Trypsinisation and Subculturing of Cells
Remove gently all medium from the culture to be passaged with a sterile Pasteur pipette or by suction from a vacuum pump. The adherent cell layer should then be rinsed one to two times with HBSS without Ca 2+ /Mg 2+ to get rid of any FBS that may interfere with the trypsinization process. Add just enough trypsin/EDTA solu- tion to cover the culture plate/tray. Keep at 37 °C for 1–2 min. Tap the culture tray against the working plate of the laminated air fl ow cabinet, and inspect the cells in an inverted microscope to ascertain that the cells are rounded up and detached from the surface. It may be necessary to increase trypsinization up to 15 min. However, prolonged incubation with trypsin may reduce cell viability.
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Add a suffi cient volume of complete medium with FBS and draw the cell suspension into a Pasteur pipet. Rinse the plate carefully with the cell suspension to dislodge any residual cells. When all cells are detached, add more complete medium with FBS to inhibit residual trypsin activity that may damage the cells.
Count the cells in a haemocytometer, alternatively in a Coulter counter or similar device. Dilute the cells in complete medium and seed them at a suitable density in the tissue culture tray of choice.
If necessary, feed subconfl uent cell layers after 3–4 days by removing most of the spent medium and replacing it with fresh new medium at 37 °C. Follow the same protocol for subsequent subcultivations. Notify the number of passages for each culture.
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 trans- formed 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
Tissue fragment
Transfer to culture
Primary culture
Subcultivation Passage
Cell line
Immortalization
Transformation Clone
Continuous
cell line Transformed
cell line Isolation of
single cells
Further sub- cultivation
Senescence and death
Fig. 1 The fi gure describes the relationship and differences between primary cells, transformed and immortalized cell lines and clones
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do not grow to confl uence, 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 × 10 6 /ml, they should be split back to 2–3 × 10 5 cells/ml. An optimal cell concentration will usually be around 10 6 /ml.
If you are going to expand the cells to obtain large cell numbers, remove the fl ask from the incubator, swirl the fl ask gently to resuspend the cells and remove a small sample to measure cell density by counting in a hemocytometer or a Coulter coun- ter. Depending on cell concentration aseptically distribute the contents into new fl asks allowing a new cell concentration of 2–3 × 10 5 cells/ml.
Add fresh medium and ascertain that the height of the medium never exceeds 2 cm above the bottom of the fl ask or tray. This is important to allow proper buffer- ing of the medium and physiological pH for the cells sedimenting to the bottom of the fl ask.
If cells are growing slowly, they might be fed by aseptically removing 1/3 of the old medium to be replenished by fresh medium.
Freezing Cells
Both adherent and suspension culture cells can be frozen and stored in liquid nitro- gen 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 × 10 6 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.
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
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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.
The Thawing and Recovery of Cells
Cells can be stored in liquid N 2 , or in the gas phase over the liquid N 2 for long periods of time. However, valuable cells should be thawed and brought back into culture once a year to ascertain viability and function. Thawing is as important as the freezing process to maintain cell viability, and thawing should be carried out according to a rigorous protocol.
Frozen vials with cells are placed on ice directly from the liquid N 2 storage tank.
The vials should be immersed in a water bath at 37 °C and thawed leaving a small lump of ice in the vial before transfer of the vial to melting ice again. When fully thawed, transfer the contents of the vial into a tube and add slowly cold medium to dilute the DMSO in the freezing medium. It is very important that the cell suspension is kept cold during this procedure.
Spin the cells carefully down in a table top centrifuge, remove the DMSO con- taining medium and add a small volume of complete medium to resuspend the cells.
Take a small sample to monitor cell viability by trypan blue exclusion and light microscopy, or similar viability test (see Sect. 9.5 ). If cell viability is below 70 % it may turn out diffi cult to obtain proper cell growth. Reduced cell viability is most likely due to improper handling of cells either before freezing, during freezing or thawing of the cells.
Dilute with complete medium to the desired cell concentration, and add the cell suspension to a suitable culture vessel. Place in incubator. Monitor cell morphology and cell growth during the following days.
Cell Viability Testing
Assessment of cell viability is fundamental in all cell culture work. Cell viability will provide information about culture conditions, in general, and may also repre- sent the fi nal read-out system for a cytotoxicity experiment. A large number of via- bility assays are reported in the literature, ranging from the simple trypan blue exclusion test to complex analysis of individual cells with Raman spectroscopy.
Cell viability assays will often overlap with cell proliferation assays. They are divided into assays that monitors viability at the single cell level and those that analyses viability at the population level. The simplest and cheapest viability test exploits the vital stain trypan blue. Trypan blue is excluded from cells and tissues with intact membranes, and the staining of dead cells is easy to detect in a standard light microscope.
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Prepare a 0.8 mM trypan blue solution in phosphate-buffered saline (PBS). Mix cells and trypan blue solution in a 1:1 ratio. Inspect and count dead and alive cells under a light microscope in a haemocytometer. Dead cells will stain blue due to trypan blue uptake, while live cells appear transparent. Cells should not be exposed to trypan blue solution for more than 20 min. Prolonged incubation will increase cell death and reduce viability.
A variety of fl uorescent dyes can also be used to monitor cell viability, typical in the combination with fl ow cytometry. Propidium iodide and 7-actinomycin D are commonly used for this purpose.
Assessing cell viability at the population level has become very popular. These assays are typically carried out by using different tetrazolium compounds. Among the most popular are MTT ((3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), MTS, XTT and WST-1. Among these MTT is positively charged and readily penetrates viable cells, while the others are negatively charged and are excluded from viable cells. The MTT assay is perhaps the most popular to assess cell viability and proliferation in a population of cells. It was the fi rst assay to be developed for high trough put screening in a 96-well format. Viable cells convert MTT into a purple colored formazan product. When cells die, they lose ability to convert MTT into formazan. The mechanism behind this process is not well under- stood, although many publications suggest that the MTT assay refl ects changes in mitochondrial activity. Commercial kits containing MTT and a solubilization reagent can be obtained from several vendors like Sigma Aldrich, Promega and Millipore, among others.