Animal Cell Culture

Animal cell culture is the in vitro maintenance and proliferation of animal cells outside the living organism, provided they are supplied with suitable nutrients and growth conditions. It involves isolating cells from animal tissue and growing them in a suitable culture medium under laboratory conditions.

Animal cell culture media are the nutrient preparations used to support the survival, growth, differentiation, and product formation of animal cells in vitro. A typical medium contains vitamins, amino acids, glucose, inorganic salts, serum, and hormones. It also helps maintain proper pH and osmolality.

Media are of two main types:

1. Natural media
- Consist of naturally occurring biological substances such as plasma, serum, tissue extract, lymph, amniotic fluid, and pleural fluid.
- Example: plasma separated from heparinised blood.
- Disadvantage: poor reproducibility and reduced uniformity because their exact composition is not known.

2. Synthetic or artificial media
- Made of a basal medium plus supplements such as serum, growth factors, and hormones.
- These can be modified according to the purpose.
- They are further classified into:
- Serum-containing media
- Serum-free media
- Xeno-free media
- Protein-free media
- Chemically defined media
- Complex media

Examples of basal or balanced media mentioned in the chapter include PBS, DPBS, HBSS, EBSS, EMEM, DMEM, RPMI-1640, and Ham’s F-12.

Advantages of serum in culture media:
- It is a good source of amino acids, proteins, vitamins, carbohydrates, lipids, hormones, and growth factors.
- It contains binding proteins such as albumin and transferrin, which help transport molecules into cells.
- It provides adhesion factors that help cells attach to the substratum before division.
- It supports cell growth and proliferation.

Disadvantages of serum in culture media:
- It contains an insufficient amount of cell-specific growth factors, so it cannot be used alone for culture.
- It may contain cytotoxic compounds and growth-inhibiting factors.
- It can carry contaminants such as viruses, fungi, and mycoplasma.
- It may interfere with the purification and isolation of cell culture products, including pharmaceutical compounds.
- Its quality may vary from batch to batch and may deteriorate within one year.

Two chemically synthesised media are:

1. Eagle’s Minimum Essential Medium (EMEM)
- A chemically defined/synthetic basal medium used for animal cell culture.
- It contains essential nutrients needed for cell growth.
- It is one of the common media listed under complex media in the chapter.

2. Dulbecco’s Minimal Essential Medium (DMEM)
- A modified version of essential medium used widely for culturing animal cells.
- It provides a suitable balance of nutrients for cell survival and proliferation.
- It is commonly used in cell culture laboratories.

Other examples mentioned in the chapter include RPMI-1640 and Ham’s F-12.

A primary cell culture is a culture obtained directly from host tissue. The cells dissociated from the parental tissue are grown in a suitable medium in a container.

Development of primary culture:
- The tissue is first dissected under sterile conditions.
- It is then mechanically or enzymatically disaggregated.
- The tissue may be chopped into small pieces and placed on a dish; cells then grow out from the fragment. Such a tissue fragment is called an explant.
- Alternatively, the tissue may be treated with enzymes such as trypsin or collagenase to form a cell suspension.
- The cells are then allowed to adhere and spread on the substrate.
- Primary cultures may grow as adherent cultures or as suspension cultures.

Primary cultures are usually heterogeneous and resemble the original tissue cells.

Subculture or passaging is the process of transferring cells from an old culture vessel to a fresh one with new growth medium so that they can continue to grow.

When a primary culture becomes crowded or reaches confluence, the cells are detached using an enzyme such as trypsin or by mechanical methods, and then transferred into fresh medium after centrifugation. This is also called splitting.

The passage number is the number of times a cell line has been sub-cultured.

Finite cell lines and continuous cell lines differ as follows:

- Finite cell lines
- Have a limited number of cell divisions.
- Have a limited life span.
- After several passages, they lose the ability to proliferate; this is called senescence.
- Cell lines derived from normal primary cultures are usually finite.

- Continuous cell lines
- Some cells from a finite line undergo transformation and regain the ability to divide indefinitely.
- They can be sub-cultured and grown indefinitely.
- They are immortal and often tumorigenic.
- They are usually faster growing and less demanding in nutrient requirements.

Cell viability measurement is used to determine whether cells are alive or dead. It is important for testing the effect of pesticides, toxins, and drugs.

There are two main types of viability assays:

1. Dye exclusion assays
- Use stains such as trypan blue, propidium iodide, 7-AAD, or acridine orange.
- Live cells exclude the dye, while dead cells allow the dye to enter because their membrane integrity is lost.

2. Metabolic viability assays
- Depend on a biochemical reaction carried out by living cells.
- A common example is the MTT assay.
- In this assay, yellow MTT is reduced by mitochondrial dehydrogenase enzymes of live cells into purple formazan crystals.
- The amount of formazan formed is proportional to the number of living cells.

Thus, viability is measured by dye exclusion or by metabolic activity.

Animal cell culture has many applications:

- It is used as a model system to study interactions between cells, diseases causing agents, and their drugs.
- It is a convenient and economic tool for virus research.
- It is useful for vaccine production on a large scale.
- It serves as a production house for medically important protein pharmaceuticals.

Examples of therapeutic products produced using animal cell culture include:
- FSH for infertility
- HGH for growth hormone deficiency
- EPO for anaemia
- Factor VIII for haemophilia A
- Factor IX for haemophilia B
- Interleukin-2 for cancer therapy
- tPA for stroke
- Monoclonal antibodies for cancer therapy and autoimmune diseases

Other important applications mentioned in the chapter:
- study of molecular genetics
- immunological analysis
- surgery and bioengineering
- pharmaceutical industry
- research and development of drugs and treatment of diseases such as cancer and genetic disorders

From the chapter, DMEM is an animal cell culture medium. MS media is plant tissue culture media, and LB media is used for bacteria, so the correct option is DMEM.

A culture prepared by inoculating directly from the tissue of an organism into culture media is called primary cell culture.