.
2 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
e.
Plates were re-incubated and maintained by complete media exchange after every
1-2 days.
Note: This method can be applied to the use of AlvetexScaffold in 12-well plate format,
AVP002. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
AlvetexScaffold 12-well inserts in 12-well plate format were prepared for seeding
by dipping in 70 % ethanol followed by media washes (twice with 4 ml per well).
b.
Wells were filled from the outside of the insert with enough medium to allow it
to rise inside the insert and cover the substrate, but not to go over the sides of
the inserts, i.e. 2.4ml 0.2ml, as described for feeding above and below separately
in the Quick Start Protocol (www.reinnervate.com/alvetex/workflow).
c.
50 l of the cell suspension was added in droplets over the surface of the
AlvetexScaffold disc, which was equivalent to 0.5 x 106 cells per well.
d.
The plate was incubated overnight at 37 C with 5 % CO2 to allow the cells to settle
into the scaffold.
e.
Media was added to each well to a total volume of 4ml the following morning taking
care not to dislodge cells from AlvetexScaffold.
f.
Plates were re-incubated and maintained by complete media exchange after every
2-3 days.
Note: This method can be applied to the use of AlvetexScaffold in 6-well insert format,
AVP004. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
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a.
41
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Figure 2. Brightfield micrographs showing the structure of SW480 cells cultured for 7
days on 15 mm diameter AlvetexScaffold discs presented in the 24-well plate format.
Cells were fixed, embedded in paraffin wax, sectioned (10 m) and counterstained with
haematoxylin and eosin.
Figure 3. Biochemical analysis of cell viability using a standard MTT assay. Data from
3 sample replicates of SW480 cells are shown (n=3, mean SD). Cells were cultured
for 3 days on 15 mm AlvetexScaffold discs presented in the 24-well plate format.
43
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Figure 4. Brightfield micrographs showing the structure of SW480 cells cultured for
7 days on 15 mm diameter AlvetexScaffold discs presented in 12-well insert in 12-well
plate format. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and
counterstained with haematoxylin and eosin.
Figure 5. Biochemical analysis of cell viability using a standard MTT assay. Data from
3 sample replicates of SW480 cells are shown (n=3, mean SD). Cells were cultured
for 3 days on 15 mm AlvetexScaffold discs presented in 12-well insert in 12-well
plate format.
44
Consolidated
Protocol Booklet
Introduction:
AlvetexScaffold is currently available in four different cell culture formats: 24-well plate
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005). 24-well
and 12-well plates are suitable for shorter term cultures and for applications where limited
cell penetration into the scaffold is required. Well insert formats generally support longer
term cultures and deeper cell penetration into the scaffold. They also provide for conveniently
tailored media set ups (see Quick Start Protocol, www.reinnervate.com/alvetex/workflow).
6-well inserts can be placed in conventional 6-well plates, while 12-well inserts can be placed
in either 6-well plates or 12-well plates, depending on media requirements. Alternatively,
both insert types can be housed in the dedicated Well Insert Holder in Deep Well Petri Dish
(AVP015) to allow for increased media volumes and prolonged cell culture. The availability of
two different AlvetexScaffold sizes enables choice on the basis of desired culture size and
cell expenditure.
Methods:
Preparation for 3D Cell Culture on AlvetexScaffold:
1.
SW480, SW620 and 3T3 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of SW480 cells (left), SW620 cells (middle)
and 3T3 cells (right) grown in conventional 2D culture plates. Images show cells at high
confluency. Scale bars: 200 m.
2.
For SW480 cells and SW620 cells, complete growth media consisted of: DMEM High
glucose supplemented with 10 % v/v heat inactivated FBS, 2 M L-glutamine and 100
U/ml Penicillin/Streptomycin.
3.
For 3T3 cells, complete growth media consisted of: DMEM High glucose supplemented
with 10 % v/v FBS, 2 M L-glutamine and 100 U/ml Penicillin/Streptomycin.
4.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
5.
HPM/14/9/2012
Note: 3T3 cells are seeded on AlvetexScaffold 7 days prior to seeding SW480 cells
(or SW620 cells).
a.
AlvetexScaffold 6-well inserts in 6-well plate format were prepared for seeding by
dipping in 70 % ethanol and washed twice with media (7 ml per well).
b.
50 l of the 3T3 cell suspension was added in the centre of the AlvetexScaffold disc,
which was equivalent to 0.5 x 106 cells per well.
c.
The plate was incubated for a minimum of 15 minutes at 37 C with 5 % CO2 to allow
the cells to settle into the scaffold.
d.
3T3 media was added to each well to a total volume of 10 ml taking care not to
dislodge cells from AlvetexScaffold.
e.
Plates were re-incubated and maintained for a further 7 days by complete 3T3 media
exchange after every 2-3 days.
f.
After 7 days, inserts were transferred to deep well Petri dish holders in deep well Petri
dishes (AVP015) and dishes were filled from the outside of the insert with enough
SW480/SW620 medium to allow the medium to rise inside the insert and cover the
substrate, but not to go over the sides of the inserts, i.e. 50ml 5ml, as described for
feeding above and below separately in the Quick Start protocol.
g.
100 l of either the SW480 cell suspension or the SW620 cell suspension was added
in droplets over the surface of the AlvetexScaffold disc, which was equivalent to 1 x
106 cells per well.
h.
The Petri dish was incubated overnight at 37 C with 5 % CO2 to allow the cells to
settle on top of the scaffold.
i.
The following morning SW480/SW620 media was added to each Petri dish to a total
volume of 70 ml taking care not to dislodge cells from AlvetexScaffold.
j.
Note: This method can be applied to the use of AlvetexScaffold in 12-well insert format,
AVP005. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
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46
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Figure 2. Brightfield micrograph showing the structure of 3T3 cells cultured for 7 days on
22 mm diameter AlvetexScaffold discs presented in 6-well insert in 6-well plate format.
Cells were fixed, embedded in paraffin wax, sectioned (10 m) and counterstained with
haematoxylin and eosin. Scale bar: 100 m.
Figure 3. Brightfield micrograph showing the structure of SW480 cells co-cultured for
7 days with established 3T3 cells on 22 mm diameter AlvetexScaffold discs presented in
6-well insert in 6-well plate format. Cells were fixed, embedded in paraffin wax, sectioned
(10 m) and counterstained with haematoxylin and eosin. Scale bar: 100 m.
47
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Figure 4. Brightfield micrograph showing the structure of SW620 cells co-cultured for 7
days with established 3T3 cells on 22 mm diameter AlvetexScaffold discs presented in
6-well insert in 6-well plate format. Cells were fixed, embedded in paraffin wax, sectioned
(10 m) and counterstained with haematoxylin and eosin. Scale bar: 100 m.
01
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White Papers
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AlvetexScaffold
technology represents a
novel tool for the scientist
working in the cell culture
field by offering an
opportunity for major
advancements in cellular
organisation over
traditional 2D cultures.
Because cells in
AlvetexScaffold form
tissue-like structures,
sophisticated techniques
traditionally reserved for
the analysis of tissues
become more appropriate
for cell visualisation.
Legend: Scanning electron microscopy image of the structure AlvetexScaffold (main picture) and populated with
TERA2.cl.SP12 cells as visualised by histological staining (right)
01
2D cell culture
03
3D cell culture
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Protocol Booklet
Figure 1. An example of how cells retain a more in-vivo-like structure when cultured in 3D within AvetexScaffold.
It is the increased volume of cells in the z plane that hinders optimal in-focus visualisation of 3D cultured cells
under standard light micrcoscope.conditions. Data generated as part of a collaborative project between
Reinnervate and LGC Standards [1].
F igure 2. Cell cultures can be visualised on AlvetexScaffold by staining with the nontoxic dye Neutral Red. CHO-K1 cells were seeded onto AlvetexScaffold (AVP002) at
a density of 0.5 million cells per scaffold. After 2 days cells were exposed to 0.5 %
Neutral red dye solution (Sigma, N6264-50ML). For full experimental details refer to
Neutral red staining protocol located at www.reinnervate.com/alvetex/protocols
02
04
Comments
2D samples
Brightfield / Phase
Contrast Microscopy
Commonly available
No cost
Easy-to-use
Routine
Best suited for 2D culture and tissue
sections
Standard Fluorescence
Microscopy
Generally available
Expensive
Moderate training required
Routine
Best suited for 2D culture and tissue
sections
Confocal Laser
Scanning Microscopy
Less available
Expensive
Training required
Less routine
Suited for both 2D and 3D cultures and
tissues
Histology
Generally available
Inexpensive
Moderate training required
Routine
Ideally suited for 3D cultures and
tissues
Transmission Electron
Microscopy (TEM)
Less available
Expensive
Substantial training required
Less routine
Suited for both 2D and 3D cultures and
tissues
Scanning Electron
Microscopy (SEM)
Less available
Expensive
Substantial training required
Less routine
Suited for both 2D and 3D cultures and
tissues
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Protocol Booklet
Method
Table 1. Comparison of common techniques available for imaging cells in 2D versus 3D cultures/tissues.
03
no cells
2 million cells
05
disc
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x4
x10
Visualisation of cells
growing in 3D is
enhanced by reagents
which produce a colour
contrast between the
cells and the scaffold:
Light Microscopy is thus
compatible with
AlvetexScaffold by
staining the cells. Cell
staining as the byproduct of a colorimetric
assay or fixation
procedure can also be
exploited for cell
visualisation and
monitoring the progress
of cultures.
x20
Figure 3. Cell cultures can be visualised on AlvetexScaffold by staining with the non-toxic dye Neutral Red. CHO-K1
cells were seeded onto AlvetexScaffold (AVP002) at a density of 0, 0.5 million and 2 million cells per scaffold. After 24
hours cells were exposed to 0.5 % Neutral Red dye solution (Sigma, N6264-50ML). Scaffolds were then transferred to a
glass microscopic slide, kept wet by adding a drop of PBS and imaged under an ICC50HD Leica microscope with LAS EZ
software (brightfield setting). For full experimental details refer to Simple staining methods for viewing cells on
AlvetexScaffold by light microscopy protocol located at www.reinnervate.com/alvetex/protocols.
04
2 million cells
disc
Figure 4. Visualisation of cells on AlvetexScaffold with Methylene Blue solution. HepG2 cells were seeded onto
AlvetexScaffold (AVP002) at a density of 0, 0.5 million and 2 million cells per scaffold. After 24 hours cells were
exposed to 0.5 % Methylene Blue dye solution (Sigma, 03978-250ML). For full experimental details refer to Simple
staining methods for viewing cells on AlvetexScaffold by light microscopy protocol located at
www.reinnervate.com/alvetex/protocols.
Figure 5: The gross location of viable cells is clearly visible on the AlvetexScaffold disc after staining
with MTT cell viability reagent. HaCat cells were cultured on AlvetexScaffold in the 12-well plate format
(AVP002) for 4 days prior to analysis (for full experimental details see separate MTT protocol available at
www.reinnervate.com/alvetex/protocols.
05
06
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no cells
07
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Figure 6. Cells can be visualised on AlvetexScaffold following fixation with the yellow coloured Bouins fixative.
SW480 colon carcinoma cells were cultured on AlvetexScaffold and removed for fixation at 4, 7, 11, 14, 18, and 21
days after seeding. The increasing number of cells over time is reflected in increasing staining intensity. A. SW480
cultures in AlvetexScaffold discs following Bouins fixation and ethanol dehydration; B. Bouins fixed and wax
embedded SW480 AlvetexScaffold cultures; C. Slide mounted cross sections (10m slices) of Bouins fixed and wax
embedded SW480 AlvetexScaffold cultures stained with haematoxylin and eosin.
Figure 7: GFP-transfected CHO-K1 cells were cultured on AlvetexScaffold 6 well inserts (AVP004) for 6 days. Captured
images were obtained every 30 minutes using a Zeiss LSM 510 confocal microscope with heated stage (for full
experimental details refer to live-cell imaging protocol located at www.reinnervate.com). In this example, a series of
integrated z-stacks is presented which shows the behaviour of a single transfected CHO-K1 cell over a period of 3 hours.
06
Fluorescence Microscopy:
Immunofluorescence uses the recognition of cellular targets
by fluorescent dyes or antigen-specific antibodies coupled to
fluorophores. Depending on the antibody or dye used,
proteins, lipids and DNA can be visualised within individual
cells and tissues. AlvetexScaffold can easily be processed
like a standard tissue sample, allowing established
immunocytochemical methods to be followed with excellent
results (Figure 8).
AlvetexScaffold cell
cultures are amenable to
highly sophisticated cell
imaging techniques such
as confocal imaging.
Confocal microscopy can
be used to visualise fixed
cells or to follow living
cultures in real time.
AlvetexScaffold cultures
can be regarded as in vitro
miniature tissues. As such
standard histological
techniques can be applied
to their analysis. These
methods include
immunohistochemistry,
histological staining,
confocal microscopy, and
electron microscopy.
Figure 8. Human keratinocyte cell line (HaCaT) grown in AlvetexScaffold (7 days air exposure). The
culture was fixed and processed for paraffin wax embedding and immunohistochemical analysis by
fluorescent microscopy. The three images from the same region show; phase contrast (top), blue
fluorescent Hoescht 33258 nuclei stain (middle) and Ki67 staining (bottom). For the full
experimental details refer to Immunocytochemistry protocol located at
www.reinnervate.com/alvetex/protocols.
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07
09
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Figure 10. HepG2 cells grown for 3 days in AlvetexScaffold 12-well plate format (AVP002). Cells were stained with
Hoechst 33342 (blue), cytokeratin 8 (green) and Nile Red (red). Pictures were taken on a Zeiss LSM 510 confocal
microscope. Note the background signal from AlvetexScaffold in the blue and green channels is very low. For full
experimental details refer to Confocal protocol located at www.reinnervate.com/alvetex/protocols.
08
Histology:
Figure 11: Cells grown in AlvetexScaffold can be fixed and processed for histological
analysis using standard methods. Both staining examples are carried out on human
keratinocytes (HaCaT) grown in AlvetexScaffold for 7 days. The sample on the top
shows a culture that was fixed and processed for resin embedding (L R White resin).
Resin sections (1 m) were stained with Toluidene Blue for structural analysis by light
microscopy. The sample on the bottom shows a culture that was fixed and processed for
paraffin embedding. Sections (10 m) were stained with Haematoxylin and Eosin for
morphological analysis by light microscopy. For full experimental details refer to the
Histology Series of protocols located at www.reinnervate.com/alvetex/protocols.
10
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Protocol Booklet
09
Electron Microscopy:
11
Consolidated
Protocol Booklet
Scanning electron microscopy (SEM) is becoming a popular method of visualisation of cultures grown in 3D. SEM is a form of
electron microscopy where images are obtained by scanning samples using a high-energy beam of electrons. As the samples
are scanned the electrons interact with the sample surface, and these interactions are detected and processed, leading to
high-resolution images depicting the sample topography and composition.
AlvetexScaffold can easily be processed like a standard tissue sample, allowing established methods to be followed with
excellent results (Figure 12).
Figure 12. Detailed structure of 3D cultures can be visualised using scanning electron microscopy. Inspection of pieces
of AlvetexScaffold at low magnification shows homogeneous coverage by cultured cells (A). Higher magnification
imaging in this transverse section reveals cells growing throughout the scaffold (B). Increasingly higher magnification
micrographs reveal how cells interact with each-other and the AlvetexScaffold (C & D). See scale bar inserts. For full
experimental details refer to SEM protocol located at www.reinnervate.com/alvetex/protocols.
10
12
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Figure 13. (A) TEM image of keratinocytes cultured at air-liquid interface without collagen or fibroblasts for 14 days. The
scaffold is indicated by a black arrow. (B) TEM image showing cells progressively flattening towards the upper surface of
the culture after 14 days at air-liquid interface. (C) High magnification image showing desmosomes, indicated with white
arrows. (D) High magnification image showing bundling of keratin filaments underneath cell membrane, indicated with
white arrows. (Scale bar 500 m) For full experimental details refer to TEM protocol located at
www.reinnervate.com/alvetex/protocols.
11
Conclusions:
13
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Protocol Booklet
As outlined throughout the document, there are many imaging techniques which can
be implemented to visualise cultures and cells grown within AlvetexScaffold in For
following culture progress, dyes stain cells contrasting them against the scaffold
background allowing visualisation via light microscopy. For more in depth culture
analysis, a range of more complex techniques can be implemented similar to those
performed on tissue samples with excellent results.
References.
1. Schutte, M., et al., Rat primary hepatocytes show
enhanced performance and sensitivity to acetaminophen
during three-dimensional culture on a polystyrene
scaffold designed for routine use. Assay Drug Dev
Technol, 2011. 9(5): p. 475-86.
2. Smith, L.E., R. Smallwood, and S. Macneil, A comparison
of imaging methodologies for 3D tissue engineering.
Microsc Res Tech, 2010. 73(12): p. 1123-33.
149
e-mail: info@reinnervate.com
web: www.reinnervate.com