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.]