This document discusses animal cell culture. It defines tissue culture as the in vitro culture of cells, tissues or organs. There are three main types of tissue culture: organ culture, tissue culture, and cell culture. Cell culture involves dispersing tissue into a single cell suspension which can then be cultured as a monolayer or in suspension. Cell culture has advantages for research, commercial production, and reducing animal use. Key factors that affect cell culture include using appropriate cell types, providing a suitable environment with optimal solid, liquid and gaseous phases, temperature, and maintaining aseptic techniques.

1. Animal Cell Culture
Submitted by :-
Mr. Sunil Saini
M.Pharm 1st
sem.
Supervised by :-
Dr. Anil Pareek
Assistant Professor,
Pharmaceutics.

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What is tissue culture?
• In vitro culture (maintain and/or proliferate)
of cells, tissues or organs
• Types of tissue culture
• Organ culture
• Tissue culture
• Cell culture

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Organ culture
• The entire embryos or organs are excised from the
body and culture
• 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.

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Tissue Culture
• Fragments of excised tissue are grown in culture
media
• Advantages
• Some normal functions may be maintained.
• Better than organ culture for scale-up but not ideal.
• Disadvantages
• Original organization of tissue is lost.

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Cell Culture
• Tissue from an explant is dispersed, mostly
enzymatically, into a cell suspension which may
then be cultured as a monolayer or suspension
culture.
• Advantages
• Development of a cell line over several generations
• Scale-up is possible
• Disadvantages
• Cells may lose some differentiated characteristics.

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Why do we need Cell culture?
• Research
• To overcome problems in studying cellular behavior
such as:
• confounding effects of the surrounding tissues
• variations that might arise in animals under experimental stress
• Reduce animal use
• Commercial or large-scale production
• Production of cell material: vaccine, antibody, hormone

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Advantages of Cell culture
• Advantages:
• Absolute control of physical environment
• Homogeneity of sample
• Less compound needed than in animal models
• Disadvantages:
• Hard to maintain
• Only grow small amount of tissue at high cost
• Dedifferentiation
• Instability, aneuploidy

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Types of Cell culture
1. Primary Cultures
• Derived directly from excised tissue and cultured
either as
• Outgrowth of excised tissue in culture
• Dissociation into single cells (by enzymatic digestion or
mechanical dispersion)
• Advantages:
• usually retain many of the differentiated characteristics of the
cell in vivo
• Disadvantages:
• initially heterogeneous but later become dominated by
fibroblasts.
• the preparation of primary cultures is labor intensive
• can be maintained in vitro only for a limited period of time.

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Types of Cell culture
2. Continuous Cultures
• derived from subculture (or passage, or transfer) of
primary culture
• Subculture = the process of dispersion and re-culture the cells
after they have increased to occupy all of the available
substrate in the culture
• usually comprised of a single cell type
• can be serially propagated in culture for several
passages
• There are two types of continuous cultures
• Cell lines
• Continuous cell lines

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Types of continuous culture
1) Cell lines
• finite life, senesce after approximately thirty
cycles of division
• usually diploid and maintain some degree of
differentiation.
• it is essential to establish a system of Master and
Working banks in order to maintain such lines for
long periods

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Types of continuous culture
2) Continuous cell lines
• can be propagated indefinitely
• generally have this ability because they have been
transformed
• tumor cells.
• viral oncogenes
• chemical treatments.
• the disadvantage of having retained very little of
the original in vivo characteristics

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Initiation of culture
Tissue
Primary cell culture
Cell line Continuous cell line
dispersion
Subculture
Finite numbers Indefinite numbers
Stored Stored

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Cell Culture Morphology
• Morphologically cell cultures take one of two forms:
• growing in suspension (as single cells or small free-floating clumps)
• cell lines derived from blood (leukaemia, lymphoma)
• growing as a monolayer that is attached to the tissue culture flask.
• cells derived from solid tissue (lungs, kidney), endothelial, epithelial, neuronal,
fibroblasts
Hela-Epithelial
MRC5-Fibroblast SHSY5Y-Neuronal
BAE1-Endothelial

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Special types of Cell culture
Cells in the culture can be grown to adopt
in vivo characteristic
• Histotypic culture
• Single cell lineage
• Organotypic culture
• Multiple cell lineages

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Biology of Culture cells
• Cell growth and differentiation in the culture
depends on:
• The nature of cells
• The culture environment
• the nature of the substrate on which cell grow
• the physicochemical and physiological constitution of
culture medium
• the constitution of gas phase
• the incubation temperature
• the cell-cell and cell-matrix interaction

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Cell cycle
M
Mitosis
G1
Gap1
G2
Gap2
G0
S
Synthesis
G1 check point
• cell big
• environment suitable
G2 check point
• DNA replicated
• cell big
• environment suitable
Metaphase check point
• chromosome align on spindle

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Cell cycle
• Interphase:
• generally lasts at least 12 to 24 hours in mammalian tissue
• the cell is constantly synthesizing RNA, producing protein and
growing in size
• Gap 0 (G0): cell will leave the cycle and quit dividing temporary or more
permanent
• Gap 1 (G1): Cells increase in size, RNA and protein synthesis, there is a
G1 Checkpoint
• S Phase: The DNA replication occurs
• Gap 2 (G2): The cell will continue to grow and produce new proteins.
There is a G2 Checkpoint
• Mitosis or M Phase:
• Cell growth and protein production stop
• the cell cycle divides into two similar daughter cell
• Mitosis last perhaps only one to two hours
• there is a Checkpoint in the middle of mitosis (Metaphase
Checkpoint) that ensures the cell is ready to complete cell division.

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Factors affecting cell proliferation
• Promotion of cell proliferation
• low cell density (leaves the cell with free
edge)
• signals from environment: Growth factors
• Inhibition of cell proliferation
• Density limitation: high cell density
• Contact inhibition: cell contact

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Factors affecting cell diferentiation
• Cell differentiation is important for normal cell
functions
• Factors promoting cell differentiations
• high cell density
• cell-cell and cell-matrix interaction
• inducers: hydrocortisone, retinoid, matrix

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Factors affecting cell adhesion
• Cell adhesion is important for cell
proliferation and differentiation (signaling
through cytoskeleton)
• Cell adhesion molecule
• Cell-cell interaction: CAMs, cadherins
• Cell-matrix interaction: integrin,
transmembrame proteoglycan
• Tight junctional complex in epithelial cells
for cell-cell interaction

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Factors affecting cell adhesion
• Enzymatic disaggregation digests the adhesion
molecule and extracellular matrix
• Most cells from solid tissues grow as adherent
monolayer
• Matrix-coated surface promotes cell
proliferation and differentiation

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Factors affect cell culture success
• Appropriate cells
• Suitable environment
• Solid phase
• substrate or phase on which the cell grow eg. glass, plastic,
collagen, agar
• Liquid phase
• physicochemical and physiological constitution of the medium
• Gaseous phase
• Temperature
• Aseptic environment

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Solid phase
• Anchorage dependent cells require a
nontoxic, biologically inert to attach and
allow movement for growth
• The most convenient vessels are
polystyrene plastic
• other growth surface such as glass, filter
wells
• The surface can be treated by
• coated with matrix substrate eg. Collagen,
poly-l-lysine, matrigel
• Feeder layers: monolayer of supporting
cells, perhaps promote cell growth and
differentiation by cell contact and
substance secreted

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Liquid phase
• Components of culture media
• Inorganic Salts
• retain the osmotic balance of the cells
• regulate membrane potential by provision of
sodium, potassium and calcium ions.
• are required in the cell matrix for cell attachment
and as enzyme cofactors.
• Carbohydrates
• Most media contain 4-20 mM glucose
• main source of energy from glycolysis

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Liquid phase
• Proteins and Peptides
• are used to replace those normally present in serum
eg. transferrin, fibronectin
• Amino acids
• important for cell proliferation and differentiation
• glutamine can enter Kreb’s cycle
• Fatty Acids and Lipids
• important in serum free media e.g. cholesterol and
steroids essential for specialized cells.

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Liquid phase
• Vitamins
• vitamins B are necessary for cell growth and
proliferation
• precursors for numerous co-factors
• The vitamins commonly used in media include
thiamine, riboflavin and biotin
• Trace Elements
• zinc, copper, selenium and tricarboxylic acid
intermediates.
• Selenium is a detoxifier and helps remove oxygen
free radicals.

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Liquid phase
• Buffering Systems
• most cells need optimal pH conditions in the range 7.2
- 7.4
• close control of pH is essential for optimum culture
conditions
• bicarbonate/CO2 buffering systems
• Chemical buffering: HEPES
• Most commercial culture media include phenol red as a
pH indicator
• yellow (acid) or purple (alkali)
• Osmolarity
• similar to plasma osmolarity 290 mOsm

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Liquid phase
• Serum
• Undefined factors: complex mix of albumins,
growth factors and growth inhibitors
• increase the buffering capacity of cultures
• able to bind and neutralize toxins
• can be important for slow growing cells or where
the seeding density is low
• Subject to batch to batch variation
• Heat inactivation of serum (incubation at 56ºC for
30 minutes) can help to reduce the risk of
contamination

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Gaseous phase
• Carbondioxide
• important for buffering system
• 5-10% CO2
• Endogenous production: pyruvate
• Oxygen
• most cells in culture require low oxygen tension
• anaerobic glycolysis
• high oxygen can produce toxic free radical
H3C C C O−
O O
C S
O
H3C CoA
HSCoA
NAD+
NADH
+ CO2
Pyruvate Dehydrogenase
pyruvate acetyl-CoA

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Temperature
• The optimum temperature depends on
• the body temperature of animals from which the
cells were obtained
• anatomical variation of temperature (skin
temperature may be lower than the rest of the
body)

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Aseptic techniques
• Microorganism remains a major problem in cell culture
• prevention of contamination
• Antibiotics
• improvement of laboratory condition
• Aseptic techniques
• Clean and tidy work surface
• Personal hygiene
• hand washing
• caps, gowns, face mask
• Reagents and media
• Culture vessels

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Cryopreservation of Cell Lines
• The aim of cryopreservation is to enable stocks of
cells to be stored to prevent the need to have all cell
lines in culture at all times
• Reduced risk of microbial contamination
• Reduced risk of cross contamination with other cell
lines
• Reduced risk of genetic drift and morphological
changes
• Work conducted using cells at a consistent passage
number
• Reduced costs (consumables and staff time)

34. Cryopreservation of Cell Lines
Method Advantages Disadvantages
Electric (-135ºC) Freezer • Ease of maintenance
• Steady temperature
• Low running costs
• Requires liquid nitrogen
back-up
• Mechanically complex
• Storage temperatures high
relative to liquid nitrogen
Liquid Phase Nitrogen
• Steady ultra-low (-196ºC)
temperature
• Simplicity and mechanical
reliability
• Requires regular supply of
liquid nitrogen
• High running costs
• Risk of cross-contamination
via the liquid nitrogen
• - 196ºC
Vapor Phase Nitrogen
• No risk of cross-
contamination from liquid
nitrogen
• Low temperatures achieved
• Simplicity and reliability
• Requires regular supply of
liquid nitrogen
• High running costs
• Temperature fluctuations
between - 135ºC and - 190ºC

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Safety aspects of cell culture
• SAFETY CONSIDERATIONS
• Assume all cultures are hazardous since they may harbor latent viruses
or other organisms
• The following safety precautions should also be observed:
• pipetting: use pipette aids to prevent ingestion
• keep aerosols down to a minimum
• no eating, drinking, or smoking
• wash hands after handling cultures and before leaving the lab
• decontaminate work surfaces with disinfectant (before and after)
• autoclave all waste
• use biological safety cabinet (laminar flow hood)
• use aseptic technique
• dispose of all liquid waste after each experiment and treat with bleach

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Biosafety cabinets
• The Class I BSC provides
personnel and environmental
protection, but no product
protection.
• The Class I BSC is hard-ducted to
the building exhaust system,
thimble-connected, or recirculated
back into the room depending on
use.
• The Class II (Types A, B1, B2, and
B3)24 biological safety cabinets
provide personnel, environmental
and product protection.
Laminar flow

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Recommended Biosafety Levels for Infectious Agents
BSL Agents Practices
Safety Equipment
(Primary Barriers)
Facilities
(Secondary Barriers)
1 Not known to consistently cause
disease in healthy adults
Standard Microbiological
Practices
None required Open bench top sink
required
2 Associated with human disease,
hazard = percutaneous injury,
ingestion, mucous membrane
exposure
BSL-1 practice plus:
• Limited access
• Biohazard warning signs
• "Sharps" precautions
• Biosafety manual defining any
needed waste decontamination
or medical surveillance policies
Primary barriers = Class I or II BSCs or
other physical containment devices used
for all manipulations of agents that cause
splashes or aerosols of infectious
materials; PPEs: laboratory coats; gloves;
face protection as needed
BSL-1 plus:
Autoclave available
3 Indigenous or exotic agents with
potential for aerosol transmission;
disease may have serious or lethal
consequences
BSL-2 practice plus:
• Controlled access
• Decontamination of all waste
• Decontamination of lab
clothing before laundering
• Baseline serum
Primary barriers = Class I or II BCSs or
other physical containment devices used
for all open manipulations of agents; PPEs:
protective lab clothing; gloves; respiratory
protection as needed
BSL-2 plus:
• Physical separation
from access corridors
• Self-closing, double-
door access
• Exhausted air not
recirculated
• Negative airflow into
laboratory
4 Dangerous/exotic agents which pose
high risk of life-threatening disease,
aerosol-transmitted lab infections; or
related agents with unknown risk of
transmission
BSL-3 practices plus:
• Clothing change before
entering
• Shower on exit
• All material decontaminated
on exit from facility
Primary barriers = All procedures
conducted in Class III BSCs or Class I or II
BSCs in combination with full-body, air-
supplied, positive pressure personnel suit
BSL-3 plus:
• Separate building or
isolated zone
• Dedicated supply
and exhaust, vacuum,
and decon systems
• Other requirements
outlined in the text

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References
• R. Ian Freshney. Culture of Animal cells a
manual of basic technique. 4th
edition. Wiley-
Liss, New York. 2000.
• en.Wikipedia.com/animal_cell_culture.

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Tissue culture area

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