GROWTH OF ANIMAL CELL IN

CULTURE

Prepared By : Ms. Priyanka Mokharkar

Assistant Professor
Anurag College of Pharmacy
✓ Animal cell culture refers to the “in vitro growth and maintenance of animal cells”
under controlled laboratory conditions.
✓ These conditions include appropriate nutrients, physiological environment, and
aseptic handling.
✓ Animal cells are delicate and require precise growth conditions for survival,
multiplication, and function.
✓ Animal cell culture is a widely used technique in biotechnology, research, and
medicine.
✓ Cells isolated from tissues grow in artificial media that supply all essential nutrients.
✓ Growth occurs through mitosis, and cells increase in number when optimal
environmental conditions are provided.
✓ Animal cell growth is important for vaccine production, drug screening, protein
expression, and tissue engineering.
✓ Animal cell culture growth is a crucial technique used in modern biotechnology and
biomedical research.
✓ Successful growth depends on strict control of environmental and nutritional
conditions.
✓ Knowledge of growth phases, requirements, and influencing factors ensures
productive and healthy cell cultures for research and industrial purposes.
 Requirements for Cell Growth
A. Physical Requirements
1. Temperature: Most mammalian cells grow at 37°C.2.
2. pH: Maintained between 7.2–7.4 using bicarbonate buffer and 5%
CO₂.
3. Osmotic Pressure: Should be isotonic (~300 mOsm).
4. Humidity: High humidity (95%) prevent drying of cultures.
5. Sterility: Strict aseptic techniques are essential to avoid contamination.

B. Nutritional Requirements
➢ Cells require a suitable culture medium, which includes:
• Amino acids
• Vitamins
• Salts and minerals
• Glucose (energy source)
• Hormones
• Growth factors
• Antibiotics (optional)
• Most cultures use serum (e.g., Fetal Bovine Serum), which provides
adhesion factors, hormones, lipids, and trace elements.
 Types of Cell Growth

1. Adherent (Anchorage-Dependent) Cells

✓ Require solid support (glass or plastic surface).

✓ Grow as a monolayer.
✓ Examples: epithelial cells, fibroblasts.

2. Suspension Cells

✓ Grow while floating in the medium.

✓ Useful for blood cells, cancer cells, hybridomas.
✓ Suitable for large-scale cultures.
 Growth Curve of Animal Cells

➢ When animal cells are cultured; they show a characteristic sigmoidal

growth curve consisting of four phases:

a) Lag Phase

✓ Cells adapt to new environment.

✓ Attach and spread on the surface (in adherent cultures).
✓ No or minimal cell division.

(b) Log / Exponential Phase

✓ Rapid mitotic division occurs.

✓ Cell number doubles at a constant rate (doubling time).
✓ Cells are metabolically most active.
✓ Ideal stage for experiments and harvesting.
c) Stationary Phase

✓ Nutrients become limited and metabolic waste increases.

✓ Growth rate slows.
✓ Contact inhibition stops further division in adherent
monolayers.

(d) Decline / Death Phase

✓ Cell death rate exceeds cell division.

✓ Medium becomes acidic and nutrients deplete.
✓ Cells lose viability.
 Mechanism of Cell Growth

✓ Growth of animal cells occurs through:

1. Attachment: Cells adhere to culture surface.

2. Spreading: Cytoskeletal reorganization occurs.

3. DNA Synthesis: Cells undergo S-phase.

4. Mitosis: Cell divides into two daughter cells.

5. Monolayer Formation: Cells proliferate until confluent.

6. Density Limitation: Cells stop dividing when overcrowded.

 Factors Affecting Cell Growth

✓ Intrinsic factors: cell type, species, genetic stability.

✓ Extrinsic factors: pH, temperature, oxygen supply, medium

composition, serum quality, contamination, and surface
properties.

✓ Mechanical factors: shear stress in suspension cultures.

 Measurement of Cell Growth

✓ Cell growth can be estimated using:

▪ Hemocytometer cell counting.

▪ Viability assays (Trypan Blue, MTT assay)
▪ Protein or DNA estimation
▪ Cell mass measurement
▪ These methods help monitor culture health and growth rate.
 Subculture (Passaging)

✓ To maintain continuous growth, cells must be transferred to

fresh vessels when they reach 70–90% confluency.

✓ Steps include:

1. Removing old medium

2. Washing cells
3. Detaching cells with trypsin (for adherent cells)
4. Neutralizing enzyme
5. Centrifuging and resuspending cells
6. Seeding into new flasks

✓ Subculturing prevents nutrient depletion and overcrowding.

 Limitations of Animal Cell Growth

✓ High risk of contamination.

✓ Requirement of expensive equipment.
✓ Limited lifespan for primary cells.
✓ Contact inhibition restricts growth.
✓ Genetic changes may occur over long passages

Applications

✓ Vaccine and therapeutic protein production.

✓ Monoclonal antibody production.
✓ Cancer and genetic research.
✓ Drug screening and toxicity testing.
✓ Regenerative medicine and tissue engineering.
✓ Study of cell physiology and metabolism
 General Procedure for
Cell Culture
✓ Cell culture is a technique used to grow and maintain cells under
controlled laboratory conditions.
✓ The general procedure involves several essential steps to ensure
proper growth, sterility, and viability of cells.
✓ The general procedure for cell culture requires strict aseptic
technique, proper selection of media, regular monitoring, and
timely passaging.
✓ Maintaining suitable environmental conditions ensures healthy,
contaminant-free cell growth.
1. Preparation of Laboratory and Equipment

✓ Perform all work in a Laminar Air Flow (LAF) cabinet to maintain sterility.
✓ Clean the cabinet, pipettes, and working area with 70% ethanol.
✓ Switch on UV lamp for 15–20 minutes before work.
✓ Prepare all materials: culture media, serum, antibiotics, flasks, pipettes, trypsin, and
cryovials.

2. Preparation of Culture Media

✓ Select an appropriate medium such as DMEM, RPMI-1640, MEM.

✓ Add Fetal Bovine Serum (FBS) (5–20%), antibiotics (penicillin-streptomycin), and
supplements like glutamine.
✓ Sterilize by filtration if required.
✓ Warm the medium to 37°C before use.
✓ Thawing of Cells (if using frozen stock).
✓ Remove frozen vial from liquid nitrogen.
✓ Thaw quickly in a 37°C water bath for 1–2 minutes.
✓ Transfer cells gently to a tube containing fresh medium.
✓ Centrifuge to remove cryoprotectant (DMSO) and resuspend in fresh medium.
4. Seeding of Cells
✓ Add an appropriate volume of cell suspension into a sterile culture flask or dish.
✓ Add sufficient culture medium to allow growth.
✓ Close the flask and place it in a CO₂ incubator (usually 5% CO₂, 37°C, 95% humidity).

5. Incubation
✓ Keep cells in the CO₂ incubator for growth.
✓ Provide optimum conditions of:
• Temperature: 37°C
• pH: 7.2–7.4
• CO₂ concentration: 5%
• Humidity: 90–95%.

6. Observation and Monitoring

✓ Examine cells regularly under an inverted microscope.
✓ Check: Cell morphology, Confluency (percentage surface covered), Contamination
(bacteria, fungi, mycoplasma).
7. Sub-culturing (Passaging)
✓ Performed when cells reach 70–80% confluency.

Steps:
1. Remove spent medium.
2. Wash cells with PBS to remove serum.
3. Add trypsin-EDTA to detach adherent cells.
4. Neutralize trypsin using fresh medium.
5. Centrifuge and resuspend the pellet.
6. Seed the cells into new flasks with fresh medium.

8. Maintenance

✓ Change the medium every 2–3 days.

✓ Maintain sterility and avoid unnecessary opening of flasks.
✓ Monitor cell health regularly.
9. Harvesting of Cells

✓ For analysis or experiments, cells can be collected:

• Adherent cells: detach using trypsin.

• Suspension cells: directly centrifuge.
• Wash with PBS and process for downstream applications
(biochemical assays, DNA/RNA extraction, etc.)

10. Storage and Cryopreservation

✓ Healthy cells can be stored long-term.

✓ Resuspend cells in medium containing 10% DMSO
(cryoprotectant).
✓ Freeze slowly at –80°C, then transfer to liquid nitrogen (–196°C).
 Primary, Established and Transformed
Cell Cultures
✓ The growth of animal cells outside the body in an artificial environment is known
as cell culture.
✓ Based on their origin, life span and growth characteristics, cell cultures are
broadly classified into primary cell culture, established (secondary) cell line, and
transformed cell line.
✓ Each type has unique properties that make them useful for research,
biotechnology and pharmaceutical applications.
✓ Primary, established and transformed cell cultures form the foundation of modern
cell biology and biotechnology.
✓ Primary cultures provide the most natural cell behavior, established lines offer
reproducibility and ease of maintenance, while transformed lines provide
unlimited growth and are widely used for research and industrial applications.
1. Primary Cell Culture

Definition

➢ Primary cell culture refers to the culture of cells directly isolated from tissues or
organs of an animal. These cells are obtained by mechanical disaggregation or
enzymatic digestion (using trypsin, collagenase, etc.) and grown in a suitable
nutrient medium.

Characteristics

✓ Closest resemblance to normal in vivo cells, both in structure and function.

✓ Finite life span: Usually survive for 5–10 passages only.
✓ Slow growth rate because cells require adaptation to in-vitro conditions.
✓ High degree of heterogeneity, as they come directly from tissue.
✓ Sensitive to contamination and changes in temperature, pH or medium.
Advantages

✓ Maintain original morphology and physiology.

✓ Useful for studying normal cell biology, metabolism, drug effects and
toxicity.

Limitations

✓ Limited growth potential.

✓ Require continuous supply of fresh tissues.
✓ More expensive and labor-intensive.
✓ Examples: Primary fibroblasts, primary hepatocytes, primary kidney
cells, lymphocytes.
2. Established (Secondary) Cell Line

Definition
➢ An established cell line is derived from a primary culture after it undergoes repeated sub-
culturing. Some cells adapt to the artificial environment and begin to proliferate continuously for
a prolonged period.

Characteristics
✓ Longer life span compared to primary cells, though not truly immortal.
✓ More uniform and stable, showing less variation.
✓ Higher growth rate and easier to maintain.
✓ Can grow as monolayer (anchorage-dependent) or suspension (anchorage-independent) cultures.
✓ Long-term subculturing allows selection of cells that survive and adapt.
✓ These adapted cells lose some primary features and become a stable cell line.

Examples: Vero cells, BHK-21, 3T3 fibroblasts.

Applications: Used in vaccine production, toxicity studies, virology, and recombinant protein
expression.
3. Transformed Cell Line

Definition

➢ A transformed cell line is a cell population that has undergone genetic transformation
- either spontaneously, chemically, physically or virally. These cells behave like
cancer cells and possess an unlimited life span, hence called immortal cell lines.

Characteristics

✓ Immortal (unlimited divisions).

✓ Loss of contact inhibition, allowing cells to pile up.
✓ Altered morphology, usually rounded or irregular.
✓ Rapid growth rate and reduced dependence on growth factors.
✓ May have chromosomal abnormalities and unstable genome.
✓ Often anchorage-independent (able to grow in soft agar).
Causes of Transformation

✓ Spontaneous mutations in long-term cultures.

✓ Chemical carcinogens (e.g., nitrosamines).
✓ Ionizing radiation.
✓ Viral infection (e.g., SV40, HPV).

Examples: HeLa cells, HEK-293, NIH-3T3 transformed line, MCF-7

cancer cells.

Applications: Used in cancer research, genetic studies, monoclonal

antibody production, and recombinant DNA technology.
 Feature Primary Cell Established Cell Transformed
Culture Line Cell Line
Origin Directly from tissue Adapted from primary Mutation/cancerous
culture
Life Span Very limited Long but finite Unlimited
(immortal)
Growth Rate Slow Moderate Rapid
Morphology Normal Adapted Abnormal
Contact Inhibition Present May be present Lost
Examples Primary fibroblasts Vero, BHK-21 HeLa, HEK-293