BT 505 Unit 1

Unit Content
Unit-I
Basics of Cell and Tissue Culture: Laboratory requirements for tissue culture,
substrates for cultures, culture media for animal cell cultures, culture procedures
and principles, freeze storing of cells and transport of cultures, Primary culture,
secondary culture; Continuous cell lines; Suspension cultures.
Characteristics of Cells in Culture: Contact inhibition, anchorage

independence/dependence, cell-cell communication, cell senescence.
Cell Culture Lines: Definition, development and maintenance, characteristics of

animal cells and their implication on process design, nutritional requirements and
serum free culture of mammalian cells, kinetics of growth and product formation,
cloning of cell lines, cell synchronization, viral sensitivity of cell lines, cell line
characterization, stem cell lines.
Dr. Harmanpreet Meehnian BT 505 Animal Cell Culture Technology Unit 1

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Laboratory Requirements for tissue culture
• Laboratory Design and Layout

Planning
1. Ventilation
a) Pressure balance
b) Laminar hood
2. Accommodation
c) Staff numbers
d) Space (culture operation, washup-preparation-sterilization, incubation and storage; 4:2:1:1)
e) Aseptic area
f) Hoods
The space between hoods should be approximately 500 mm (2 ft), to allow access for
maintenance and to minimize interference in airflow between hoods.
11/17/2022 Dr. Harmanpreet Meehnian BT 505 Animal Cell Culture Technology Unit 1 2
e) Incubation area
f) Preparation area
Washing and sterilization are should be close to aseptic area and near to outside wall
remove the heat generated
g) Servicing aseptic areas
h) Storage
3. Renovations
4. Access (doorways and space)
5. Quarantine
• CONSTRUCTION AND SERVICES

 The rooms should be designed for easy cleaning. Furniture should fit tightly to the
floor or be suspended from the bench, with a space left underneath for cleaning.
 Cover the floor with a vinyl or other dustproof finish, and allow a slight fall in the
level toward a floor drain located outside the door of the room (i.e., well away from
the sterile cabinets).
 This arrangement allows liberal use of water if the floor has to be washed.
 If possible it is preferable for the tissue culture lab to be separated from the
preparation, washup, and sterilization areas, while still remaining adjacent
LAYOUT OF ASEPTIC ROOM OR SUITE
• Six main functions need to be accommodated in the laboratory: sterile

handling, incubation, preparation, washup, sterilization, and storage
• If a single room is used, create a ‘‘sterility gradient’’; the clean area for sterile
handling should be located at one end of the room, farthest from the door,
and washup and sterilization facilities should be placed at the other end, with
preparation, storage, and incubation in between.
• The preparation area should be adjacent to the washup and sterilization

areas, and storage and incubators should be readily accessible to the sterile
working area
• Sterile Handling Area

- Restricted to tissue culture (i.e., not shared with chemical work or with work on other
organisms such as bacteria or yeast), and there should be no through traffic or other
disturbance that is likely to cause dust or drafts.
- Facilitate the observation of cultures, dissection, etc., and to allow an accurate reading
of pH when phenol red is used as an indicator
• Laminar Flow
- Individual freestanding hoods are preferable, as they separate operators and can be
moved around.
Quarantine and Containment

• If sufficient space is available, it is worth designating a separate room as a
quarantine and/or containment room.
• This is a separate aseptic room with its own laminar-flow hood (Class II
microbiological safety cabinet), incubators, freezer, refrigerator, centrifuge,
supplies, and disposal. Negative pressure
Service Bench
• It may be convenient to position a bench for a cell counter, microscope,
etc., close to the sterile handling area and either dividing the area or
separating it from the other end of the lab.
• The service bench should also provide for the storage of sterile glassware,
plastics, pipettes, screw caps, syringes, etc., in drawer units below and open
shelves above
INCUBATION
The requirement for cleanliness is not as stringent as that for sterile handling,
but clean air, a low disturbance level, and minimal traffic will give your
incubation area a better chance of avoiding dust, spores, and the drafts that
carry them
Incubators
• Incubation may be carried out in separate incubators or in a thermostatically
controlled hot room
• As a rough guide, you will need 0.2 m3 (200 L, 6 ft3) of incubation space
with 0.5 m2 (6 ft2) shelf space per person.
• Extra provision may need to be made for one or more humid incubators with
a controlled CO2 level in the atmosphere
INCUBATION
Hot Room
If you have the space within the laboratory area or have an adjacent room or walk-in
cupboard readily available and accessible, it may be possible to convert the area into a
hot room.
Heaters. Heat is best supplied via a fan heater, domestic or industrial, depending on the
size of the room. Approximately 2–3 kW per 20 m3 (700 ft3) will be required (or two
heaters could be used, each generating 1.0–1.5 kW), depending on the insulation .
Air circulation. A second fan, positioned on the opposite side of the room and with the
airflow opposing that of the fan heater, will ensure maximum circulation. If the room
is more than 2 × 2 m (6 × 6 ft), some form of ducting may be necessary.
Thermostats. Thermostats should be of the ‘‘proportional controller’’ type, acting via a
relay to supply heat at a rate proportional to the difference between the room
temperature and the set point.
Overheating, Access, thermometer
INCUBATION
Hot Room
PREPARATION AREA
Media Preparation
• The need for extensive preparation of media in small laboratories can be avoided if
there is a proven source of reliable commercial culture media.
• Large enterprise (approximately 50 people doing tissue culture) may still find it more
economical to prepare its own media, smaller laboratories may prefer to purchase
readymade media.
• Washup
Washup and sterilization facilities are best situated outside the tissue culture lab, as
the humidity and heat that they produce may be difficult to dissipate without
increasing the airflow above desirable limits.
• The washup area should have plenty of space for soaking glassware and space for an
automatic washing machine, should you require one. There should also be plenty of
bench space for handling baskets of glassware, sorting pipettes, and packaging and
sealing packs for sterilization
PREPARATION AREA
• Washup
PREPARATION AREA
• Storage
Storage must be provided for the following items ensuring sterile and nonsterile are
kept separate and clearly labeled:
(1) Sterile liquids, at room temperature (salt solutions, water, etc.), at 4◦C (media),
and at -20◦C or -70◦C (serum, trypsin, glutamine, etc.)
(2) Sterile and nonsterile glassware, including media bottles and pipettes
(3) Sterile disposable plastics (e.g., culture flasks and Petri dishes, centrifuge tubes
and vials, and syringes)
(4) Screw caps, stoppers, etc., sterile and nonsterile
(5) Apparatus such as filters, sterile and nonsterile
(6) Gloves, disposal bags, etc.
(7) Liquid nitrogen to replenish freezers; the liquid nitrogen should be stored in two
ways:
a) in Dewars (25–50 L) under the bench, or
b) in a large storage vessel (100–150 L) on a trolley or in storage tanks (500–1000 L)
permanently sited in a room of their own with adequate ventilation or, preferably,
outdoors in secure, weatherproof housing
PREPARATION AREA
• Storage
(8) Cylinder storage for carbon dioxide, in separate cylinders for transferring to the
laboratory as required
9) A piped supply of CO2 can be taken to work stations, or else the CO2 supply can
be piped from a pressurized tank of CO2 that is replenished regularly (and must
therefore be accessible to delivery vehicles).
EQUIPMENT
EQUIPMENT
Aseptic Area
• Laminar-Flow Hood
• Pipette Cylinders
• Aspiration Pump
• Service Carts
• Inverted Microscope
• Centrifuge
• Sterile Liquid Handling—Pipetting and Dispensing
• Cell Counter
• CCD Camera and Monitor & Dissecting Microscope

EQUIPMENT
EQUIPMENT
EQUIPMENT
EQUIPMENT
EQUIPMENT
EQUIPMENT
EQUIPMENT
Incubation
• Incubator
• Humid CO2 Incubator
• Temperature Recorder
• Roller Racks
• Magnetic Stirrer
EQUIPMENT
EQUIPMENT
Preparation and sterilization

• Washup
• Water Purifier
• Sterilizing and Drying Ovens
• Steam Sterilizer (Autoclave)
• Balances
• pH Meter
• Hot Plate Magnetic Stirrer
• Automatic Dispensers
• Conductivity Meter, Osmometer & Glassware Washing Machine

EQUIPMENT
EQUIPMENT
EQUIPMENT
EQUIPMENT
EQUIPMENT
Storage
• Refrigerators and Freezers
• Cryostorage Containers
• Controlled-Rate Freezer
• Low-Temperature Freezer
EQUIPMENT
Laboratory Backup
• Computers and Networks
• Upright Microscope
• Low-Temperature Freezer
• Confocal Microscope
• PCR Cycler
EQUIPMENT
Specialized Equipment
• Microinjection Facilities
• Colony Counter
• Centrifugal Elutriator
• Flow Cytometer
EQUIPMENT
Consumable Items
• Pipettes
• Hemocytometer
• Sterile Containers
• Syringes and Needles
• Sterilization Filters
• Paper Towels and Swabs
• Disinfectants
Substrates for culture
Attachment and Growth
• The majority of vertebrate cells cultured in vitro grow as monolayers on an artificial

substrate.
• Hence the substrate must be correctly charged to allow cell adhesion, or at least to
allow the adhesion of cell-derived attachment factors, which will, in turn, allow cell
adhesion and spreading.
• Cells shown to require attachment for growth are said to be anchorage dependent
• Cells that have undergone transformation frequently become anchorage independent

and can grow in suspension when stirred or held in suspension with semisolid media
such as agar.
SUBSTRATE MATERIALS
Glass
• This was the original substrate because of its optical properties and surface charge,
but it has been replaced in most laboratories by synthetic plastic (usually
polystyrene), which has greater consistency and superior optical properties.
• Glass is now rarely used, although it is cheap, is easily washed without losing its
growth-supporting properties, can be sterilized readily by dry or moist heat, and is
optically clear.
SUBSTRATE MATERIALS
Disposable plastic
• Single-use sterile polystyrene flasks provide a simple, reproducible substrate for

culture.
• They are usually of good optical quality, and the growth surface is flat, providing
uniformly distributed and reproducible monolayer cultures.
• As manufactured, polystyrene is hydrophobic and does not provide a suitable surface

for cell attachment, so tissue culture plastics are treated by gas plasma, or γ -
irradiation, or chemically, to produce a charged, wettable surface.
• Although polystyrene is by far the most common and cheapest plastic substrate, cells
may also be grown on polyvinylchloride (PVC), polycarbonate,
polytetrafluorethylene (PTFE; Teflon), Melinex, Thermanox (TPX), and a number of
other plastics.
SUBSTRATE MATERIALS
Choice Of Culture Vessel
Several factors govern the choice of culture vessel, including
(1) The cell mass required,
(2) Whether the cells grow in suspension or as a monolayer,
(3) Whether the culture should be vented to the atmosphere or sealed,
(4) The frequency of sampling,
(5) The type of analysis required, and
(6) The cost
SUBSTRATE MATERIALS
Cell Yield
• For monolayer cultures, the cell yield is proportional to the available surface area of the
flask.
• Small volumes and multiple replicates are best performed in multi-well plates, which
can have a large number of small wells (e.g., microtitration plates with 96 or 144 wells,
0.1–0.2 mL of medium, and 0.25-cm2 growth area or 24-well ‘‘cluster dishes’’ with 1–2
mL medium in each well, 1.75-cm2 growth area) up to 4-well plates with each well 5
cm in diameter and using 5 mL of culture medium
• The middle of the size range embraces both Petri dishes and flasks ranging from 10
cm2 to 225 cm2. Flasks are usually designated by their surface area (e.g., 25 cm 2 or 175
cm2, and sometimes T25 or T175, respectively), whereas Petri dishes are referred to by
diameter (e.g., 3.5 cm or 9 cm)
SUBSTRATE MATERIALS
Suspension Culture
• Cells that grow in suspension can be grown in any

type of flask, plate, or Petri dish that, although
sterile, need not be treated for cell attachment.
• Stirrer bottles are used when agitation is required to

keep the cells in suspension.
• These bottles are available in a wide range of sizes,

usually in glass (Bellco, Techne). Agitation is
usually by a suspended paddle containing a magnet,
whose rotation is driven by a magnetic stirrer
SUBSTRATE MATERIALS
Uneven Growth
• Sometimes, cells can be inadvertently distributed nonuniformly across the growth

surface.
• Vibration, caused by opening and closing of the incubator, a faulty fan motor, or
vibration from equipment, can perturb the medium, which can result in resonance or
standing waves in the flask that, in turn, result in a wave pattern in the monolayer,
creating variations in cell density
SUBSTRATE MATERIALS
Cost
• Cost always has to be balanced against convenience; for example, Petri dishes are
cheaper than flasks with an equivalent surface area, but require humid, CO 2-controlled
conditions and are more prone to infection.
• Cheap soda glass bottles, although not always of good optical quality, are often better
for culture than higher-grade Pyrex, or optically clear glass, which usually contains
lead.
• A major disadvantage of glass is that its preparation is labor intensive, because it must
be carefully washed and resterilized before it can be reused. Most laboratories now use
plastic, because of its convenience, optical clarity, and quality
SUBSTRATE MATERIALS
Specialized Systems
Permeable Supports
• Semipermeable membranes are used as gas-permeable substrates and will also allow
the passage of water and small molecules (<500–1000 da), a property that is exploited
in some large-scale bioreactors
• Filter wells: The permeability of the surface to which the cell is anchored may induce
polarity in the cell by simulating the basement membrane. Such polarity may be vital to
full functional expression in secretory epithelia and many other types of cells.
Hollow fibers. Knazek et al. [1972] developed a technique for growing cells on the
outer surface of bundles of plastic microcapillaries. The plastic allows the diffusion of
nutrients and dissolved gases from a medium perfused through the capillaries.
SUBSTRATE MATERIALS
Treated Surfaces
Matrix Coating
i) Conditioning
ii) Polylysine
iii) Collagen and gelatin
iv) Matrigel (eg. Becton Dickinson, laminin, fibronectin, vitronectin, Pronectin F)
v) Extracellular matrix (Gospodarowicz et al. [1980] were able to grow endothelium on

extracellular matrix (ECM) derived from confluent monolayers of 3T3 cells that had been
removed with Triton X-100)
SUBSTRATE MATERIALS
Treated Surfaces
• Feeder layer: Although matrix coating may help attachment, growth, and
differentiation, some cultures of more fastidious cells, particularly at low cell densities
[Puck & Marcus, 1955], require support from living cells (e.g., mouse embryo
fibroblasts)
• Three-Dimensional Matrices
• Metallic Substrates (palladium)
• Non-adhesive Substrates
Cell Culture Media
• Media is generally defined as the medium which contains all the necessary
elements requires for the growth of cell or tissue culture.
• Besides meeting the basic nutritional requirement of the cells, the culture medium
should also have any necessary growth factors, regulate the pH and osmolality, and
provide essential gases (O2 and CO2).
• The ‘food’ portion of the culture medium consists of amino acids, vitamins,
minerals, and carbohydrates. These allow the cells to build new proteins and other
components essential for growth & function as well as providing energy necessary
for metabolism.
Types of Cell Culture Media
Biological Fluids
Eg. plasma,
serum, lymph
Natural
Media Tissue Extracts
Eg. Extract of
liver, spleen,
Cell tumors, leucocytes
and bone
Culture
Media Balanced Salt
Solutions
Eg. Eagle’s BS,
Artificia Hank’s BS,
l Media Basal media
Eg. MEM,
DMEM
Complex media
Eg. RPMI-
1640, IMDM
Ingredients of Media
Culture media (as a powder or as a liquid) contains:

• Amino acids
• Sugar
• Inorganic Salts
• Vitamins
• Natural buffering system Ex-HEPES
• Phenol red
• Proteins and Peptides (important in serum-free media).
• Trace Elements
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Unit 1
Amino Acids (CO1)
• The essential amino acids are required by cultured cells.
• Other non-essential amino acids are often added as well.

• The concentration of amino acids usually limits the
maximum cell concentration attainable, and the balance
may influence cell survivalSugar
and growth rate.
• Glucose is included in most media as a source of energy.

• It is metabolized principally by glycolysis to form pyruvate,
which may be converted to lactate or acetoacetate and may
enter the citric acid cycle and be oxidized to form CO2 and
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water. Unit 1
Vitamins
• Amino acids, vitamin requirements have been derived

empirically and often relate to the cell line originally used in
their development.
• Eagle’s MEM contains only the water-soluble vitamins
excluding biotin
• Biotin is present in most of the more complex media,
including the serum free recipes.
• All the fat-soluble vitamins (A, D, E, and K)are present only
in M199, whereas vitamin A is present in LHC-9 and vitamin
E in MCDB 110
Inorganic Salts
• The salts are chiefly those of Na+, K+, Mg2+, Ca2+, Cl−, SO42-, PO43-, and
HCO3- and are the major components contributing to the osmolality of the
medium.
• Ca2+, are required by some cell adhesion molecules, such as the cadherins
• Ca2+ also acts as an intermediary in signal transduction and the concentration
of Ca2+ in the medium can influence whether cells will proliferate or
differentiate.
• Na+, K+, and Cl− regulate membrane potential.
• SO42-, PO43-, and HCO3- have roles as anions required by the matrix and
nutritional precursors for macromolecules, as well as regulators of
intracellular charge.
• The sodium bicarbonate concentration is determined by the concentration of
CO2 in the gas phase and has a significant role in buffering capability.
Proteins & Peptides
• The most commonly used proteins are:
1) Albumin,
• Albumin binds to water, salts, free fatty acids, hormones, and vitamins, and
transport them between tissues and cells.
• Also remover of toxic substance from media.
2) Aprotinin
• Aprotinin is a protective agent in cell culture systems, stable at neutral and acidic
pH and resistant to high temperatures and degradation by proteolytic enzymes.
• Inhibit proteases such as trypsin
3) Fibronectin
• Fibronectin -in cell attachment.
Trace Elements
• Trace elements are often supplemented to serum-free media to replace

those normally found in serum.
• Trace elements like copper, zinc, selenium are needed in minute amounts
for proper cell growth.
• These micronutrients are essential for many biological processes, e.g. the
maintenance of the functionality of enzymes.
Phenol Red
• Most of the commercially available culture media include phenol red as a

pH indicator, which allows constant monitoring of pH During the cell
growth, the medium changes colour as pH is changed due to the
metabolites released by the cells.
• At low pH levels, phenol red turns the medium yellow, while at higher pH
levels it turns the medium purple.
• Medium is bright red for pH 7.4, the optimum pH value for cell culture.
Buffering System
• Regulating pH is critical for optimum culture conditions and is generally

achieved by one of the two buffering systems: Natural buffering system and
Chemical Buffering System
• In a natural buffering system, gaseous CO2 balances with the CO3/HCO3
content of the culture medium.
• Cultures with a natural buffering system need to be maintained in an air
atmosphere with 5-10% CO2, usually maintained by an CO2 incubator
• Natural buffering system is low cost and non-toxic.
• Chemical buffering using a zwitterion, HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid).
• HEPES, has a superior buffering capacity in the pH range 7.2-7.4 and does
not require a controlled gaseous atmosphere.
• HEPES is relatively expensive and toxic at a higher concentration for some
cell types.
Oxygen
• The other major significant constituent of the gas phase is oxygen.

• Whereas most cells require oxygen for respiration in vivo, cultured cells often
rely mainly on glycolysis, a high proportion of which, as in transformed cells,
may be anaerobic.
• Cultures vary in their oxygen requirement, the major distinction lying
between organ and cell cultures.
• Although atmospheric or lower oxygen tensions are preferable for most cell
cultures, some organ cultures, particularly from late-stage embryos, newborns,
or adults, require up to 95% O2 in the gas phase
• Most dispersed cell cultures prefer lower oxygen tensions, and some systems
(e.g., human tumor cells in clonogenic assay and human embryonic lung
fibroblasts ) do better in less than the normal level of atmospheric oxygen
tension.
Osmolarity
• Most cultured cells have a fairly wide tolerance for osmotic

pressure.
• As the osmolality of human plasma is about 290 mosmol/kg, it is
reasonable to assume that this level is the optimum for human
cells in vitro, although it may be different for other species (e.g.,
around 310 mosmol/kg for mice).
• In practice, osmolalities between 260 mosmol/kg and 320
mosmol/kg are quite acceptable for most cells but, once selected,
should be kept consistent at ±10 mosmol/kg.
• Slightly hypotonic medium may be better for Petri dish or open-
plate culture to compensate for evaporation during incubation.
Temperature
• The optimal temperature for cell culture is dependent on:

(1) the body temperature of the animal from which the cells were
obtained,
(2) any anatomic variation in temperature (e.g., the temperature of
the skin and testis may be lower than that of the rest of the body),
and
(3) the incorporation of a safety factor to allow for minor errors in
regulating the incubator.
• Thus the temperature recommended for most human and warm-
blooded animal cell lines is 37◦C, close to body heat, but set a
little lower for safety, as overheating is a more serious problem
than underheating.
Surface Tension and Foaming
• The effects of foaming have not been clearly defined, but the
rate of protein denaturation may increase, as may the risk of
contamination if the foam reaches the neck of the culture
vessel.
• Foaming will also limit gaseous diffusion if a film from a
foam or spillage gets into the capillary space between the cap
and the bottle, or between the lid and the base of a Petri dish.
• Foaming can arise in suspension cultures in stirrer vessels or
bioreactors when 5% CO2 in air is bubbled through medium
containing serum.
• The addition of a silicone antifoam (Dow Chemical) or
Pluronic F68 (Sigma), 0.01–0.1%, helps prevent foaming in
this situation by reducing surface tension and may also protect
cells against shear stress from bubbles.
Balance Salt Solution
• Balanced salt solution (BSS) is composed of inorganic salts and may

include sodium bicarbonate and, in some cases glucose.
• HEPES buffer (5–20 mM) may be added to these solutions if necessary and
the equivalent amount of NaCl omitted to maintain the correct osmolality.
• BSS forms the basis of many complete media
• Commercial suppliers will provide Eagle’s MEM with Hanks’s salts or

Eagle’s MEM with Earle’s salts, indicating which BSS formulation was
used;
Balance Salt Solution Uses
• BSS is also used as a diluent for concentrates of amino acids

and vitamins to make complete media
• As an isotonic wash or dissection medium, and for short
incubations up to about 4 h (usually with glucose present).
• Used to maintain pH and osmatic pressure of the medium
Antibiotics Growth Supplements
• Antibiotics and/or antimycotic agents are added to

prevent contamination, as a cure once contamination is
found, to induce the expression of recombinant proteins,
or to maintain selective pressure on transfected cells.
• Routine use of antibiotics or antimycotics for cell
culture is not recommended unless they are specifically
required.
• Antibiotics can mask contamination by mycoplasma
and resistant bacteria.
• Further, they can interfere with the metabolism of
sensitive cells.
Fetal Bovine Serum
• Sera serve as a source for amino acids, proteins, vitamins ,

carbohydrates, lipids, hormones, growth factors, minerals, and
trace elements.
• Serum buffers the culture medium, inactivates proteolytic
enzymes, increases medium viscosity, and conditions the growth
surface of the culture vessel.
• Sera from fetal and calf bovine sources are commonly used to
support the growth of cells in culture.
• Fetal serum is a rich source of growth factors and is appropriate
for cell cloning and for the growth of fastidious cells.
• Calf serum, because of its lower growth-promoting properties, is
used in contact-inhibition
Serum Free Media
• Presence of serum in the media lead to misinterpretations in immunological studies;

•  serum-free media have been developed .
•  These media are specifically formulated to support the culture of a single cell
type and incorporate defined quantities of purified components.
• Advantages:
• Given pure constituents are used, a given medium formulation can be standardized
regardless of where it is used and by whom.
• Selective Media
•  One of the major advantage- control over growth promoting activity by serum-
free media. This ability to make a medium selective for a particular cell type.
•  Fibroblastic overgrowth can be inhibited (in breast and skin cultures) by using
MCDB 170 and 153
• Regulation of Proliferation and Differentiation
Selection of medium and serum
• The choice of cell culture media is extremely important, and significantly affects the
success of cell culture experiments.
• The selection of the media depends on the:
• 1. Type of cells to be cultured
• 2. The purpose of the culture
• 3. Resources available in the laboratory .
• Different cell types have highly specific growth requirements, therefore, the most
suitable media for each cell type must be determined experimentally
• Example; MEM for adherent cells and RPMI-1640 for suspension cells.
Preparation of Cell Culture Media
• Culture medium is available in three forms from the commercial suppliers:

• Powdered form: It need to prepared and sterilized.
• Concentrated form: Needs to be diluted.
• Working solution: Used directly without further manipulation.
• Powder medium is least expensive but needs to be sterilized.
• It is advisable to filter and sterilize it prior to addition of serum as the foaming that
occurs in presence of serum denatures protein.
• Serum should be added after filter sterilization.
• Media should be tested for sterility by placing it in a incubator for 72 hours prior to
utilization to ensure that the lot is contamination free.
• Media after preparation should be stored at 4 oC.
Sterilization of Cell Culture Media
• Media that cannot be autoclaved must be

sterilized through a 0.22 μm pore size
membrane filter.
• These are obtainable in various designs to
allow a wide range of volumes to be
filtered (e.g., Millipore, Gelman).
• Culture media, enzymes, hormones,
cofactors and bicarbonate buffers are
examples of non-autoclavable substances.
Conditioned Media
• Conditioning medium adds undefined components and should be eliminated if

possible after the active constituents are determined
• Hauschka & Konigsberg showed that the conditioning of culture medium that was
necessary for the growth and differentiation of myoblasts was due to collagen
released by the feeder cells.
• Using feeder layers and conditioning the medium with embryonic fibroblasts or
other cell lines remains a valuable method of culturing difficult cells
• Conditioned medium contains both substrate-modifying matrix constituents, like
collagen, fibronectin, and proteoglycans, and growth factors, such as those of the
heparin-binding group (FGF, etc.), insulin-like growth factors (IGF-I and -II),
PDGF, and several others, in addition to the intermediary metabolites previously
proposed.
Common Cell Culture Media
• Eagle’s Minimum Essential Medium (EMEM)

• Dulbecco’s Modified Eagle’s Medium (DMEM)
• RPMI-1640
• Ham’s Nutrient Mixtures
• DMEM/F12
• Iscove’s Modified Dulbecco’s Medium (IMDM)
Culture procedures and principles
Culture procedures and principles
Freeze storing of cells
• As cell culture develops within a laboratory a number of cell lines

will be developed or acquired, unless the work is exclusively with
primary cultures.
• The use of each cell line adds to its provenance, and each one
becomes a valuable resource.
• If unique, the cell line might be impossible to replace; at best

replacement would be expensive and time-consuming.
• It is, therefore, essential to protect this considerable investment by

preserving cell lines.
RATIONALE FOR FREEZING

There are many reasons, therefore, for freezing down a validated stock of cells;
these reasons can be summarized as follows:
(1) Genotypic drift due to genetic instability

(2) Senescence and the resultant extinction of the cell line
(3) Transformation of growth characteristics and acquisition of malignancy-
associated properties
(4) Phenotypic instability due to selection and dedifferentiation
(5) Contamination by microorganisms
(6) Cross-contamination by other cell lines
(7) Misidentification due to careless handling
(8) Incubator failure
(9) Saving time and materials maintaining lines not in immediate use
(10) Need for distribution to other users
PRINCIPLES OF CRYOPRESERVATION
• Theoretical Background to Cell Freezing
• Cell concentration
• Freezing Medium (glycerol, DMSO, etc.)
• Cooling Rate (1oC/min)
• Cryofreezers
• Freezer records
• Thawing stored ampoules
Transport of cultures
Cultures may be transferred from one laboratory to another as frozen ampoules or

as living cultures. In either case:
(1) Advise the recipient as to when the cells are to be shipped;
(2) Fax or E-mail instructions on the following:
a) what to do on receipt,
b) medium or serum required,
c) any special supplements, and
d) subculture regimen;
(3) Tape the data sheet for the cells and a copy of the instructions to the outside of
the package so that the recipient knows what to do before opening it.
Frozen Ampoules
• Ship frozen ampoules in solid CO2, in a thick-walled polystyrene foam container.

Transfer the ampoule from the liquid nitrogen freezer to the solid CO 2 as quickly as
possible, as it will warm up at ∼10–20◦C/min and must not rise above -50◦C.
• Place a sealable polypropylene centrifuge tube on solid CO 2 in an insulated box.

Remove the ampoule from the freezer, quickly wrap it in absorbent paper tissue (in
case of leakage), and place it in the polypropylene tube and seal with a tight cap.
• The box should be about 30 × 30 × 30 cm (12 × 12 × 12 in.) with a wall thickness

of 5 cm (2 in.) and a central space of about 20 × 20 × 20 cm (8 × 8 × 8 in.).
• You will need about 5 kg (12 lb) of solid CO2 to fill the central space. Usually, cells
will remain frozen for up to 3 days if properly packed, but if they thaw slowly their
viability will decline rapidly. The carrier must be informed when cells are shipped
in solid CO2
Living Cultures
Alternatively, cells may be shipped as a growing culture. The cells should be at the mid-
to late log phase; confluent or post-confluent cultures will exhaust the medium more
rapidly and may tend to detach in transit.
• The flask should be filled to the top with medium, taped securely around the neck
with a stretch-type waterproof adhesive tape, and sealed in a small polythene bag.
• The bagged flask is packed within a rigid container filled with absorbent packing.
This container is then sealed in a polythene bag and then in plastic foam or bubble
wrap.
• Alternatively, there is an inflatable bag that is ideal for transporting cells (Air
Packaging Technologies, Inc). The flask is sealed in an inner envelope and cushioned
by the inflatable outer jacket. Place on the container a label that says ‘‘fragile’’ and,
in large letters: DO NOT FREEZE!
Primary cell culture
• Once live cells are isolated and separated from an individual and
placed into a suitable culture environment, these cells will attach,
proliferate and grow.
• Primary cell culture is the maintenance and growth of cells dissociated

from the primary or original tissue like liver, blood or kidney.
• Cell are isolated by using enzymatic and /or mechanical methods and
cultures in specialized nutrient rich culture medium in suitable glass or
plastic containers.
• Cells grown in primary cell culture usually have the same morphology
and karyotype as the tissue they have been originated from.
• Examples: Mouse embryos, Chick embryos, Human biopsy materials

Types of Primary Culture
• Depending on the type of cells growing in culture, primary cell culture can be
of two types- Adherent and Suspension:
1. Adherent cell culture:

• In this type of culture, adherent cells are generally obtained from tissues of
immobile organs such as liver or kidney where these were immobile and are
fixed in connective tissue.
• These cells shows the requirement of attachment for growth as these cells need
solid surface for attachment and only then can grow. These cells are called as
anchorage dependent cells.
2. Suspension cell culture:

• Suspension cultures are obtained from cells of the free floating organ as blood
system since these cells are suspended in plasma for e.g. lymphocytes.
• These cell types do not require any attachment or surface for growth and are
known as anchorage independent cells or suspension cells.
Mechanical Disaggregation
• Process of disaggregation in which mechanical/physical forces

are applied for the dissociation of tissue into individual cells.
• This technique basically involves careful chopping or slicing of
tissue into pieces and collection of spill out cells.
• It can be achieved by:
• Pressing the tissue pieces through a series of sieves with a
gradual reduction in mesh size.
• Forcing the tissue fragment through a syringe and needle.
• Less expensive, quick and simple method.
• This process effects cell viability.
Enzymatic Disaggregation
• Process of disaggregation of tissue with the help of

enzymes.
• Enzymatic disaggregation is mostly used when high
recovery of cells is required from a tissue.
• Mostly used for embryonic tissues or tissues with fibrous
connective tissue and extracellular matrix.
• There are two important enzymes used in tissue
disaggregation- collagenase and trypsin.
Warm Trypsinization
• This method is widely used for disaggregation of cells.
• The chopped tissue is washed with dissection basal salt solution (DBSS),
and then transferred to a flask containing warm trypsin (37°C).
• The contents are stirred, and at an interval of every thirty minutes, the
supernatant containing the dissociated cells can be collected.
• After removal of trypsin, the cells are dispersed in a suitable medium and
preserved (by keeping the vial on ice)
Cold Trypsinization
•This technique is more appropriately referred to as trypsinization with cold pre-

exposure. The risk of damage to the cells by prolonged exposure to trypsin at 37°C
(in warm trypsinization) can be minimized in this technique.
•After chopping and washing, the tissue pieces are kept in a vial (on ice) and soaked
with cold trypsin for about 6-24 hours.
•The trypsin is removed and discarded. However, the tissue pieces contain residual
trypsin.
•These tissue pieces in a medium are incubated at 37°C for 20-30 minutes. The cells
get dispersed by repeated pipettings.
•The dissociated cells can be counted, appropriately diluted and then used.
Disaggregation by Collagenase
• Collagen is the most abundant structural protein in higher animals.

• It is mainly present in the extracellular matrix of connective tissue and muscle.
• The enzyme collagenase (usually a crude one contaminated with non-specific
proteases) can be effectively used for the disaggregation of several tissues (normal
or malignant) that may be sensitive to trypsin.
• The desired tissue suspended in basal salt solution, containing antibiotics is chopped
into pieces.
• These pieces are washed by settling, and then suspended in a complete medium
containing collagenase.
• After incubating for 1-5 days, the tissue pieces are dispersed by pipetting.
• The clusters of cells are separated by settling. The epithelial cells and fibroblastic
cells can be separated.
Subculturing of monolayer
Criteria for Subculture
• Density of Culture
• Exhaustion of Medium
• Time Since Last Subculture
• Requirements for Other

Procedures
Secondary culture
• When primary culture is passaged or sub-cultured, it becomes

secondary culture or cell line.
• Passaging or Subculture means removal of medium and
transfer of cells from previous culture vessel to another fresh
culture vessel with fresh growth medium.
• It is regularly required for the supply of fresh nutrients and
increasing space for continuously dividing cells.
• Sub-culturing of cells from one culture vessel to another
involves disassociating the adhered cells by using enzyme
trypsin and adding fresh growth media.
• It depends on the characteristics and growth rates of particular
cell types to decide the subculture or splitting ratio.
Characteristics of secondary culture
Types of secondary culture
Finite Cell Lines:

• Cell lines which grows for a limited life span and have limited number of cell
divisions (around 20 - 80 population doublings or subcultures) are known as finite
cell lines.
• The factors which manage the division of these cells in vitro are related to the type
and differentiation level of the starting cell type.
Continuous Cell Lines:

• Those cell line which get transformed or immortalized either automatically or
manually during subcultures under in vitro culture conditions changes into
continuous cell lines.
• These types of cell lines gains potential for sub culturing indefinitely, becomes
anchorage independent, lack contact inhibition, and accumulate aneuploidy or
heteroploidy.
Continuous cell lines
Cell
Culture
Secondary
Primary cell cell
culture culture/Cell
Line
Adherent Suspension Finite cell Continuous

cell culture cell culture culture cell culture
Continuous cell lines
Suspension culture
Characteristics of Cells in Culture
Anchorage Independence/dependence
• Normal cells produces cell adhesion molecules (CAM) and adhere

to the surface which promotes there cell growth and division. They
are basically anchorage dependence cell.
• Transformed cells may lack specific CAMs (e.g., L-CAM), which,

when transfected back into the cell, regenerate the normal,
noninvasive phenotype and, as such, they may be recognized as
tumor suppressor genes.
• CAM: Integrin, fibronectin, surface glycoproteins etc.
Contact inhibition
Cell- Cell Communication
Cell- Senescence
• Normal cells can divide a limited number of times; hence, cell lines derived
from normal tissue will die out after a fixed number of population
doublings.
• This is a genetically determined event involving several different genes and

is known as senescence. It is thought to be determined, in part, by the
inability of terminal sequences of the DNA in the telomeres to replicate at
each cell division.
• The result is a progressive shortening of the telomeres until, finally, the cell
is unable to divide further.
• Exceptions to this rule are germ cells, stem cells, and transformed cells,
which often express the enzyme telomerase, which is capable of replicating
the terminal sequences of DNA in the telomere and extending the life span
of the cells, infinitely in the case of germ cells and some tumor cells
Cells Culture lines
Cells Culture lines
Development of Cells Culture lines
Maintenance of Cells Culture lines
Selecting the appropriate Cells lines
Some commonly used Cells lines
Growth of Animal Cells in Culture
References
1. Freshney, R. I. (2015). Culture of animal cells: a manual of

basic technique and specialized applications. John Wiley &
Sons.
2. Butler, M. (2004). Animal cell culture and technology. Taylor
& Francis.
Thank You

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Unit Content

Characteristics of Cells in Culture: Contact inhibition, anchorage

Cell Culture Lines: Definition, development and maintenance, characteristics of

Dr. Harmanpreet Meehnian BT 505 Animal Cell Culture Technology Unit 1

• Laboratory Design and Layout

g) Servicing aseptic areas

4. Access (doorways and space)

• CONSTRUCTION AND SERVICES

LAYOUT OF ASEPTIC ROOM OR SUITE

• Six main functions need to be accommodated in the laboratory: sterile

• The preparation area should be adjacent to the washup and sterilization

LAYOUT OF ASEPTIC ROOM OR SUITE

• Sterile Handling Area

LAYOUT OF ASEPTIC ROOM OR SUITE

Quarantine and Containment

• Sterile Liquid Handling—Pipetting and Dispensing

• CCD Camera and Monitor & Dissecting Microscope

• Humid CO2 Incubator

Preparation and sterilization

• Sterilizing and Drying Ovens

• Steam Sterilizer (Autoclave)

• Hot Plate Magnetic Stirrer

• Conductivity Meter, Osmometer & Glassware Washing Machine

• Computers and Networks

• Syringes and Needles

• Paper Towels and Swabs

Attachment and Growth

• The majority of vertebrate cells cultured in vitro grow as monolayers on an artificial

• Cells that have undergone transformation frequently become anchorage independent

• Single-use sterile polystyrene flasks provide a simple, reproducible substrate for

• As manufactured, polystyrene is hydrophobic and does not provide a suitable surface

Choice Of Culture Vessel

Several factors govern the choice of culture vessel, including

(1) The cell mass required,

(2) Whether the cells grow in suspension or as a monolayer,

(3) Whether the culture should be vented to the atmosphere or sealed,

(4) The frequency of sampling,

(5) The type of analysis required, and

(6) The cost

• Cells that grow in suspension can be grown in any

• Stirrer bottles are used when agitation is required to

• These bottles are available in a wide range of sizes,

• Sometimes, cells can be inadvertently distributed nonuniformly across the growth

iii) Collagen and gelatin

iv) Matrigel (eg. Becton Dickinson, laminin, fibronectin, vitronectin, Pronectin F)

v) Extracellular matrix (Gospodarowicz et al. [1980] were able to grow endothelium on

Culture media (as a powder or as a liquid) contains:

Dr. Harmanpreet Meehnian BT 505 Animal Cell Culture Technology

• The essential amino acids are required by cultured cells.

• Other non-essential amino acids are often added as well.

• Glucose is included in most media as a source of energy.

• Amino acids, vitamin requirements have been derived

• The most commonly used proteins are:

• Trace elements are often supplemented to serum-free media to replace

• Most of the commercially available culture media include phenol red as a

• Regulating pH is critical for optimum culture conditions and is generally

• The other major significant constituent of the gas phase is oxygen.