Introduction Terminologies Types of tissue culture Applications Culturing Sub-culturing Cryopreservation Detection of contaminants In vitro transformation of cells Cell viability Rules for working in the Lab Advantages Limitations

1. BASIC TECHNIQUES OF
MAMMALIAN CELL CULTURE
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By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

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CONTENTS
Introduction
Terminologies
Types of tissue culture
Applications
-Culturing
- Sub-culturing
-Cryopreservation
- Detection of contaminants
- In vitro transformation of cells
- Cell viability
Rules for working in the Lab
Advantages
Limitations

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INTRODUCTION
Cell culture can be defined as the process of cultivating cells and tissues outside
the body of an organism(in vitro) in an artificial environment, which stimulates
the in vivo conditions such as temperature, nutrition and protection from
microorganisms.
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.

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TERMINOLOGIES
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 primary culture is sub-cultured and they show an ability to
continually propagate.
Anchorage Dependency : cells grow an monolayers adhreing to the
substrate(glass/plastic).
Passaging /Sub-culturing : the process of splitting the cells.
Finite Cells : when the cells has finite life span.
Continues Cell Line : when the cell grow upto infinite life span.

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TYPES OF TISSUE CULTURE
CULTURE
MEANING
ADVANTAGES
DISADVANTAGES
cell tissue organ
Tissue from an explant is
dispersed, mostly
enzymatically, into cell
suspension which may
then be cultured as a
monolayer or suspension
culture.
•Scale-up is possible
•Control of physical
condition
•Homogeneity of sample
•Cells may lose
differentiated
characteristics
•instability
Growth of tissue separate
from the organism.
Facilitated using liquid or
semi-solid growth medium
such as broth or agar.
•Some normal functions may
be maintained.
•Better than organ culture
for scale-up but not ideal.
•Original organization of
tissue is lost.
Entire embryo or organs are
excised from body and
cultured.
•Normal physiological
functions are maintained.
•Cells remain fully
differentiated.
•Scale-up is not
recommended.
•Growth is slow.

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APPLICATIONS
Excellent model system for studying
•The normal phenotype, 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
Virology, Genetic Engineering, Gene therapy
Large scale manufacturing of biological compounds
like vaccines, insulin, interferon, other therapeutic protein.

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Culturing :-
•Cells when surgically or enzymatically removed from an organism and placed
in suitable culture environment will attach and grow are called as primary
culture
•Primary cells have a finite life span
•Sub culturing of primary cells leads to the generation of cell lines
•Cell lines have limited life span, they passage several times before they
become senescent
Cells are cultured as anchorage dependent or independent
Cell lines derived from normal tissues are considered as anchorage-dependent
grows only on a suitable substrate e.g. tissue cells
Suspension cells are anchorage-independent e.g. blood cells
Transformed cell lines either grows as monolayer or as suspension

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Culturing :-

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Continues cell lines
Cell lines which either occur spontaneously or induced virally or chemically
transformed into Continuous cell lines
Characteristics of continuous cell lines
-smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio
-Fast growth and
-reduced serum and anchorage dependence and grow more in suspension
conditions
-ability to grow up to higher cell density
-different in phenotypes from donor tissue
-stop expressing tissue specific genes

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Types of cells
On the basis of morphology (shape & appearance) or on their functional
characteristics. They are divided into three.
•Epithelial like-attached to a substrate and appears flattened and polygonal in
shape
•Lymphoblast like- cells do not attach remain in suspension with a spherical
shape
•Fibroblast like- cells attached to an substrate appears elongated and bipolar

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Sub-Culturing :-
Once the available substrate surface is covered by cells (a confluent culture)
growth slows & ceases.
Cells to be kept in healthy & in growing state have to be sub-cultured or
passaged
It’s the passage of cells when they reach to 80-90% confluency in
flask/dishes/plates
Enzyme such as trypsin, dipase, collagenase in combination with EDTA breaks
the cellular glue that attached the cells to the surface

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Cryopreservation :-
Storage at freeze condition.

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Detection of contaminants :-

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Invitro transformation of cells :-

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Cell Viability :-
Cell viability is determined by staining the cells with trypan blue
As trypan blue dye is permeable to non-viable cells or death cells whereas it is
impermeable to this dye
Stain the cells with trypan dye and load to haemocytometer and calculate % of
viable cells
- % of viable cells= Nu. of unstained cells x 100
total nu. of cells

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Rules for working in the Lab
• If working on the bench use a Bunsen flame to heat the air surrounding the
Bunsen
• Swab all bottle tops & necks with 70% ethanol
• Flame all bottle necks & pipette by passing very quickly through the hottest
part of the flame
• Avoiding placing caps & pipettes down on the bench; practice holding bottle
tops with the little finger
• Work either left to right or vice versa, so that all material goes to one side, once
finished
• Clean up spills immediately & always leave the work place neat & tidy

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ADVANTAGES
• control of the environment
• characterization and homogeneity of the sample
•Economy, scale and mechanization
• Invitro modeling of Invivio conditions

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LIMITATIONS
• To grow cells outside their normal environment, three
major controls are involved :
- keen observation
- providing right kind of physic-chemical environment
- nutrients in its simples absorbable form
• culturing technique needs a great deal of expertise.
• tissue sample consists a mixture of heterogeneous cell
population
• continually growing cells often show genetic instability
• differences in behavior of cells cultured or in its natural
form
• should include proper balance of hormones

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CONCLUSION
Although animal tissue culture is benefiting mankind but there are many
ethical reasons as well as requirement of expertise in the field and a well
established lab is required, it becomes difficult for practical
implementation of it with ease.