UNIT 12 – Animal Cell Culture (Q&A) | MZO-001 MSCZOO | IGNOU

SAQ 1

Fill in the blanks:

(i) The technique of isolating cells from an organism and allowing them to develop in a suitable artificial environment is known as ...................... .
Answer: cell culture

(ii) Hybridoma technique for the production of monoclonal antibodies was developed by ...................... .
Answer: Kohler and Milstein

(iii) A cell line obtained from primary cell culture is called ................... .
Answer: secondary cell culture

(iv) One example of coagulant is ..................... .
Answer: plasma from heparinized blood

(v) Full form of DME medium is ................... .
Answer: Dulbecco's Modified Eagle's

SAQ 2

State True or False:

i) Finite cell lines are those with a fixed maximum number of cell generations and growth.
Answer: True

ii) In the Lag phase, the cell metabolic activity is the highest.
Answer: False 

iii) Enzymes like proteases are better to detach the cells off the growing surface than mechanical methods.
Answer: True 

iv) Protease release assay is meant to analyze cell viability.
Answer: True

v) Hayflick's phenomenon is the number of times a normal cell population multiplies prior to entering the senescence phase.
Answer: True

TERMINAL QUESTIONS

1. Distinguish between primary and secondary cell culture.

To distinguish between primary and secondary cell culture, we must first understand what each term represents and how they differ in terms of origin, growth behavior, life span and application.

Primary cell culture refers to the initial culture that is directly derived from animal or plant tissues by mechanical or enzymatic disaggregation. The cells in this culture closely resemble the in vivo state both genetically and functionally. However, these cells have a limited capacity to divide and usually undergo senescence after a few passages.

On the other hand, secondary cell culture is derived from the subculturing or passaging of primary cells. After several passages, the cells that adapt better to in vitro conditions may give rise to a more stable population. In some cases, these cells may undergo transformation and acquire the ability to proliferate indefinitely, forming a cell line.

Now, based on several key features, we can distinguish between primary and secondary cell culture as follows:

1. Based on Origin:

Primary cell culture: Directly derived from tissues by enzymatic or mechanical means.

Secondary cell culture: Obtained by subculturing primary cells after their initial growth.

2. Based on Cell Characteristics:

Primary cells culture: Maintain morphology and functional properties close to original tissue.

Secondary cells culture: May show changes in morphology, reduced dependency on growth factors, or altered gene expression with passaging.

3. Based on Growth Capacity:

Primary cells culture: Have limited growth potential; usually stop dividing after a few divisions due to senescence.

Secondary cells culture: May exhibit extended growth potential and some may transform into continuous cell lines.

4. Based on Stability:

Primary cells culture: Genetically more stable but less consistent in behavior.

Secondary cells culture: May become genetically unstable over time but are more consistent in vitro.

5. Based on Applications:

Primary cell culture: Used when the goal is to study natural cell behavior, signaling, or drug response in a near-native environment.

Secondary cell culture: Used when long-term experiments or large-scale assays are required, as these cells grow more reliably.

2. Discuss the steps of animal cell culture.

Animal cell culture is a well-controlled laboratory technique where cells from animal tissues are grown outside the organism in a nutrient-rich artificial environment. This method is highly useful in various biological and medical research areas like cancer studies, genetic engineering, drug testing and vaccine production. The whole process requires strict aseptic conditions, proper nutritional media, and controlled temperature, pH and gas levels. To establish a successful culture of animal cells, a stepwise procedure is followed. These steps help in obtaining viable, healthy and contaminant-free cells that can divide and grow properly in laboratory conditions.

In general, the animal cell culture procedure involves three main steps: tissue isolation, disaggregation into single cells and culturing in growth media.

1. Isolation of Tissue and Explant Preparation:

The first and very crucial step is to obtain a suitable tissue sample from an animal, which could be from organs like kidney, liver, skin and even tumor tissue. The animal is either sacrificed ethically or biopsy is taken under sterile conditions. The tissue sample is immediately transferred to a sterile container with cold buffered saline (like PBS) that contains antibiotics and antifungals to reduce chances of contamination. The tissue is then washed multiple times to remove any remaining blood, serum, or external microorganisms. After that, it is chopped into very small pieces called explants using sterile scissors or scalpels. These explants are used for the next step of the process.

2. Disaggregation into Single Cells:

After preparing explants, the next step is to convert these tissue fragments into individual cells so that they can grow properly in culture vessels. This disaggregation can be done using the following two methods:
  • Mechanical Method: In this method, the explants are broken into smaller fragments manually by pipetting, shaking, or using sterile blades. This is simple but often does not give a uniform single-cell suspension.
  • Enzymatic Method: This is more effective and commonly used. Special enzymes like trypsin, collagenase, or dispase are added to digest the extracellular matrix (ECM) and release the cells from the tissue. Trypsin breaks peptide bonds, collagenase targets collagen fibers and dispase works on the basement membrane. After digestion, the cell suspension is filtered and centrifuged to remove debris.

3. Culturing and Incubation of Cells:

Now, the single-cell suspension is transferred into sterile culture flasks or Petri dishes containing a nutrient-rich culture medium like DMEM and RPMI, which provides glucose, amino acids, vitamins, minerals, salts and serum. The vessels are then incubated at 37°C in a CO₂ incubator maintaining about 5% carbon dioxide and high humidity. The cells start attaching to the surface of the flask and begin dividing. This is the phase where the culture starts to establish. Daily microscopic observation is needed to check cell shape, attachment, and any contamination. Once the culture reaches confluence (complete surface coverage), it is considered established.

[Note: Sometimes, many textbooks also mention further steps like subculturing (passaging), cryopreservation (freezing for storage), and revival. But these are part of long-term culture maintenance, not the basic establishment. So, when the question asks for "steps of animal cell culture," only the above three steps are considered relevant.]

3. Enumerate the applications of animal cell culture.

Animal cell culture refers to the process of growing animal cells in artificial conditions outside the body. These cells are maintained in nutrient-rich media under strictly controlled conditions of temperature, pH and oxygen. This technique plays a vital role in many areas of biological research and biotechnology. The wide applications of animal cell culture span from basic research to industrial production, clinical therapy and diagnostics.

There are following major applications of animal cell culture which are:

1. Vaccine Production

1. Vaccine Production
One of the most common applications of animal cell culture is in the production of vaccines. Viruses are grown in cultured animal cells to produce vaccines safely and in large quantities. For example, Vero cells (taken from monkey kidney) were used in the development of Covaxin during the COVID-19 pandemic in India. Similarly, vaccines for rabies, polio, measles and hepatitis B are also made using animal cell cultures. This method ensures that vaccines are free from contamination and have good quality.

2. Production of Monoclonal Antibodies and Therapeutic Proteins

Animal cells such as CHO (Chinese Hamster Ovary) cells and hybridoma cells are used for large-scale production of monoclonal antibodies and proteins like insulin, erythropoietin and interferons. These are used in the treatment of cancer, autoimmune diseases and infections. For example, Herceptin used in breast cancer treatment is produced using animal cell cultures.

3. Drug Testing and Toxicity Studies

New medicines and chemicals are first tested on cultured animal cells to check whether they are safe and effective. It helps to reduce the use of animals in early testing. For example, HepG2 liver cell lines are used to test for liver toxicity. This step is very important in pharmaceutical industries and drug discovery research.

4. Research on Cancer and Genetic Disorders

Many types of cancer cells like HeLa, MCF-7 and A549 are cultured in labs to study how cancer grows, spreads and responds to different treatments. Scientists also use cultured cells with genetic disorders to understand diseases like muscular dystrophy and cystic fibrosis. This helps in discovering new treatments and understanding disease progression.

5. Tissue Engineering and Regenerative Medicine

In this application, cells are grown to form tissues which can be used to replace damaged body parts. For example, skin cells are cultured for burn patients and cartilage cells are used in joint repair. It is also used in making artificial organs and tissues for transplantation.

6. Virology and Viral Pathogenesis Studies

Animal cells are used to study how viruses infect cells, multiply and affect the immune system. This helps scientists to develop antiviral drugs and understand viral diseases. For example, many COVID-19 related studies used cultured lung cells to study SARS-CoV-2 behavior.

4. What are the different methods to monitor cell viability?

Different Methods to Monitor Cell Viability
Monitoring cell viability is a critical aspect of animal cell culture experiments. It helps in assessing the health, growth and physiological condition of cultured cells. Cell viability refers to the proportion of living cells in a population, and its measurement is important for applications like cytotoxicity testing, drug screening, vaccine production and bioprocess optimization. Viability assays work on different principles such as membrane integrity, metabolic activity, enzyme function, and dye uptake or exclusion. There are several reliable methods available to evaluate cell viability and each has its own advantages and limitations.

Here are the six most widely used and experimentally validated methods to monitor cell viability in animal cell culture:

1. Trypan Blue Exclusion Assay

This is one of the most classical and basic methods. Trypan blue is a dye that cannot enter live cells due to intact cell membranes. Only dead cells with compromised membranes allow the dye to enter and appear blue under the microscope. The live cells remain unstained. Using a hemocytometer, both live and dead cells are manually counted. Though this method is simple, quick, and inexpensive, it lacks sensitivity and is not ideal for high-throughput screening.

2. MTT, XTT, and MTS Assays (Metabolic Activity Assays)

These are colorimetric assays based on the reduction of tetrazolium salts by mitochondrial dehydrogenase enzymes present in metabolically active cells. MTT turns into purple formazan crystals, XTT and MTS into soluble formazan products. The amount of formazan formed is directly proportional to the number of viable cells and is measured using a spectrophotometer. These assays are widely used in drug screening and cytotoxicity studies.

3. Alamar Blue (Resazurin Reduction Assay)

This is a fluorometric and colorimetric method where resazurin, a blue non-toxic dye, is reduced to pink and fluorescent resorufin by viable cells. This assay is non-destructive, allows continuous monitoring, and is more sensitive than MTT. It is also suitable for long-term assays and small-scale experiments where cell preservation is important.

4. ATP-Based Luminescence Assay

This assay measures the level of intracellular ATP, which is an indicator of metabolically active and live cells. The luciferase enzyme converts ATP into light, which is then measured using a luminometer. The amount of light produced is directly proportional to the number of viable cells. This method is highly sensitive and quick but more expensive than others.

5. Annexin V and Propidium Iodide (PI) Staining by Flow Cytometry

This method allows detection of different cell death stages. Annexin V binds to phosphatidylserine, which is externalized on apoptotic cells, while PI enters only dead or late apoptotic cells. By using flow cytometry, cells can be classified as live, early apoptotic, or necrotic. This method provides very detailed results but requires advanced instrumentation and technical skill.

6. Calcein-AM and Ethidium Homodimer-1 (Live/Dead Assay)

This is a dual-fluorescence assay where live cells convert non-fluorescent Calcein-AM to green fluorescent calcein, while Ethidium Homodimer-1 stains the nuclei of dead cells red. This method is accurate, provides visual confirmation under fluorescence microscopy, and is often used in 3D cell cultures or tissue imaging.

[Note: Some researchers also include gelatin exclusion assay, LDH release assay and clonogenic assay under viability assays, but they are generally considered supportive or indirect methods.]

5. Write a brief account on primary cell culture.

Primary cell culture refers to the process of isolating cells directly from animal tissues and growing them in a suitable artificial environment under controlled laboratory conditions. These cells are taken from a living organism and maintained in vitro for a limited period. Since they closely mimic the in vivo state, primary cells are considered more physiologically relevant than immortalized cell lines. However, they have limited lifespan and can divide only for a few generations. Primary cell culture is widely used in cell biology, pharmacology, toxicology, cancer research and vaccine production because of its close resemblance to natural cell behavior in organisms.

Types of Primary Cells (Based on Growth Behaviour):

There are two main types of primary cells depending on how they behave during growth in culture:

1. Adherent Cells / Anchorage-Dependent Cells:

  • Adherent cells, also known as anchorage-dependent cells, are those which require a solid surface to attach, spread and grow. These cells are mostly derived from organs and tissues like kidney, liver, fibroblasts and epithelial tissues. They grow in monolayers and cannot survive or divide without proper attachment to a substrate like tissue culture-treated plastic or glass. Growth stops when the surface area is completely occupied (contact inhibition). Subculturing them involves enzymatic digestion using trypsin or EDTA to detach them. These cells are widely used in drug testing, virology and cancer research.

2. Suspension Cells / Anchorage-Independent Cells:

  • Suspension cells, also called anchorage-independent cells, can grow and divide freely in suspension without needing any solid surface to attach. These are usually isolated from blood or lymphoid organs, such as lymphocytes or certain myeloma cells. These cells grow as single cells or small clumps floating in the culture medium. They are ideal for large-scale cultures in bioreactors because they do not require enzymatic harvesting. These are especially useful in immunological research, vaccine production and monoclonal antibody generation. Their handling is easier and they can be passaged simply by dilution.

Steps Involved in Primary Cell Culture Preparation:

1. Tissue Collection:

  • A fresh tissue sample is collected from an animal under sterile conditions. Common sources include liver, kidney, lung, or skin. The sample is transported quickly to the lab in a suitable transport medium

2. Tissue Washing and Trimming:

  • The tissue is washed multiple times with sterile PBS (phosphate-buffered saline) to remove blood, fat and debris. It is then cut into small pieces using sterile instruments.

3. Tissue Disaggregation:

  • To release individual cells, the tissue is disaggregated either by enzymatic digestion using enzymes like trypsin, collagenase, or dispase, or by mechanical methods like pipetting or mincing.

4. Filtration and Centrifugation:

  • The cell suspension is filtered to remove large clumps and debris. Then it is centrifuged to collect viable cells at the bottom.

5. Seeding and Culturing:

  • The collected cells are suspended in a suitable culture medium and seeded into culture flasks or plates. Adherent cells attach to the surface, while suspension cells remain in the medium.

6. Incubation and Maintenance:

  • The cultures are incubated at 37°C with 5% CO₂. The medium is changed regularly to provide nutrients and remove waste. Cells are monitored for growth and contamination.

Benefits of Primary Cell Culture:

  • High physiological relevance: They closely resemble cells inside the body, so results are more accurate for in vivo studies.
  • Useful in toxicity and drug screening: Since they respond naturally to stimuli, they are preferred in pharmacological testing.
  • Vaccine and protein production: Many vaccines, like polio and rabies, are made using primary cell cultures.
  • Genetic studies: They are used to study gene expression in normal cells before transformation.
  • Model for disease study: Cells from diseased tissues can be cultured to study pathological conditions directly.

Drawbacks of Primary Cell Culture:

  • Limited lifespan: They divide only a few times before entering senescence.
  • More variation: Each preparation may show variability depending on the donor or isolation method.
  • Sensitive to conditions: They are highly sensitive to culture conditions like temperature, pH, and contamination.
  • High cost and effort: Isolation and maintenance require sterile conditions, time, and expensive equipment.
  • Ethical issues: Tissue collection from animals may raise ethical concerns.











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