Primary culture

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A Primary Culture is that stage of a cell culture following the isolation of the cells, but before the first subculture. There are three stages to consider:
1. Isolation of the tissue
2. Dissection and /or disaggregation , and
3. Culture following seeding in the culture vessel.

Following isolation, a primary culture may be obtained either by allowing cells to migrate out from the fragment of tissue adhering to a suitable substrate or by disaggregating the tissue mechanically or enzymatically to produce a suspension of cells, some of which will ultimately attach to the substrate.

Normal animal tissues (e.g., skin, kidney, liver) or whole embryos commonly are used to establish primary cell cultures. To prepare tissue cells for culture (or to remove adherent cells from a culture dish for biochemical studies), trypsin or another protease is used to destroy the proteins in the junctions that normally interconnect cells. For many years, most cell types were difficult, if not impossible, to culture. But the identification and preparation of various protein growth factors that stimulate the replication of specific cell types, as well as other recent modifications in culture methods, now permit experimenters to grow various types of specialized cells.

Dr. K.C Many studies with vertebrate cells, however, still are performed with those few cell types that grow most readily in culture. These are not cells of a defined type; rather, they represent whatever grows when a tissue or an embryo is placed in culture. The cell type that usually predominates in such cultures is called a fibroblast because it secretes the types of proteins associated with fibroblasts in fibrous connective tissue of animals. Cultured fibroblasts have the morphology of tissue fibroblasts, but they retain the ability to differentiate into other cell types; thus they are not as differentiated as tissue fibroblasts.

1. Isolation of Tissue:

For tissue isolation sterilize the site with 70% alcohol, remove the tissue aseptically. The isolated tissue may be stored in a refrigerator before transferring it to BSS or to a culture medium, because viable cells may be recovered from chilled tissue several days after explantation The first step in isolating cells of a uniform type from a tissue that contains a mixture of cell types is to disrupt the extra cellular matrix that holds the cells together. The best yields of viable dissociated cells are usually obtained from fetal or neonatal tissues. The tissue sample is typically treated with proteolytic enzymes (such as trypsin and collagenase) to digest proteins in the extra cellular matrix and with agents (such as ethylenediaminetetraacetic acid, or EDTA) that bind, or chelate, the Ca2+ on which cell-cell adhesion depends. The tissue can then be teased apart into single living cells by gentle agitation. Several approaches are used to separate the different cell types from a mixed cell suspension. One exploits differences in physical properties. Large cells can be separated from small cells and dense cells from light cells by centrifugation, for example. Another approach is based on the tendency of some cell types to adhere strongly to glass or plastic, which allows them to be separated from cells that adhere less strongly. An important refinement of this last technique depends on the specific binding properties of antibodies. Antibodies that bind specifically to the surface of only one cell type in a tissue can be coupled to various matrices such as collagen, polysaccharide beads, or plastic to form an affinity surface to which only cells recognized by the antibodies can adhere. The bound cells are then recovered by gentle shaking, by treatment with trypsin to digest the proteins that mediate the adhesion, or, in the case of a digestible matrix (such as collagen), by degrading the matrix itself with enzymes (such as collagenase).

Enzymatic Disaggregation:

The mechanical or enzymatic disaggregation gives a much higher yield of representative cells in a short duration. The embryonic tissue disperses more readily and gives higher yield than the newly born or adult. In several tumors, the tissue (being fragile) is mostly destroyed during enzyme treatment and viable cells are obtained with difficulty.

1. Disaggregation by Trypsin:

Trypisinization may be of two types: (1) Warm trypsinization and (2) cold trypsinization.

Crude trypsin is the most common enzyme used for disaggregation. It can be tolerated by a variety of cells and is effective for many tissues. Its residual activity is neutralized by serum of the medium or by a trypsin inhibitor (eg. Soyabean trypsin inhibitor) in case of serum free medium. The cells are exposed to warm enzyme (36.5 °C) for a minimum period. The dissociated cells are collected every half an hour. The trypsin is removed by centrifugation after 3-4 hours. This time is required for complete dissaggregation. Alternatively cold trypisinazation may be used which involves soaking of tissue in trypsin in 40 °C (to allow penetration of enzyme), followed by incubation at 36.5 °C for a shorter period.

2. Disaggregation by collagenase (for embryonic, normal and malignant tissues)

Sometimes disaggregation by trypsin may be damaging for epithelial cells or it becomes ineffective for fibrous tissues. The intracellular matrix contains collagen; therefore, collagenase has proved effective for disaggregation for several normal and malignant tissues. These tissues may be sensitive to trypsin. Crude collagenase (with contamination of nonspecific proteases) is often used with a finely chopped tissue in complete medium. When tissue is disaggregated, collagenase is removed by centrifugation, and the cells may be seeded and cultured at a high concentration.

Mechanical Disaggregation:

The cell suspension is more quickly obtained than in the enzymatic disaggregation. The mechanical disaggregation is force based technique in which the tissue is carefully sliced and the cells, which spill out, are collected. In this technique the cells are either pressed through a syringe and needle, or repeatedly pipetted. Sometimes there may be mechanical damage of cells. The mechanical disaggregation provides good yield of cells in shorter time.

2. Separation of viable and Non-viable cells:

The dissociated cells obtained as above, usually described as primary cells. They grow well when seeded on culture plates at high density. These are known as adherent primary cultures in which the non-viable cells are removed at the first change of medium. The primary cultures can also be maintained in suspension in which the non-viable cells are gradually diluted out, when cell proliferation starts. The non-viable cells can be removed from primary disaggregate using centrifugation technique using a mixture of Ficoll and sodium metrizoate.