The culture animal material is washed in balanced salt solution to avoid contamination.
The tissue to be
cultured should be properly sterilized with 70% ethanol and removed surgically under aseptic conditions.
Disaggregation of tissue To obtain the cell suspension for primary cell culture, the tissue is
disintegrated either mechanically or by using enzymes.
(i) Physical or mechanical disaggregation- After removing the tissue under aseptic conditions, it is
pressed through a sieve of 100 micrometer. It is then kept in a sterile Petri dish containing buffered
medium with balanced salt solution. The cells are then alternately passed through the sieve of
decreasing pore size (50 micrometer and 20 micrometer mesh). The debris which remains on the sieve
is discarded and the medium containing cells is collected and cells are counted by using
haemocytometer. This method is cheap and quick but it damages a lot of cells.
(ii) Enzymatic disaggregation- In this method, enzymes are used for dislodging the cells of tissues. The
two important enzymes used in tissue disaggregation are-collagenase and trypsin. -a) Collagenase- The
intracellular matrix contains collagen therefore collagenase is used for disaggregation of embryonic,
normal as well as malignant tissues. The tissues are kept in medium containing antibiotics and then
dissected into pieces in basal salt solution. After washing the chopped tissue with distilled water, it is
transferred to complete medium containing collagenase. After a few days (around 5 days), the mixture
is pipetted so that the medium gets dispersed. The whole treatment is left for sometimes during which
the epithelial cells settle on bottom of test tubes. The enzyme collagenase is removed by centrifugation.
Suspension consists of cells which are then plated out on the medium. (b) Trypsin- Use of trypsin for
disaggregation is called trypsinization. On the basis of role of temperature on trypsin, the activity of
trypsin is of two types- Cold trypsinization and warm trypsinization.
Cold trypsinization- The sample tissue to be disaggregated is chopped into 2-3 small pieces and kept in
sterile glass vial. The tissues are subsequently washed with sterile water and dissected and then kept in
BSS. The whole content is then placed on ice and soaked in cold trypsin for 4-6 hours to allow the
penetration of enzymes in tissue. After this the trypsin is removed and the tissue is incubated at 36.50C
for 20-30 minutes. About 10 ml of medium containing serum is added to the vials containing the cells
and the cells are dispersed by repeated pipetting. The cells are counted by haemocytometer and are
plated and incubated for 48-72 hours for cell growth.
Warm trypsinization- The initial steps are the same as in cold trypsinization however, in this case the
tissue pieces are treated with warm trypsin (36.50C). The tissues are stirred for 4 hours and then pieces
are allowed to settle down. The disassociated cells are collected at every 30 minutes. The process is
repeated by adding fresh trypsin back to pieces and incubating the contents. The trypsin is removed by
centrifugation after 3-4 hours during which the complete disaggregation of tissues takes place. The glass
vials containing dispersed cells are then placed on ice. The cells are counted using haemocytometer and
cell density is maintained at an appropriate number. The cells are then plated on medium and incubated
for 48-72 hours for cell growth.
(iii) Treatment with chelating agents- The tissues like epithelium (which needs Ca2+ and Mg2+ ions for
its integrity are treated with chelating agents such as citrate and ethylene-diamine-tetra-acetic acid
(EDTA). Chelating agents are mainly used for production of cell suspensions from established cultures of
epithelial type.
