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Passaging of Cells in Suspension Culture
Although most primary cells and cell lines are plastic adherent, some cell types are non-adherent. Typically, primary immune cells like B and T lymphocytes and transformed lymphoid cells, NK cells and granulocytes grow in suspension. It is easier to passage cells growing in suspension than adherent cells since no trypsinization or detachment from the tissue culture plastic is necessary. Cells in suspension culture
Fig. 2 The drawing shows a rubber policeman (left) and a typical cell scraper to collect adherent cells from the surface of the tissue culture plastic ware
do not grow to confluence, and different cells may tolerate growing at different densities. Thus, optimal cell concentration has to be determined in each instance. Also, seeding densities may vary. Some cells are able to grow at very low densities while others need support from accompanying cells and have to be reseeded at higher densities. As a rule of thumb, when the cells reach a density of around 2 × 106/ml, they should be split back to 2–3 × 105 cells/ml. An optimal cell concentration will usually be around 106/ml.
If you are going to expand the cells to obtain large cell numbers, remove the flask from the incubator, swirl the flask gently to resuspend the cells and remove a small sample to measure cell density by counting in a hemocytometer or a Coulter counter. Depending on cell concentration aseptically distribute the contents into new flasks allowing a new cell concentration of 2–3 × 105 cells/ml.
Add fresh medium and ascertain that the height of the medium never exceeds 2 cm above the bottom of the flask or tray. This is important to allow proper buffering of the medium and physiological pH for the cells sedimenting to the bottom of the flask.
If cells are growing slowly, they might be fed by aseptically removing 1/3 of the old medium to be replenished by fresh medium.
Both adherent and suspension culture cells can be frozen and stored in liquid nitrogen for extended periods of time. In general, the same protocol can be used for both cell types.
Harvest the cells and spin them down in a table top centrifuge. Resuspend the cells carefully with a Pasteur pipette in cold medium containing 10 % FBS to a concentration of around 5 × 106 cells/ml. Keep tube or bottle with cells on ice in the sterile hood. Add a similar volume of cold freezing medium dropwise to the tube with cells while gently swirling the tube.
Distribute the cell suspension in 1 ml aliquots in 2 ml cryovials. Tighten caps properly. Place vials in a styrofoam box and place in a −80 °C freezer overnight before transfer to liquid nitrogen storage tank.
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