Animalcellculturetechniques

Animal Cell Culture Techniques &
Its Applications
1

Introduction
•Cell culture can be defined as the process of
cultivating cells and tissues outside the body of an
organism(invitro) in an artificial environment.
•Cell culture was first successfully undertaken by
Ross Harrison in 1907.
•Roux in 1885 for the first time maintained
embryonic chick cells in a cell culture.
2

Historical events in the
development of cell culture
• 1878: Claude Bernard proposed that physiological systems
of an organism can be maintained in a living system after
the death of an organism.
• 1897: Loeb demonstrated the survival of cells isolated from
blood and connective tissue in serum and plasma.
• 1907: Harrison cultivated frog nerve cells in a lymph clot
held by the 'hanging drop' method and observed the growth
of nerve fibers in vitro for several weeks. He was
considered by some as the father of cell culture.
3

• 1916: Rous and Jones introduced proteolytic enzyme trypsin
for the subculture of adherent cells.
• 1940s: The use of the antibiotics penicillin and streptomycin in
culture medium decreased the problem of contamination in cell
culture.
• 1952: George Gey established a continuous cell line from a
human cervical carcinoma known as HeLa (Helen Lane) cells.
• Dulbecco developed plaque assay for animal viruses using
confluent(merge) monolayers of cultured cells.
• 1955: Eagle studied the nutrient requirements of selected cells
in culture and established the first widely used chemically
defined medium.
• 1961: Hayflick and Moorhead isolated human fibroblasts
and showed that they have a finite lifespan in culture. 4

Major development’s in cell culture
technology
5
• First development was the use of antibiotics which inhibits
the growth of contaminants.
• Second was the use of trypsin to remove adherent cells to
subculture further from the culture vessel.
• Third was the use of chemically defined culture medium.

Terminologies
6
• Primary Cell Culture: When cells are surgically removed from an
organism and placed into a suitable culture environment they will
attach, divide and grow.
• Cell Line: When the primary culture is subcultured and they show an
ability to continuously propagate.
• Anchorage dependency: Cells grow as monolayers adhering to the
substrate (glass/ plastic)(refers to the need for cells to be adhered
to or in contact with another layer of cells.)
• Passaging/ subculturing: The process of splitting the cells.
grow upto infinite
• Finite cells: When the cells has finite life span.
• Continuous cell lines: When the cells can
lifespan.

Equipping Tissue Culture Laboratory
• Laminar cabinet - Vertical LAF are
preferable. They are fitted with HEPA
filters and UV.
• Incubation facilities - Temperature of
37°C.
7

• Refrigerators- Liquid media kept at 4°C, enzymes
(e.g. trypsin) & media components (e.g.
glutamine & serum) at -20°C.
• Microscope- An inverted microscope with 10x to
100x magnification(light sources and condenser
on the top)
• Tissue culture ware- Culture plastic ware treated
by polystyrene (thermocol)
8

Tissue Culture Media
• Cells have complex nutritional requirements that must be met to
permit their propagation in vitro.
• Previously, scientists employed chick embryo extract, plasma,
sera, lymph etc.,
• However, they varied in their growth promoting characteristics
and thus hampering the reproducibility of the experiments.
• Today, a number of chemically-defined formulations have been
developed that support the growth of a variety of established
cell lines
11

• Eagle’s basal media
• Eagles’s Minimum Essential Media (MEM)
• Dulbecco’s Modified Essential Media (DMEM)
• Iscove’s Modified Dulbecco’s Medium (IMDM)
• Roosevelt Park Memorial Institute (RPMI 1640)
• HAM’s F12
• The various nutrients required are:
• glucose,
• fats and fatty acids,
• lipids, phospholipids and sulpholipids(lipid containing sulphur)
• ATPand amino acids
• Vitamins
• Minerals
• Serum:
Serum can provide various growth factors, hormones, cell
adhesion factor and other factors needed by the most
mammalian cells for their long term growth and metabolism.
13

Properties and Special Requirements of
Media
• pH: Optimum pH between 7.2 to 7.4 is generally needed for
mammalian cells. Phenol red is used as an internal indicator.
(pH indicator)
S.
f
pH Colour of the medium
7.8 Pink / purple
7.6 Pink / purple
7.4 Red
7.0 Orange
5.5 Yellow
< 6.5 Lemon Yellow
• CO2 and Bicarbonate: NaHCO3 and 20 mM HEPE
(maintaining physiological Ph).
14

• Oxygen
• Cells depend upon glycolysis for the supply of O2
• Selenium controls O2 diffusion(Selenium helps your body make special
proteins, called antioxidant enzymes)
• Glutathione acts as free radical scavenger(cleaning)
• Temperature
• The optimum temperature of mammal is 37oC.
• Change of ± 5°C is acceptable.
• Humidity
• For cell growth 100% humidity is essential to reduce evaporation of the
media.
• Antibiotics
• penicillin (100 U/ml) for bacteria,
• streptomycin (100 mg/ml) for bacteria,
• or gentamycin (50mg/ ml) for bacteria,
• and nystatin (50mg/ml) for fungi and yeast 14

15
Serum Free Media
• welldefined and controlled culture media that has been process
ed in some form (e.g.,filtered to remove inhibitors and/or growth
factors).
•Serum-free media are not necessarily protein-
free or chemically defined; most are optimised for the growth
requirements of cell-specific applications.
•Serum-free media do not contain or require the addition of
serum

Types of tissue culture
• CELLCULTURE
• Tissue from an explant is dispersed,
mostly enzymatically, into a cell
suspension which may then be
cultured as a monolayer or
suspension culture
Advantages
cell line over
physical
than in
• Development of a
several generations
• Scale-up is possible
• Absolute control of
environment
• Homogeneity of sample
• Less compound needed
animal models
Disadvantages
• Cells may lose some differentiated
characteristics.
• Hard to maintain
• Only grow small amount of tissue at
high cost
• Dedifferentiation
• EXPLANT CULTURE
• Is the growth
of tissues or cells separate from the
organism.
• This is typically facilitated via use of
a liquid, semi-solid, or solid growth
medium, such as broth or agar.
Advantages
• Some normal functions may be
maintained.
• Better than organ culture forscale-up
but not ideal.
Disadvantages
• Original organization of tissue islost.
ORGAN CULTURE
The entire embryos or organs are
excised from the body and culture
16
• Instability, aneuploidy
Advantages
Normal physiological functions
are maintained.
Cells remain fully differentiated.
Disadvantages
Scale-up is not recommended.
Growth is slow.
Fresh explantation is required for
every experiment.

Suspension culture- the culture of tissue and
cells cultured in liquid medium, producing a
suspension of single cells and cell clump.
In cell culture a monolayer refers to a layer of
cells in which no cell is growing on top of
another, but all are growing side by side and
often touching each other on the same growth
surface.

Primary Cultures
20
• When cells are surgically removed from an organism and
placed into a suitable culture environment they will attach,
divide and grow.
• Most of the primary culture cells have a finite lifespan of 50-
60 divisions invitro.
• Primary cells are considered by many researchers to be more
physiologically similar to in vivo cells
• Due to their limited lifespan, one cannot do long-term
experiments with these cells

Cell lines
 A cell culture developed from a single cell and therefore
consisting of cells with a uniform genetic make-up.
 Cell culture and cell lines have assumed an important
role in studying physiological, pathophysiological and
the differentiation processes of specific cells. It allows
the examination of stepwise alterations in the structure,
biology, and genetic makeup of the cell under controlled
environments.
 This is especially valuable for complex tissues, such as
the pancreas, which is composed of various cell types,
where in vivo examination of individual cells is difficult,
if not impossible.

Tissue explants are excised using sharp scalpel.
Mechanical disruption by pestle and mortar. Then filtered using a 0.22µ Filter fitted
to a syringe.
Enzymatic digestion by Trysin or collagenase
Cells are counted on a Haemocytometer. 1-2 × 10^5 cells / mL is seeded in to the
media.
5mL of cells is suspended into 25cm^2 flask.
The flasks are incubated in a CO2 incubator.
The flasks are observed daily for their normal growth characteristics.
Media is changed every 2-3 days until the cells attain 80% confluent. 20

Isolation of cell lines for in vitro culture
Resected Tissue
Cell or tissue culture in vitro
Primary culture
Sub-culture
Secondary culture
Sub-culture
Cell Line
Immortalization
Successive sub-cultureSingle cell isolation
Clonal cell line Senescence
Immortalised cell line
Loss of control
of cell growth
Transformed cell line

Isolation of the tissue
When work with human or animal tissues, medical
ethical rules should be observed
Embryonic or fetal animal tissues more than half term
also needs guidelines
Work with human tissue should be carried out at
Containment Level 2 in a Class II biological safety
cabinet
Sterilization of the site
70% alcohol
Remove the tissue aseptically
Transfer to lab in dissection BSS(borate buffered saline-
pH maintain) or collection medium

Isolation of mouse
embryo
Convenient source of cells for undifferentiated mesenchymal (multipotent) cell
cultures
Mouse embryo fibroblasts are used as feeder layers(a popl of connective tissue)
Full term- 19-21 days
Optimal age for preparing culture 13 days
Since isolation of above half term embryo needs license, 9-10 day embryo is
preferable
Kill the mouse by cervical dislocation

Swabbing with alcohol
Tearing the skin to expose the abdominal wall

Cut longitudinally along median line of exposed abdomen with
sterile scissors

Revealing the uterus
Dissecting the uterus and transferring to
DBSS(dissection balanced salt solution)

Dissection of embryos
from the uterus by
tearing the uterus
Take the intact uteri to
tissue culture lab and
transfer to fresh petri dish

Free the embryos
from the membrane
and placenta, cut the
head if necessary

Chopping the embryos
Primary
explants
Disaggregation and
suspension culture
enzymatic
mechanical

Primary explants
Primary culture refers to the stage of the culture after the cells are
isolated from the tissue and proliferated under the appropriate conditions
until they occupy all of the available substrate (i.e., reach confluence).
A fragment of tissue transplanted from its original site and maintained in
artificial medium
Original method developed by Harrison, Carrel and others
A fragment of tissue was embedded in blood plasma
Mixed with heterologous serum and embryo extract
Placed on a coverslip that was inverted over a concavity slide.
The clotted plasma held the tissue in place
The explant could be examined with a conventional microscope

Cells migrate out from fragments of
tissue that is adhered to a suitable
substrate
The tissue is chopped finely and
rinsed
Pieces are seeded onto culture flask
surfaces
Small volume of medium is added
with high concentration of serum
After the tissue adheres, increase the
volume of medium
Primary explants

 Suitable for small amount of
tissue
 Risk of losing cells during
mechanical or enzymatic
disaggregation
 Disadvantages
 Poor adhesiveness of some
tissue
 Only cells capable of
migration outgrow
Primary explants
 To promote attachment
Place a glass coverslip on top of
explant
 Coating the dish with
polylysine, fibronectin or other
ECM proteins
 Purified fibrinogen or thrombin
 Small amount of medium with
high concentration of serum.
 Increase the number of
positively charged sites
 fibronectin - cell adhesion
molecule

Disaggregation using enzymes
Trypsin, collagenase, dispase, hyaluronidase
Alone or in combinations (collagenase with dispase, dispase with
trypsin)
In combination with EDTA (chelating agent for Ca2+)(stable-water soluble
complex)
Crude preparations or purified enzyme preparation
Latter(end) generally less toxic and more specific
Dispase is a protease which cleaves fibronectin, collagen IV, and
to a lesser extent collagen I.
PRONASE- Mix of protease from ECF- denature proteins

NOTE
The five most common types of collagen are:
Type I: skin, organs, bone (main component of
the organic part of bone)
Type II: cartilage (main collagenous component
of cartilage)
Type III: reticulate (main component of reticular
fibers
Type IV: forms basal lamina, the epithelium-
secreted layer of the basement membrane.
Type V: cell surfaces, hair and placenta

Some of the cell-cell adhesion
molecules are calcium
dependant. Also integrins which
bind to ECM components have
calcium ion binding domains. So
all these interactions affected by
Ca depletion.
Proteoglycans need
hyaluronidase(degradation of
hyaluronic acid (HA)) and
glycoproteins are protease
sensitive
Enzymes varies in effect
Trypsin and pronase gives most complete
disaggregation but may damage the cells
Collagenase and dispase give incomplete
disaggregation but are less harmful
Non mammalian enzymes
Trypzean, recombinant maize derived trypsin
TrypLE, recombinant and microbial in origin.
Disaggregation of tissue yields higher number of
cells that are more representative of the whole
tissue in a shorter time
Embryonic tissue disperses more readily and gives
more proliferating cells
DISPASE- CLEAVES FIBRONECTIN, COLLAGEN IV
AND COLLAGEN I

 Enzymatic disaggregation is mostly used when high recovery of
cells is required from a tissue. Disaggregation of embryonic
tissues is more efficient with higher yield of cells by use of
enzymes.
 This is due to the presence of less fibrous connective tissue and
extracellular matrix. Enzymatic disaggregation can be carried
out by using trypsin, collagenase or some other enzymes.

Trypsin
The term trypsinization is commonly used for disaggregation of tissues
by the enzyme, trypsin.
Many workers prefer to use crude trypsin rather than pure trypsin for
the following reasons:
i. The crude trypsin is more effective due to the presence of other
proteases
ii. Cells can tolerate crude trypsin better.
iii. The residual activity of crude trypsin can be easily neutralized by the
serum of the culture media (when serum-free media are used, a trypsin
inhibitor can be used for neutralization).
Disaggregation of cells can also be carried out by using pure trypsin
which is less toxic and more specific in its action. The desired tissue is
chopped to 2-3 mm pieces and then subjected to disaggregation by
trypsin. There are two techniques of trypsinization-warm trypsinization
and cold trypsinization

Warm Trypsin
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).
• The process of addition of fresh trypsin (to the tissue pieces),
incubation and collection of dissociated cells (at 30 minutes
intervals) is carried out for about 4 hours. The disaggregated cells
are pooled, counted, appropriately diluted and then incubated.

Trypsinization with cold pre exposure
Particularly suitable for small amounts of tissue
Cold trypsin method gives a higher yield of viable cells
Improved survival after 24 h
Preserves more different cell types than warm method
No stirring or centrifugation needed
Incubation at 4oC can be left overnight unattended

Treatment with 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.
 Highly purified grades of collagenase have been tried, but they
are less effective when compared to crude collagenase.

 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.
 Collagenase disaggregation has been successfully used for human
brain, lung and several other epithelial tissues, besides various
human tumors, and other animal tissues. Addition of another
enzyme hyaluronidase (acts on carbohydrate residues on cell
surfaces) promotes disaggregation.
 Collagenase in combination with hyaluronidase is found to be
very effective for dissociating rat or rabbit liver. This can be done
by per-fusing the whole organ in situ. Some workers use
collagenase in conjunction with trypsin, a formulation developed
in chick serum, for disaggregation of certain tissues.

Mechanical
disaggregation
To avoid the risk of proteolytic damage
Methods
The cells that spill out when the tissue is carefully sliced or
scraped can be collected
The dissected tissue can be pressed through a series of
sieves(strain solid from liquid) for which the mesh is gradually
reduced in size
The tissue fragments can be forced through a syringe (with or
without a wide-gauge needle)
Pipette the tissue repeatedly
Due to a risk of proteolytic damage to cells during enzymatic
digestion

Gives a cell suspension more quickly than does
enzymatic digestion
Scraping (left over reprocessing) and sieving are the
gentlest mechanical methods
Mostly suitable only for soft tissues, such as spleen,
embryonic liver, embryonic and adult brain, and some
human and animal soft tumors
The viability of the resulting suspension is lower
Suitable when the availability of tissue is not a limitation
and the efficiency of the yield is unimportant

Separation of viable and non viable
cells
Adherent primary culture
nonviable cells are removed at the first change of medium
Primary cultures maintained in suspension
Nonviable cells are gradually diluted out when cell
proliferation starts
Centrifuge the cells on a mixture of Ficoll(hydrophilic polsac
dissolved in aq sol) and sodium metrizoate(radiopaque occurs
in Na salt )
viable cells collect at the interface between the medium
and the Ficoll/ metrizoate the dead cells form a pellet at
the bottom of the tube.

Cell Suspension Culture
Suspension culture is a type of culture in which single cells or small aggregates of
cells multiply while suspended in agitated liquid medium. It is also referred to as cell
culture or cell suspension culture.
Suspension cells do not attach to the surface of the culture vessels. These cells are
also called anchorage independent or non-adherent cells which can be grown
floating in the culture medium.
Hematopoietic stem cells (derived from blood, spleen and bone marrow) and tumor
cells can be grown in suspension. These cells grow much faster which do not require
the frequent replacement of the medium and can be easily maintained. These are of
homogeneous types and enzyme treatment is not required for the dissociation of
cells; similarly these cultures have short lag period.

Continuous cell lines
• Most cell lines grow for a limited number of generations after which
they ceases.
• Cell lines which either occur spontaneously or induced virally or
chemically gets transformed into Continuous cell lines.
• Characteristics of continuous cell lines
-smaller, more rounded, less adherent with a higher nucleus
/cytoplasm ratio
-Fast growth and have aneuploid chromosome number
-reduced serum and anchorage dependence and grow more in
suspension conditions
-ability to grow upto higher cell density
-different in phenotypes from donar tissue
-stop expressing tissue specific markers
- Loss of Contact inhibition
22

Common cell lines
Human cell lines
• MCF-7
• HL60
• HEK-293
• HeLa
breast canc
Leukemia
Human em
Henrietta lac
ryonic kidney
ks
liPrimate cell
• Vero
nes
African green Monkey kidney ephelial cells
• Cos-7 African green monkey kidney cells
• And others such as CHO from hamster, sf9 & sf21 from insect
cells
Hela-Epithelial
53
MCF-7 breast
MRC5-FibroblastHT1080- kidney 3LL - lungs

er BAE1-Endothelial
b
ac
SHSY5Y-Neuronal
m it

Subculturing
Incubate the cells.
Remove spent media from the culture vessel.
Add the pre-warmed dissociation reagent such as trypsin. Gently rock the
container to get complete coverage of the cell layer.
Incubate the culture vessel at room temperature for approximately 2
minutes.
Add equivalent of 2 volumes of pre-warmed complete growth
medium. Disperse the medium by pipetting over the cell layer surface
several times.
Then split the cells into 2 or 3 flasks containing complete media.
55

Cryopreservation
Next day transfer the cryovials to Liquidnitrogen.
Remove the growth medium, wash the cells by PBS and remove the PBS by
aspiration.
Dissociate the cells by trypsin
Dilute the cells with growthmedium.
Centrifuge at 200g for 5 min at RTand remove the growth medium byaspiration
Resuspendthe cells in 1-2ml of freezing medium containingDMSO(dimethylsulfoxide).
Transfer the cells to cryovials, incubate the cryovials at -80°Covernight
25

Detection of contaminants
• In general indicators of contamination are turbid culture media,
change in growth rates, abnormally high pH, poor attachment, multi-
nucleated cells, graining cellular appearance, vacuolization,
inclusion bodies and cell lysis
• Yeast, bacteria & fungi usually shows visible effect on the culture
(changes in medium turbidity or pH)
• Mycoplasma(lack cell wall)detected by direct DNA staining with
intercalating fluorescent substances e.g. Hoechst 33258
• Mycoplasma also detected by enzyme immunoassay by specific
antisera or monoclonal abs or by PCR amplification of
mycoplasmal RNA
• The best and the oldest way to eliminate contamination is to discard
the infected cell lines directly
26

Invitro Transformation of Cells
• Transformed, Infinite or Established Cells
• Changed from normal cells to cells with many of the properties of cancer
cells
• Some of these cell lines have actually been derived from tumors or are
transformed spontaneously in culture by mutations
• Chemical or gamma ray treated cells can become infinite with loss of growth
factors
• Viral infection with SV40 T antigen can insert oncogenes and lead togene
alteration
• No matter how transformation occurred, the result is a cell with altered
functional, morphological, and growth characteristics

Advantages of Tissue Culture
59
Control of the environment.
Characterization and Homogenity of sample.
Economy, Scale and Mechanization.
Invitro modelling of Invivo Conditions.

Limitations
60
• To grow cells outside their normal environment, three major controls are
involved.
• Observing strict asepsis
• Providing the right kind of physic-chemical environment
• Nutrients in its simplest absorbable form
• Culturing technique needs a great deal of expertise.
• Tissue samples consists of a mixture of heterogenous cell populations
• Continuously growing cells often show genetic instability.
• Differences in the behavior or cells in cultured and in its natural form.
• Should include proper balance of the hormones.

Applications of Cell Culture
61
• Excellent model systems for studying:
 The normal physiology, cell biology and biochemistry of cells
 The effects of drugs, radiation and toxic compounds on the cells
 Study mutagenesis and carcinogenesis
• Used for gene transfer studies.
• Large scale manufacturing of biological compounds
• (vaccines, insulin, interferon, other therapeutic protein)

Products
Monoclonal
Antibodies
(Mab’s)
Immuno-
biological
Regulators
Virus
vaccines
Hormones
Produced by hybridoma cell
Usedfor diagnostic assaysystems
(determine drugs, toxins & vitamin);
therapeutic purposes & biological
separations – chromatographic
separations to purify protein
molecules
Interferon – anticancer
glycoprotein (secreted
animal cell or recombinant
bacteria)
Lymphokines
Interleukines (anticancer
agent)
Prophylactics(prevent disease)
Virus is collected, inactivated
and used asvaccine
Aweakened form will induce a
protective response but no
disease
Largemolecules: 50-200 amino
acids
Produce by hormone-
synthesizing organ
May also produce by chemical
synthesis
Example: Erythropoietin(s-
kidney)

Products
Enzymes
InsecticidesWhole
cellsand
tissue
culture
Urokinase, rennin, asparaginase,
collaginase, pepsin, trypsin, etc..
Production of someinsect
viruses that are highly
specific and safeto
envirionmentArtificial organs and semi
synthetic bone anddental
structure

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