To obtain a suspension culture, disruption of cells has to be done and these cells grow to form primary culture. Primary culture is the original culture or the culture before first sub-culturing. The culture obtained after subculturing are cell lines and the products of cell line are referred to as secondary cultures.
Methods of Disruption
1. Physical or mechanical methods
2. Enzymatic methods
3. Chemical methods
1. Physical methods
Following are the steps involved in disruption of cells by physical methods
1. After careful removal of tissue from the spot, the tissue is kept aseptically in a sieve of 100 μm mesh size.
2. The sieve is placed in a petridish containing buffer medium Balanced or Basal salt solution.
3. Cells are alternately passed through sieves of decreasing pore size (50μm and 20μm mesh size). If desired the process of sieving is repeated to get more disaggregation of cells.
4. The debris remaining in the sieve is discarded and the medium containing cells is collected and cells are counted using haemocytometer.
5. Medium is diluted with serum to lower the level of cells to 104cells/mL. The viable dissociated cells are the primary cells. These are seeded on culture medium in high density and grown well. These primary viable cells of primary culture are known as adherent cells and the culture as adherent cell culture. The non-viable cells can be removed by centrifugation using ficoll and sodium metrizoate. Although this method is quick and cheap but it cause damage to many live cells.
2. Enzymatic methods
In these methods enzymes are used like mucase, papain, trypsin and collagenase. Among these trypsin and collagenase are most commonly used.
Enzymatic treatment by Trypsin : The use of Trypsin for cell disruption is known as trypsinization. There are two types of trypsinization
a) Warm Trypsinization
b) Cold Trypsinization
a) Warm Trypsinization Cells are exposed to warm enzyme (36.5°C) for one minute. The dissociated cells are collected every half an hour. The trypsin is removed by centrifugation after 3-4 hours which is required for complete disaggregation.
Steps involved in warm trypsinization
1. In this one tissue sample is chopped into two to three pieces and washed in distilled water keeping in glass vial. The pieces are transferred to 250ml flask containing 100ml of warm trypsin at 36.5°C.
2. The content is stirred for 4 hours; thereafter the pieces are allowed to settle. The dissociated cells are collected at every 30 minute. This facilitates the minute exposure of cell to warm enzyme. The process may be repeated by adding fresh trypsin back to pieces and incubating the contents. The trypsin is removed by centrifugation after 3-4 hours after which complete tissue may be disaggregated.
3. The glass vials containing dispersed cell pellets in medium are placed on ice. Cells counted using haemocytometer and cell density is maintained at 104cells/ml. These are plated and incubate for 48-72 hours for cell growth.
b) Cold Trypsinization It involves soaking of tissue at 4°C to allow penetration of enzyme followed by incubation at 36.5°C for a shorter period for the disaggregation of cells.
Steps involved in cold trypsinization
1. The tissue sample to be disaggregated is chopped into two to three pieces and kept in a small sterile glass vial. If necessary these may be washed with sterile water.
2. The pieces are removed from vials dissected while keep in BSS. The whole content is transferred in glass vial and is placed on ice and soaked in cold trypsin for 4-6 hours. This allows penetration of enzyme in tissue. Further trypsin is removed and tissue is incubated at 36.5°C for 20-30 minutes.
3. The vials that contain tissue pieces are poured with 10ml of medium containing serum and cells are dispersed by repeated pipetting. The cells are counted using haemocytometer. Cells density is maintained at 104cells/ml with dilution in growth medium. These are plated and incubated for 48-72 hours for cell growth.
Enzymatic treatment by Collagenase is used where trypsin has damaging effects like epithelial cells or fibrous tissue. Therefore, disaggregation of several embryonic normal and malignant tissues is better achieved by collagenase which readily digests away materials containing collagen. The different steps involved in disaggregation of tissue using collagenase are as follows
1. Biopsy tissues are kept in a medium containing antibiotics. It is then dissected into pieces in basal salt solution containing antibiotics.
2. The chopped tissue is washed properly with sterile distilled water and transfer to complete medium containing collagenase.
3. After 5 days of incubation the mixture is pipetted so that the medium may get dispersed when the whole treatment is left for sometimes. The cluster of cells settles at the bottom of the test tube.
4. The cluster present in test tube is washed either by setting or is centrifuged to make the dispersed cell suspension free of cells.
5. The cells suspension now consists of enriched fibroblast fraction which is plated out on medium.
6. The cluster which is washed by settling contains enriched epithelial fraction. It is also plated out on medium.
3.Chemical methods- EDTA treatment Some tissues like epithelium require calcium and magnesium ions for their integrity. Disaggregation of such tissues is readily achieved by treatment with EDTA (ethylene diamine tetraacetic acid) prepared in a basal salt solution. This treatment is used for the separation of cells from established cultures of epithelial tissue.