ANIMAL TISSUE

CULTURE AND ITS

APPLICATIONS.
Suspension cultures are usually grown either:

In magnetically rotated spinner flasks or shaken rlenmeyer

flasks where the cells are kept actively suspended in the
medium;

In stationary culture vessels such as T-flasks and bottles

where, although the cells are not kept agitated, they are
unable to attach firmly to the substrate.
• Many cell lines, especially those derived from normal
tissues, are considered to be Anchorage-Dependent, that is,
they can only grow when attached to a suitable substrate.

• Some cell lines that are no longer considered normal

(frequently designated as Transformed Cells) are
frequently able to grow either attached to a substrate or
floating free in suspension; they are Anchorage-
Independent.

• In addition, some normal cells, such as those found in the

blood, do not normally attach to substrates and always grow
in suspension
 Types of Cells
Cultured cells are usually described based on their morphology
(shape and appearance) or their functional characteristics.
There are three basic morphologies:

1. Epithelial-like: cells that are attached to a substrate and

appear flattened and polygonal in shape.
2. Lymphoblast-like: cells that do not attach normally to a
substrate but remain in suspension with a spherical shape.
3. Fibroblast-like: cells that are attached to a substrate and
appear elongated and bipolar, frequently forming swirls in
heavy cultures
 CELL FUSION:
• culture conditions play an important role in determining shape and that
many cell cultures are capable of exhibiting multiple morphologies Using
cell fusion techniques, it is also possible to obtain hybrid cells by fusing
cells from two different parents. These may exhibit characteristics of either
parent or both parents.

• This technique was used in 1975 to create cells capable of producing

custom tailored monoclonal antibodies. These hybrid cells (called
Hybridomas)  fusing two different but related cells.

a. spleen-derived lymphocyte  producing the desired antibody

b. rapidly dividing myeloma cell (a type of cancer cell)  antibodies but is
not programmed to produce any antibody.

• These antibodies, called Monoclonal Antibodies due to their purity, have

many important clinical, diagnostic, and industrial applications
 Functional Characteristics
1. Biochemical markers can be used to determine if cells are still carrying on
specialized functions that they performed in vivo (e.g., liver cells
secreting albumin).
2. Morphological or ultra structural markers can also be examined (e.g.,
beating heart cells).
3. Frequently, these characteristics are either lost or changed as a result of
being placed in an artificial environment.
4. Cell line may be:
a. Finite.
b. Continuous
c. Transformed Cells are usually easier and faster growing, may often have
extra or abnormal chromosomes and frequently can be grown in
suspension.
d. If the cells form tumors when they are injected into animals, they are
considered to be Neoplastically Transformed.
 TYPES OF ANIMAL CELL
CULTURES
1. Anchorage dependent culture
Anchorage dependent cell lines can be grown by the following
methods
a. conventional methods
b. new trends
 multiple surface tissue prapagator

 capillary semi permeable membranes

 as aggregates

 encapsulation cells
 packed bed reactor
 Suspension culture:
 Bioreactor
 Hollow fiber reactor
 Air lift fermentors
 Chemostates

Modes of suspension culture

 Batch culture

 Fed batch culture

 Continuous culture

 Perfusion culture
 Fermentation technology for the growth of animal cells
and their products:

 Fermentors have been used for the cultivation of bacteria and

yeasts for a long time. Initially, fermentation was synonymous
with alcohol production. Later, bacteriologists learnt to use the
same principles for the preparation of vitamins, organic acids,
antibiotics etc. This led to the rapid development of a variety
of fermentors and methodologies.
 WORK AREA AND EQUIPMENT
 Laminar flow hoods.
 Vertical LFH
 Horizontal LFH

 B. CO2 Incubators: cells are grown in an atmosphere of 5-10%

CO2

 Microscopes

 Preservation. Cells are stored in liquid nitrogen

 E. Vessels.
 Media and growth
requirements
1. Physiological parameters
A. temperature - 37C for cells from homeother
B. pH - 7.2-7.5 and osmolality of medium must be maintained
C. humidity is required
D. gas phase - bicarbonate conc. and CO2 tension in equilibrium
E. visible light - can have an adverse effect on cells; light induced
production of toxic compounds can occur in some media; cells
should be cultured in the dark and exposed to room light as little
as possible
2. Medium requirements: (often empirical)
 A. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO 2

B. Trace elements - iron, zinc, selenium

C. sugars - glucose is the most common
D. amino acids - 13 essential
E. vitamins - B, etc.
F. choline, inositol
G. serum - contains a large number of growth promoting activities such as
buffering toxic nutrients by binding them, neutralizes trypsin and other
proteases, has undefined effects on the interaction between cells and
substrate, and contains peptide hormones or hormone-like growth factors
that promote healthy growth.
H. antibiotics - although not required for cell growth, antibiotics are often
used to control the growth of bacterial and fungal contaminants.

 3. Feeding - 2-3 times/week

 Measurement of growth and
viability
 The viability of cells can be observed visually using an
inverted phase contrast microscope. Live cells are phase
bright; suspension cells are typically rounded and somewhat
symmetrical; adherent cells will form projections when they
attach to the growth surface.

 Viability can also be assessed using the vital dye, trypan blue,
which is excluded by live cells but accumulates in dead cells .

Cell numbers are determined using a hemocytometer
TISSUE CULTURE PROCEDURES

1. TRYPSINIZING AND SUBCULTURING CELLS FROM

A MONOLAYER
 A primary culture is grown to confluency in a 60-mm petri

plate or 25-cm2 tissue culture

 flask containing 5 ml tissue culture medium. Cells are

dispersed by trypsin treatment and

 then reseeded into secondary cultures. The process of

removing cells from the primary

 culture and transferring them to secondary cultures constitutes

a passage, or subculture.
2. Materials

 Primary cultures of cells

 HBSS without Ca2+ and Mg, 37°C
 Trypsin/EDTA solution 37°C
 Complete medium with serum: e.g., supplemented DMEM (APPENDIX
2A) with 10%
 to 15% (v/v) FBS (complete DMEM-10 or -15), 37°C
 Sterile Pasteur pipets
 37°C warming tray or incubator
 Tissue culture plasticware or glassware including pipets and 25-cm2 flasks
 or 60-mm petri plates, sterile
 NOTE: All culture incubations should be performed in a humidified 37°C,
5% CO2
 incubator unless otherwise specified Some media (e.g., DMEM) may
require altered
 levels of CO2 to maintain pH 7.4.
 Procedure:
 Remove all medium from primary culture with a sterile Pasteur pipet.
Wash adhering
 cell monolayer once or twice with a small volume of 37°C HBSS without
Ca2+ and
 Mg2+ to remove any residual FBS that may inhibit the action of trypsin.
 .Use a buffered salt solution that is Ca2+ and Mg2+ free to wash cells.
Ca2+ and Mg2+ in the
 salt solution can cause cells to stick together.
 .If this is the first medium change, rather than discarding medium that is
removed from
 primary culture, put it into a fresh dish or flask. The medium contains
unattached cells that
 may attach and grow, thereby providing a backup culture.
 Add enough 37°C trypsin/EDTA solution to culture to cover
adhering cell layer.

 Place plate on a 37°C warming tray 1 to 2 min. Tap bottom of

plate on the countertop to dislodge cells.

 Check culture with an inverted microscope to be sure that cells

are rounded up and detached from the surface.

 If cells are not sufficiently detached, return plate to warming

tray for an additional minute or two.
 Add 2 ml 37°C complete medium. Draw cell suspension into a
Pasteur pipet
 rinse cell layer two or three times to dissociate cells and to
dislodge any remaining adherent cells.
 As soon as cells are detached, add serum or medium
containing serum to inhibit further trypsin activity that might
damage cells.
 If cultures are to be split 1/3 or 1/4 rather than 1/2, add
sufficient medium such that 1 ml of cell suspension can be
transferred into each fresh culture vessel.
 Add 4 ml fresh medium to each new culture. Incubate in a
humidified 37°C, 5% CO2 incubator.

 If using 75-cm2 culture flasks, add 9 ml medium per flask.

 Some labs now use incubators with 5% CO2 and 4% O2. The
low oxygen concentration is thought to simulate the in vivo
environment of cells and to enhance cell growth.

 For some media it is necessary to adjust the CO2 to a higher or

lower level to maintain the pH at 7.4.

7. If necessary, feed subconfluent cultures after 3 or 4 days by

removing old medium and adding fresh 37°C medium.
8. Passage secondary culture when it becomes confluent by
repeating steps 1 to 7, and continue to passage as necessary
 Add an equal volume of cell suspension to fresh plates or
flasks that have been appropriately labeled.
 Alternatively, cells can be counted using a hemacytometer or
Coulter counter and diluted to the desired density so a specific
number of cells can be added to each culture vessel.
 A final concentration of ∼5 104 cells/ml is appropriate for
most subcultures.
 For primary cultures and early subcultures, 60-mm petri plates
or 25-cm2 flasks are generally used; larger vessels (e.g., 150-
mm plates or 75-cm2 flasks) may be used for later subcultures.
 Cultures should be labeled with date of subculture and passage
number.
 If necessary, feed sub confluent cultures after 3 or 4 days by
removing old medium and adding fresh 37°C medium.

Passage secondary culture when it becomes confluent by

repeating steps 1 to 7, and continue to passage as necessary
 CELL FEEDING
Use prewarmed media and have cells out of the incubator for
as little time as possible. Feeding and sub culturing suspension
cultures are done simultaneously. About every 2-3 days, dilute
the cells into fresh media. The dilution you use will depend on
the density of the cells and how quickly they divide, which
only you can determine. Typically 1:4 to 1:20 dilutions are
appropriate for most cell lines. b. Adherent cells. About every
2-3 days, pour off old media from culture flasks and replace
with fresh media. Subculture cells as described below before
confluency is reached.
 SUBCULTURING OF ADHERENT
CELLS
When adherent cells become semi-confluent, subculture using 2
mM EDTA or trypsin/EDTA.

TRYPSIN EDTA
a. Remove medium from culture dish and wash cells in a balanced salt solution
without Ca++ or Mg++. Remove the wash solution.
b. Add enough trypsin-EDTA solution to cover the bottom of the culture
vessel and then pour off the excess.
c. Place culture in the 37°C incubator for 2 minutes.
d. Monitor cells under microscope. Cells are beginning to detach when they
appear rounded.
e. As soon as cells are in suspension, immediately add culture medium
containing serum. Wash cells once with serum containing medium and
dilute as appropriate (generally 4-20 fold).
 EDTA ALONE
a. Prepare a 2 mM EDTA solution in a balanced salt solution (i.e.,
PBS without Ca++ or Mg++).
b. Remove medium from culture vessel by aspiration and wash the
monolayer to remove all traces of serum. Remove salt solution
by aspiration.
c. Dispense enough EDTA solution into culture vessels to completely
cover the monolayer of cells.
d. The coated cells are allowed to incubate until cells detach from
the surface. Progress can be checked by examination with an
inverted microscope. Cells can be gently nudged by banging the
side of the flask against the palm of the hand.
e. Dilute cells with fresh medium and transfer to a sterile centrifuge
tube.
f. Spin cells down, remove supernatant, and resuspend in culture
medium (or freezing medium if cells are to be frozen). Dilute as
appropriate into culture flasks.
THAWING FROZEN CELLS
a. Remove cells from frozen storage and quickly thaw in a 37°C
water bath by gently agitating vial.
b. As soon as the ice crystals melt, pipette gently into a culture
flask containing prewarmed growth medium.
c. Log out cells in the "Liquid Nitrogen Freezer Log" Book.
 FREEZING CELLS
 a. Harvest cells as usual and wash once with complete medium.
 b. Resuspend cells in complete medium and determine cell
count/viability.
 c. Centrifuge and resuspend in ice-cold freezing medium: 90%
calf serum/10% DMSO, at 106 - 107 cells/ml. Keep cells on ice.
 d. Transfer 1 ml aliquots to freezer vials on ice.
 e. Place in a Mr. Frosty container that is at room temperature
and that has sufficient isopropanol.
 f. Place the Mr. Frosty in the -70°C freezer overnight. Note:
Cells should be exposed to freezing medium for as little time as
possible prior to freezing
 g Next day, transfer to liquid nitrogen (DON'T FORGET) and
log in the "Liquid Nitrogen Freezer Log" Book.
 VIABLE CELL COUNT

USING A HEMOCYTOMETER TO DETERMINE TOTAL

CELL COUNTS AND VIABLE CELL NUMBERS

Trypan blue is one of several stains recommended for use in

dye exclusion procedures for viable cell counting. This
method is based on the principle that live cells do not take up
certain dyes, whereas dead cells do.
 MAINTENANCE

Cultures should be examined daily, observing the morphology,

the color of the medium and the density of the cells. A tissue
culture log should be maintained that is separate from your
regular laboratory notebook. The log should contain: the name
of the cell line, the medium components and any alterations to
the standard medium, the dates on which the cells were split
and/or fed, a calculation of the doubling time of the culture
(this should be done at least once during the semester), and
any observations relative to the morphology, etc.
 A. Growth pattern. Cells will initially go through a quiescent
or lag phase that depends on the cell type, the seeding density,
the media components, and previous handling. The cells will
then go into exponential growth where they have the highest
metabolic activity. The cells will then enter into stationary
phase where the number of cells is constant, this is
characteristic of a confluent population (where all growth
surfaces are covered).
 B. Harvesting. Cells are harvested when the cells
have reached a population density which suppresses
growth. Ideally, cells are harvested when they are in a
semi-confluent state and are still in log phase. Cells
that are not passaged and are allowed to grow to a
confluent state can sometime lag for a long period of
time and some may never recover. It is also essential
to keep your cells as happy as possible to maximize
the efficiency of transformation. Most cells are
passaged (or at least fed) three times a week.
1. Suspension culture. Suspension cultures are fed by
dilution into fresh medium.

2. Adherent cultures. Adherent cultures that do not

need to be divided can simply be fed by removing
the old medium and replacing it with fresh medium.
3. When the cells become semi-confluent, several
methods are used to remove the cells from the
growing surface so that they can be diluted:
 Mechanical - A rubber spatula can be used to physically
remove the cells from the growth surface. This method is
quick and easy but is also disruptive to the cells and may result
in significant cell death. This method is best when harvesting
many different samples of cells for preparing extracts, i.e.,
when viability is not important.
 Proteolytic enzymes - Trypsin, collagenase, or pronase,
usually in combination with EDTA, causes cells to detach
from the growth surface. This method is fast and reliable but
can damage the cell surface by digesting exposed cell surface
proteins. The proteolysis reaction can be quickly terminated
by the addition of complete medium containing serum
 EDTA - EDTA alone can also be used to detach cells and
seems to be gentler on the cells than trypsin.
The standard procedure for detaching adherent cells is as follows:
1. Visually inspect daily
2. Release cells from monolayer surface by the following
procedure.
Release cells from monolayer surface
 wash once with a buffer solution
 treat with dissociating agent
 observe cells under the microscope. Incubate until cells
become rounded and loosen when flask is gently tapped
with the side of the hand.
 Transfer cells to a culture tube and dilute with medium
containing serum.
 Spin down cells, remove supernatant and replace with fresh
medium.
 Count the cells in a hemacytometer, and dilute as
appropriate into fresh medium.
 PRESERVATION AND STORAGE.
 Liquid N2 is used to preserve tissue culture cells, either in the
liquid phase (-196°C) or in the vapor phase (-156°C). Freezing
can be lethal to cells due to the effects of damage by ice
crystals, alterations in the concentration of electrolytes,
dehydration, and changes in pH. To minimize the effects of
freezing, several precautions are taken.
 SAFETY CONSIDERATIONS
 Assume all cultures are hazardous since they may harbor
latent viruses or other organisms that are uncharacterized. The
following safety precautions should also be observed:
 pipetting: use pipette aids to prevent ingestion and keep
aerosols down to a minimum
 no eating, drinking, or smoking
 wash hands after handling cultures and before leaving the lab
 decontaminate work surfaces with disinfectant (before and
after)
 autoclave all waste
 use biological safety cabinet (laminar flow hood) when
working with hazardous organisms. The cabinet protects
worker by preventing airborne cells and viruses released
during experimental activity from escaping the cabinet; there
is an air barrier at the front opening and exhaust air is filtered
with a HEPA filter make sure cabinet is not overloaded and
leave exhaust grills in the front and the back clear (helps to
maintain a uniform airflow)
 use aseptic technique
 dispose of all liquid waste after each experiment and treat with
bleach
 Some of the Problems Faced by Cultured Cells

Avoiding Contamination
 Two types of contamination

 Biological e.g. viruses, bacteria, fungi etc

 Chemical e.g. endotoxins metal ions and traces of disinfectants.

Methods:
 1. proper training in and use of good aseptic technique on
the part of the cell culturist.

 Secondly properly designed maintained and sterilized

equipments, plastic wares, glass wares and media .The careful
and selective (limited) use of antibiotics designed for use in
tissue culture can also help avoid culture loss due to biological
contamination.
 Finding A “Happy” Environment
 A happy environment is the one which

 Allows the cell to survive.

 Allows the cell to divide and redivide i.e. increased

mitosis.

 Carrying out imp in vivo physiological or biochemical

functions such as muscle contractions or secretion of
enzymes or hormones.
 BASIC ENVOIRNMENTAL
REQUIREMENTS FOR HAPPY CELLS
 Controlled temperature (depends upon host organism) cold
blooded vertebrates temp is 18 to 25C. While mammalian its
36 to 37C.

 Good substrate for cell attachment such as collagen, gelatin,

fibronectin and laminin.

 Appropriate medium and incubator that maintains the correct

pH and osmolality.
 How to Decide if Cultured Cells Are
“Happy”

 The Morphology or cell shape usualy staining is done to

find out any problem. Also a difficult characteristic to
quantify and measured precisely.

 Growth rate determination.

 Plating Efficiency.

 Expression of a specialized function.

 What is Cell Culture Used
For?
Cell culture has become one of the major tools used in cell and
molecular biology. Some of the important areas where cell
culture is currently playing a major role are briefly described
below:

 Model Systems
 Toxicity Testing
 Cancer Research
 Virology
 Cell-Based Manufacturing
 Genetic Counseling
 Genetic Engineering
 Gene Therapy
 Drug Screening and Development
 Model Systems
 Cell cultures provide a good model system for studying
1) basic cell biology
and biochemistry,
 2) the interactions between disease-causing agents and cells,
 3) the effects of drugs on cells,
 4) the process and triggers for aging, and 5) nutritional
studies.
 Toxicity Testing

Cultured cells are widely used alone or in conjunction

with animal tests to study the effects of new drugs,
cosmetics and chemicals on survival and growth in a
wide variety of cell types. Especially important are
liver- and kidney-derived cell cultures.
 Cancer Research
Since both normal cells and cancer cells can be grown in
culture, the basic differences between them can be closely
studied. In addition, it is possible, by the use of chemicals,
viruses and radiation, to convert normal cultured cells to
cancer causing cells. Thus, the mechanisms that cause the
change can be studied. Cultured cancer cells also serve as a
test system to determine suitable drugs and methods for
selectively destroying types of cancer.
 Virology
One of the earliest and major uses of cell culture is the
replication of viruses in cell cultures (in place of
animals) for use in vaccine production. Cell cultures
are also widely used in the clinical detection and
isolation of viruses, as well as basic research into how
they grow and infect organisms.
 CELL BASED MANUFACTURING

 Use of cells as replacement tissue and organ.

 large scale production of viruses for use in vaccines.
 Large scale production if genetically engineered cell
for the production of commercially imp protein.
 Genetic Counseling
Amniocentesis, a diagnostic technique that enables
doctors to remove and culture fetal cells from pregnant
women, has given doctors an important tool for the early
diagnosis of fetal disorders. These cells can then be
examined for abnormalities in their chromosomes and
genes using karyotyping, chromosome painting and other
molecular techniques.
 Genetic Engineering
The ability to transfect or reprogram cultured cells with
new genetic material (DNA and genes) has provided a
major tool to molecular biologists wishing to study the
cellular effects of the expression of theses genes (new
proteins). These techniques can also be used to produce
these new proteins in large quantity in cultured cells for
further study. Insect cells are widely used as miniature
cells factories to express substantial quantities of proteins
that they manufacture after being infected with
genetically engineered baculo viruses.
 Drug Screening and Development

Cell-based assays have become increasingly important for

the pharmaceutical industry, not just for cyto toxicity testing
but also for high throughput screening of compounds that
may have potential use as drugs. Originally, these cell culture
tests were done in 96 well plates, but increasing use is now
being made of 384 and 1536 well plates.
 Gene Therapy
The ability to genetically engineer cells has also led to their use
for gene therapy. Cells can be removed from a patient lacking a
functional gene and the missing or damaged gene can then be
replaced. The cells can be grown for a while in culture and then
replaced into the patient. An alternative approach is to place the
missing gene into a viral vector and then “infect’’ the patient with
the virus in the hope that the missing gene will then be expressed
in the patient’s cells.
THANKS FOR
YOUR
PATIENCE