Cell culture

IN THIS PRESENTATION I HAVE DISCUSSED THE BASIC OF CELL CULTURE

1. CELL CULTURE By Y asharth agrahari Mpharm 1 st year

2. CONTENT TERMINOLOGY BASIC EQUIPMENT CELL CULTURE MEDIA TYPES OF CELL CULTURE PROCEDURE OF CELL CULTURE ISOLATION OF CELL

3. TERMINOLOGY Cell culture :- It refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been already established. Cell Line :- After the first subculture, the primary culture is known as a cell line or sub clone. Cell lines derived from primary cultures have a limited life span

4. BASIC EQUIPMENT Cell culture hood (i.e., laminar-flow hood or biosafety cabinet) It is used to provide sterile environment and protect the laboratory worker from exposure to aerosols from cell culture. Air is filtered through a HEPA (high efficiency particulate air) filter before exiting the cabinet Incubator (humid CO2 incubator recommended) A cell culture incubator is designed to maintain a constant and high humidity for the growth of tissue culture cells under a CO2 atmosphere. Water bath A 37 o c water bath is required for a cell culture facility to warm up the media and other reagents used for cell culture. Centrifuge It is used to separate cells from culture media

5. Refrigerator and freezer (–20°C) Cell counter (e.g., Countess® Automated Cell Counter or hemacytometer ) Inverted microscope A simple inverted microscope is essential to look at cultures regularly to detect morphological changes and the possibility of microbiological contamination. Liquid nitrogen (N2) freezer or cryostorage container Filter Sterilizer It is used for heat sensitive materials such as culture media through a membrane with 0.2 micron or smaller pore size

6. CELL CULTURE MEDIA Cell culture media are complex mixtures of salts, carbohydrates, vitamins, amino acids, metabolic precursors, growth factors, hormones, and trace elements. Carbohydrates are supplied in the form of glucose. The pH is maintained by one or more buffering systems; CO₂/sodium bicarbonate, phosphate, and HEPES are the most common. Commonly used culture media include the following: Eagle’s basal medium (BME) has salt solution, non- essential amino acids, and sodium pyruvate. It is formulated with a reduced sodium bicarbonate concentration (1,500 mg/l) for use with 5% CO₂ Dulbecco’s Modified Eagle’s Medium (DMEM) has twice the concentration of amino acids and four times the amount of vitamins as EMEM. The original formulation contained 1,000 mg/L of glucose, but in the more commonly used variations this amount was increased to 4,500 mg/L.

7. Iscove’s Modified Dulbecco’s Medium (IMDM) was formulated for growth of lymphocytes and hybridomas . Compared to DMEM, it has additional amino acids, vitamins and inorganic salts. Potassium nitrate was substituted for ferric nitrate. It also contains HEPES and selenium, a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO₂ . McCoy’s 5A and RPMI-1640 was originally used to grow Novikoff hepatoma cells and will support the growth of primary cultures. RPMI-1640 is a modification of McCoy’s 5A and was developed for the long-term culture of peripheral blood lymphocytes. Leibovitz’s L-15 is formulated for use without CO₂ incubation . The standard sodium bicarbonate/CO₂ buffering system is replaced by a combination of phosphate buffers, free-base amino acids, higher levels of sodium pyruvate, and galactose.

8. TYPES OF CELL CULTURE Cell culture is classified into three : Primary cell culture Adherent cell culture Suspension cell culture Secondary cell culture Cell line Finite cell line Continuous cell line

9. Primary cell culture : Culture obtained straight from the cells of a host tissue . Such cultures comprises mostly heterogeneous cells and most of the cells divide only for a limited time. However, these cells are much similar to their parents. Adherent cells Anchorage dependent and propagate as a monolayer. These adhere to the culture vessel with the use of an extracellular matrix which is generally derived from tissues of organs that are immobile and embedded in network of connective tissues . EX- Fibroblasts and epithelial cells. Suspension culture Anchorage independent which can be grown floating in the culture medium. Do not attach to the surface of culture vessel. Ex- Hematopoietic stem cells, tumor cells.

10. Secondary cell culture and cell line : When primary culture is sub-cultured, it is known as secondary culture or cell line or sub clone. The process involves removing the growth media and disassociating the adherent cells. During the passage cells with the highest growth capacity predominates, resulting in degree of genotype and phenotypic uniformity in the population. However , as they are sub cultured serially they become different from the original cell.

11. Finite cell line : The cell lines which go through a limited number of cell divisions having a limited life span are known as finite cell line. After several passages cells lose their ability to proliferate, which is a genetically determined event known as senescence. Cell lines derived from primary cultures of normal cells are finite cell lines . Continuous cell lines : When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line . Such transformation can be spontaneous or chemically induced. These cells are less adherent, fact growing and grow more in suspension . They also have tendency grow on top of each other in multilayer culture vessel system.

12. PROCEDURE OF CELL CULTURE Harvest Cells Isolate cells with the use of appropriate enzymes Transfer the isolated cells on to an appropriate growth media in a culture dish Culture cells by placing the culture dish in a cell incubator Subculture cells to obtain a pure culture or to bypass some problems ( such as senescence ) Verify the cultured cells are type of the cell type of interest Cells are ready to be manipulated or modified for experimental procedures

13. ISOLATION OF CELL

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