ANIMAL

TISSUE
CULTURE
ANAND CHINTAKRINDI
COLLABORATION STUDENT
DEPT. OF VIROLOGY
HAFFKINE INSTITUTE
 WHAT IS TISSUE
CULTURE
• The term tissue culture refers to the culture of whole
organs, tissue fragments as well as dispersed cells on a
suitable nutrient medium. It can be divided into

– Organ culture - whole embryonic organs or small tissue

fragments are cultured in vitro in such a manner that they
retain their tissue architecture.

– Cell culture - obtained either by enzymatic or mechanical

dispersal of tissues into individual cells or by spontaneous
migration of cells from explants; they are maintained as
attached monolayers or as cell suspensions.
 HISTORY
• In 1880 Arnold showed that leucocyte can divide outside the
body.

• Later in 1903, Jolly studied the behaviour of animal tissue

explants immersed in serum, lymph or ascites fluid.

• In 1907 Harrison cultured frog tadpole spinal cord in a lymph

drop hanging over a cover slip into the cavity on a microscope
slide

• In 1913, Carrel developed methodology for maintaining

cultures free from contamination, especially by bacteria.

• Subsequently, suitable culture media were developed and the

techniques of cell culture were refined.
 WHAT IS NEEDED?
• Appropriate tissue (some tissues culture better than
others).

• A suitable growth medium containing energy sources and

inorganic salts to supply cell growth needs. This can be
liquid or semisolid.

• Aseptic (sterile) conditions.

• Frequent sub-culturing to ensure adequate nutrition and to

avoid the build up of waste metabolites.
 GENERAL REQUIREMENTS
OF TISSUE CULTURE LAB

• Clean and sterile.

• Preparation facilities.

• Animal house.

• Microbiology laboratory.

• Storage facilities.
 GENERAL REQUIREMENTS OF
TISSUE CULTURE LAB
• The lab should have only
one door and have a glass
window, but must not be a
transit area to other rooms.

• Windows should be large

enough to permit sufficient
amount of light.

• Sterile room should be large

enough for 2 persons to work
at the same time.

• The tissue culture lab should

have a UV light.
 BASIC INSTRUMENTS
• Bio safety cabinet
• Water bath
• CO2 Incubator
• Autoclave
• Hot air oven
• Refrigerator
• Freezers of –20°C and –70°C
• Liquid nitrogen container
• Centrifuge
• Inverted microscope
• Water purifier
• Hemocytometer count
• Deep washing sink
 MATERIALS USED FOR
TISSUE CULTURE
• Glass: used initially but now discontinued due to alternative and
suitable substrates

• Disposable Plastics – synthetic plastic materials with good

consistency and optical properties. Commonly used are
– Polystyrene,
– PVC,
– Polycarbonate
 TYPES OF CULTURE
VESSELS
• Multiwell Culture Plates
• Petri dishes
• Flasks
• Stirrer bottles

• Choice of selecting a culture vessel depends on:

– Growth of cells in culture – monolayer or suspension

– Quantity of cells required

– Purpose for which cells are grown

– Cost factor.
 Multiwell
Culture Petri dishes
Plates
Multiwell culture plates are Plastic Petri dishes with specially
available in 6-. 12-, 24-, 48- and treated surfaces to which cells can
96 well formats, and tissue culture attach are called culture dishes.
treated for cell culture of They are often coated with a thin
anchorage dependent cells and film of usually collagen or
non-treated for suspension cell fibronectin or basement
culture applications. membrane
 Flasks
• Tissue Culture Flasks are designed for superior
performance and ease of use. 

• In particular, great importance has been placed on

optimizing the culture growth properties,
accessibility to the entire growth surface, and
convenience for the researcher.

• Total access to the entire growth surface for 100%

cell retrieval

• The filtered cap has a 0.22µm hydrophobic

membrane which prevents contamination while
allowing optimal gas exchange.

• Easy-to-see white marking area and graduations

 CONTAMINATION
• Various materials
– Glassware, Pipettes

• Equipments
– Incubators, refrigerators, laminar – flow hoods

• Reagents
– Media, Solutions

• Contaminated Cell Lines

• Poor techniques
 ASEPTIC CONDITIONS
• Strict adherence to standard sterile techniques and code of
practices.

• Checking of reagents and media for sterility before use.

• Checking of cultures by eyes, microscopes every time they

are used.

• Use of media and separate bottles for each line is advised.

• Maintenance of clean and tidy conditions at work places.

• Personal hygiene of the staff is very important.

• Primary cell culture - Freshly isolated cell cultures are called
primary cultures; they are usually heterogeneous and slow growing,
but are more representative of the tissue of their origin both in cell
type and properties.

• Primary cell line - Once a primary culture is subcultured, it gives rise

to cell lines, which may either die after several subcultures (such cell
lines are known as finite cell lines) or may continue to grow
indefinitely (these are called continuous cell lines).

• Usually, normal tissues give rise to finite cell lines, while tumours
give rise to continuous cell lines. But there are several examples of
continuous cell lines, which were derived from normal tissues and are
themselves nontumorigenic, e.g., MDCK dog kidney, 3T3 fibroblasts,
etc.

• The evolution of continuous cell lines from primary cultures is

supposed to involve a mutation, which alters their properties as
compared to those of finite lines.
 TYPES OF CELL LINES
Adherent cell lines [monolayer] Non – adherent cell lines
– Cells attach to substratum of flask [suspension]
or plate. – Cell remains in suspension.
– Cells need to be detached from – No such need.
substratum before use. – Do not show such property.
– Show property of contact – E.g. Peripheral Blood mononuclear
inhibition. cells (PBMCs).
– E.g. Madin Darby canine kidney
[MDCK] cell lines, Vero cell lines.
 CULTURE MEDIA
• The nutrient media used for culture of animal cells and tissues
must be able to support their survival as well as growth, i.e.,
must provide nutritional and hormonal factors.

• The choice of medium depends mainly on the type of cells to be

cultured (normal, immortalized or transformed), and the
objective of culture (growth, survival, differentiation, production
of desired proteins).
• The various culture media developed for cell cultures may be
grouped into the following four classes:

(i) serum containing media,

(ii) serum free media,

(iii) chemically defined media, and

(iv) protein free media.

 COMPONENTS OF MEDIA
• Amino acids – serve as energy and carbon source
• Vitamins
• Salts
• Glucose
• Hormones and growth factors – added to serum free
media
• Antibiotics
• Serum
• Amino acids: glutamine and/or glutamate are added since they serve
as good energy and carbon source.

• Vitamins: vitamin A, Folic Acid, B-complex, choline, inositol.

• Salts: cations like Na+, K+, Mg2+, Ca2+, etc. and anions like Cl-, HCO3−,
SO42-, PO34-
– Ca2+ ions required for cell adhesion, signal transduction, cell
proliferation and differentiation.
– Na+, K+, Cl- ions regulate membrane potential.
– HCO3−, SO42-, PO34- involved in maintenance of intracellular
charge. Also serve as precursors for production of certain
compounds. E.g. PO34- required in ATP synthesis.

• Glucose/Glutamine: energy source, carbon for the operation of citric

acid cycle.
 EXAMPLES OF CELL CULTURE
MEDIA
• Eagle's minimal essential medium
(EMEM) is a cell culture medium by
Harry Eagle.

• A variation of this EMEM, called

Dulbecco’s modified Eagle's medium
(DMEM), (Dulbecco/Vogt modified
Eagle's minimal essential medium).

• RPMI 1640 (Roswell Park Memorial

Institute medium), for lymph cells

• GMEM (Glasgow's Minimal Essential

Medium)
 Antibiotics
• They are added to reduce contamination.

• Commonly added are gentamycin,

kanamycin, penicillin, erythromycin. Usually
Pen-Strep is added as100U/ml & 0.1mg/ml.

• Disadvantages by use of antibiotics:

– Develop antibiotic resistant cells

– Hamper proliferation by causing

antimetabolic effects.

– Possibility of hiding several infections

temporarily.

– May encourage poor aseptic conditions.

 Serum
• Natural biological fluid.

• Rich in various components to support

cell proliferation.

• Commonly used are Calf Serum, Fetal

Bovine Serum, Horse Serum and human
serum.
 Functions of Serum

• It provides the basic nutrients for cells.

• It provides several hormones, e.g., insulin, which is essential for growth of

nearly all cells in culture

• A major role of serum is to supply proteins, e.g., fibronectin, which promote

attachment of cells to the substrate.

• It provides several binding proteins, e.g., albumin, transferrin, etc., which

carry other molecules into the cell. For example, albumin carries into cells
lipids, vitamins, hormones, etc.
Disadvantages of Serum in the Culture
Medium
• Serum may contain some cytotoxic or potentially cytotoxic
constituents.

• There is a large variation in serum quality from one batch to another.

• Some growth factors may be inadequate for specific cell types and
may need supplementation.

• It interferes with downstream processing when cell cultures are used

for production of biochemicals.

• The supply of serum is always lower than its demand.

ADVANTAGES OF SERUM FREE
MEDIA
• Improved reproducibility of results from different
laboratories and over time since variation due to batch change
of serum is avoided.

• Easier downstream processing of products from cultured

cells.

• Toxic effects of serum are avoided

• There is no danger of degradation of sensitive proteins by

serum proteases.

• They permit selective culture of differentiated and producing

cell types from the heterogenous cultures.
DISADVANTAGES OF SERUM FREE
MEDIA
• Most serum free media are specific to one cell type. Therefore,
different media may be required for different cell lines.

• Reliable serum free preparations, for most of the media

formulations are not available commercially. This necessitates
time consuming task of preparing the desired formulations in the
laboratory.

• A greater control of pH, temperature, etc. is necessary as

compared to that with serum containing media.

• Growth rate and the maximum cell density attained are lower
than those with serum containing media.
 INITIATION OF CELL
CULTURES
The initiation of cell cultures involves the following condition:

1. Preparation and sterilization of the substrate (culture

vessels)
2. Preparation and sterilization of the medium
3. Isolation of explant
4. Disaggregation of the explant
5. Subculture and cloning
PREPARATION AND STERILIZATION
OF SUBSTRATE
• Plastic ware are supplied sterilized and
ready for use.

• Glassware must be carefully washed with

non-toxic detergent following,
preferably, on overnight soak.

• They should be thoroughly rinsed in tap

water and finally in deionized or distilled
water.

• Glassware are wrapped in aluminium foil

and usually sterilize in by autoclaving

• The quality of glassware washing and

sterilization should be checked
periodically by examining the glassware
with the eye after washing.
 SUBCULTURE/PASSAGING
• Subculture refers to transfer of cells from one culture vessel to
another vessel.

• It is also termed as passage.

• Passage number: the number of times a culture has been subcultured

• 2 types of subcultures:
– Monolayer subculture

– Suspension subculture
 PASSAGING OF MONOLAYER
CULTURES

• Attachment of cells to themselves and to substrate is due to calcium

ions and surface glycoproteins i.e. cell adhesion molecules.

• It is necessary to degrade the cell adhesion molecules and also remove

the ions.

• Methods of cell dissociation

– Physical by mechanical shaking and scraping.

– Enzymatic
 PASSAGING OF MONOLAYER
CULTURES
• After the monolayer is achieved in the tissue culture bottle, the
medium is discarded from the SAME SIDE of the glass bottle
containing the cells.

• Appropriate Conc. Of 0.25%Trypsin, 0.02% EDTA is used.

• The pH is most important during trypsinisation. Below pH 7.0 trypsin

is virtually inactive whereas above pH 8.0 the cells are progressively
damaged .

• After addition of Trypsin EDTA the bottle should be placed in the

incubator for 1 min after which the Trypsin EDTA is discarded from
the bottle and again placed in the incubator for 5 mins so as to allow
cell fall .
• After the cells have fallen or lost contact with the bottle appropriate
quantity of medium and serum are added.
• This is then distributed into an appropriate number of bottles and then
allowed to grow at 37 0c or cryopreserved.

Bottle For 1 min, then

containing discard the TPVG
cells and again keep
TPVG for 5 mins / until
cells start falling
 The cells will be
distributed into
appropriate number
Or cryopreserved in
of cells as per the cell liq. nitrogen for
count future use

Bottle
containing
cells
 PASSAGING OF SUSPENSION
CULTURES
• Advantages
– Propagation is faster
– Lag period shorter
– Homogeneous suspension of cells
– Treatment with trypsin not required
– Scale up convenient
– No frequent replacement of medium
– Maintenance is easy
– Bulk production of cells achieved.

• Technique
– Cells suspended in culture flask with desired medium.
– Continuous stirring with a magnetic pendulum.
– Periodical examination important to check for contamination and deterioration
of cells.
 FREEZING OF CELLS
• Trypsinise for detaching the
adhered cells in to the medium.

• Check under the microscope.

• Count the cells: counted by

using Neuber’s Counting
chamber (Haemocytometer),
Cell counting is also done by
electronic particle counter and
Coulter particle counter.
• Centrifuge at low speed i.e. at 600 rpm for 10 mins to pellet the cells.
• Freezing Mixture (medium, serum, DMSO various ratio.) is added
dropwise to the pellet and then mixed slowly.
• Note : The medium containing cells should be cool before
drop by drop addition of DMSO else DMSO is toxic at room
temp.
• Approximately 1 ml of the above mixture is aliqouted to each
cryovial.
• Freezing done by 2 methods:
• Use of Mr. Frosty
• Stepwise freezer using liquid nitrogen
 Bottle
containing
cells

Liq N2 MEM SERUM DMSO

 Use of Mr. Frosty
• Provides the critical, repeatable -1°C/minute cooling rate required for
successful cell cryopreservation and recovery.
• Requires only 100% isopropyl alcohol and mechanical freezer.
 Stepwise freezing
method
• It is done by use of liquid nitrogen
tank.

• Liquid Nitrogen is chilled,

condensed gaseous nitrogen.

• It is also colorless, odorless, non-

flammable, and non-toxic.

• Handling this material requires the

use of protective gloves and goggles.
 REVIVAL
• The vials are thawed by gradually swirling in a water bath at
37°C.

• Centrifuge at lower speed (600-1000 RPM) to eliminate the

supernatant containing DMSO.

• The pellet is resuspended in 1 ml of the growth medium and

then added to the tissue culture bottle at a concentration of
1.2x106 cells/ml.

• This is then allowed to grow at 37°C.

MAINTENANCE OF CELL LINES
• Maintenance at -196°c.
• Examination of cell morphology and change of medium are
very important.
• Change of medium important under following conditions:
• Cell concentration
• A decrease in pH
• Cell type
• Morphological changes
 BIOLOGY OF THE CELL IN TISSUE
CULTURE

Phase of attachment – lag phase

Phase of multiplication – log phase

Phase of Plateau – stationary phase

Phase of degeneration
 ADVANTAGES
• Controlled physiochemical environment (pH, temperature, oxygen, carbon
dioxide, osmotic pressure, etc.)

• Controlled and defined physiological conditions (constitution of medium,

etc.)

• Homogeneity of cell types (achieved through serial passages).

• Economical, since smaller quantities of reagents are needed than in vitro.

• It is comparatively easier to maintain in bulk.

• Results can be obtained in lesser time as compare to those from animal

models.

• No base level immunity will exist as in case of animals which may inhibit
viral replication.

• It is cost effective
 DISADVANTAGES
• Expertise is needed.

• 10 times more expensive for the same quantity of animal

tissue.

• Requirement of controlled and defined physiological

conditions
 APPLICATION OF TISSUE
CULTURE
• Isolation of virus, study viral replication and viral genetics, to
study transformation of cells by viruses.

• To analyze antiviral efficacy of the drugs.

• Studies on intracellular activity, e.g. cell cycle and

differentiation, metabolism, cell-cell interaction, e.g. cell
adhesion and motility, metabolic cooperation.

• Evaluation of environmental interactions, e.g. cytotoxicity,

mutagenesis.
• Laboratory production of medical, pharmaceutical compounds
for a wide range of applications, e.g. vaccines interferons,
hormones.

• Making monoclonal antibodies, used for diagnosis and research.

• S‘Knockout’ technology -inactivating certain genes and tracing

their effects.
 SAFETY REGULATIONS

• Strict adherence to recommendations of regulatory bodies.

• Periodical meetings and discussions of local safety committees.

• Regular monitoring of the laboratories.

• Periodical training of the personnel through seminars and workshops.

• Printing and making the standard operating procedures (SOPs)

available to all staff.

• Good record keeping.

• Limited access to the laboratory (only for the trained personnel and
selected visitors)

• Appropriate waste disposal system for biohazards, radioactive wasters,

toxins and corrosives.