Animal Biotechnology - Tissue Culture and Cell Culture

Definitions

Tissue Culture is the technique involving the removal of cells, tissues, or organs from an animal or plant and these are later placed into an artificial environment that is specifically modified for growth of the cells and tissues.

This environment usually consists of a suitable glass or plastic culture vessel containing a liquid or semi-solid nutrient medium that supplies the nutrients essential for survival and growth of the cells and tissues.

Organ Culture is the culture of whole organs or intact organ fragments with the intent of study and their continued function and organ or tissue development.

Cell Culture is defined as when the cells are removed from the organ fragments before or during cultivation, thus disrupting their actual relationships with neighboring cells by enzymatic, mechanical, or chemical disaggregation.

How Are Cell Cultures Obtained?

Primary Culture in Animal Tissue Culture

In a cell culture, when cells are removed from an organism surgically and then they are placed into a suitable culture environment, they will attach themselves to the medium, divide and grow in the controlled conditions. This culture of cells is called a Primary Culture.

There are two basic methods for doing this,

First, for Explant Cultures, small pieces of tissue are attached to a glass or treated plastic culture vessel and bathed in culture medium. After a few days, individual cells will move from the tissue explant out onto the culture vessel surface or substrate where they will begin to divide and grow.

The second method is also called as Enzymatic Dissociation. It is a more widely used method and is also less time consuming as it involves the addition of digesting (proteolytic) enzymes, such as trypsin or collagenase, to the tissue fragments to dissolve the proteins that hold the cells together. This creates a suspension of single cells. These single cells from the suspension are placed into the culture bottles or tubes containing culture medium. Here in the controlled conditions the cells grow and divide.

Subculturing in Animal Tissue Culture

When the cells in the primary culture bottles or tubes have grown and occupied or used up all of the available culture substrate, they need to be Subcultured to give them more space for further continued growth. This is mostly done by separating them from the substrate or medium as gently as possible by the use of proteolytic enzymes used to break the protein bonds between the cells and the substrate.

Some cell lines can be removed simply by gently scraping the cells off the walls of culture bottles and tubes. After this the cell cluster or the cell suspension can be subdivided and placed into new culture bottles. When sufficient amount of cells is obtained, the cells can be treated with cryoprotective agents like the dimethyl sulfoxide (DMSO) or glycerol. These treated cells are then carefully frozen and stored at cryogenic temperatures (below -127°C).

The cells can be recovered again from cryopreserved cultures whenever necessary.

An alternative method for setting up a primary culture is to buy established cell cultures from research Institutes, Organizations or Laboratories. The authenticity of cell cultures is well maintained in reputed organizations and laboratories but there is still one major drawback, there is a high probability that the cell cultures obtained in this way will not be too healthy cultures. This is mostly due to mixing up or contamination with other cell lines. It may also be due to contamination with microorganisms like mycoplasmas, bacteria, fungi or yeast.

Significance of Cell Culture in relation to Animal Biotechnology

Cell culture has become an important tool used in cell and molecular biology. The important areas where cell cultures play an important role are described in short, as follows:

1.      Toxicity Testing

Cultured cells are widely used alone or in conjunction with animal tests to study the effects of new drugs, cosmetics and chemicals on survival and growth in a wide variety of cell types. Especially important are liver- and kidney-derived cell cultures.

2.      Model Systems

Cell cultures provide a good model system for studying

          i.            Basic cell biology and biochemistry

        ii.            The interactions between disease-causing agents and cells

      iii.            The effects of drugs on cells

       iv.            The process and triggers for aging

         v.            Nutritional studies.

3.      Cancer Research

Since both normal cells and cancer cells can be grown in culture, the basic differences between them can be closely studied. In addition, it is possible, by the use of chemicals, viruses and radiation, to convert normal cultured cells to cancer causing cells. Thus, the mechanisms that cause the change can be studied. Cultured cancer cells also serve as a test system to determine suitable drugs and methods for selectively destroying types of cancer.

4.      Cell-Based Manufacturing

While cultured cells can be used to produce many important products, three areas are generating the most interest. The first is the large-scale production of viruses for use in vaccine production. These include vaccines for polio, rabies, chicken pox, hepatitis B and measles. Second is the large-scale production of cells that have been genetically engineered to produce proteins that have medicinal or commercial value. These include monoclonal antibodies, insulin, hormones, etc. Third is the use of cells as replacement tissues and organs. Artificial skin for use in treating burns and ulcers is the first commercially available product. However, testing is underway on artificial organs such as pancreas, liver and kidney. A potential supply of replacement cells and tissues may come out of work currently being done with both embryonic and adult stem cells. These are cells that have the potential to differentiate into a variety of different cell types. It is hoped that learning how to control the development of these cells may offer new treatment approaches for a wide variety of medical conditions.

5.      Virology

One of the earliest and major uses of cell culture is the replication of viruses in cell cultures (in place of animals) for use in vaccine production. Cell cultures are also widely used in the clinical detection and isolation of viruses, as well as basic research into how they grow and infect organisms.

6.     Genetic Counseling

Amniocentesis, a diagnostic technique that enables doctors to remove and culture foetal cells from pregnant women, has given doctors an important tool for the early diagnosis of foetal disorders. These cells can then be examined for abnormalities in their chromosomes and genes using karyotyping, chromosome painting and other molecular techniques.

7.      Genetic Engineering

The ability to transfect or reprogram cultured cells with new genetic material (DNA and genes) has provided a major tool to molecular biologists wishing to study the cellular effects of the expression of these genes (new proteins). These techniques can also be used to produce these new proteins in large quantity in cultured cells for further study. Insect cells are widely used as miniature cells factories to express substantial quantities of proteins that they manufacture after being infected with genetically engineered baculoviruses.

8.     Gene Therapy

The ability to genetically engineer cells has also led to their use for gene therapy. Cells can be removed from a patient lacking a functional gene and the missing or damaged gene can then be replaced. The cells can be grown for a while in culture and then replaced into the patient. An alternative approach is to place the missing gene into a viral vector and then “infect’’ the patient with the virus in the hope that the missing gene will then be expressed in the patient’s cells.

9.     Drug Screening and Development

Cell-based assays have become increasingly important for the pharmaceutical industry, not just for cytotoxicity testing but also for high throughput screening of compounds that may have potential use as drugs. In the beginning, the cell culture tests were done in 96 well plates, but increasing use is now being made of 384 and 1536 well plates.