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Subculturing Adherent Cells
Materials (All Materials/Solutions Are Sterile Unless Otherwise Noted)
1. Growth medium
2. Phosphate-buffered saline (PBS) (Ca2+/Mg2+ free)
3. Trypsin solution (0.05% (w/w) tissue-culture-grade trypsin, 0.53 mM EDTA; unless otherwise noted, this is the concentration used)
4. Culture dishes (100 mm)
5. Hemacytometer or electronic particle counter Procedure
1. Remove medium from the plate. If the cell line adheres tightly, the medium may be discarded. If many cells in the plate are floating or only loosely attached, the plate should be gently shaken or washed with a pipette and the loose cells saved and recombined with the trypsin-dispersed cells before replating. If this is not done, the overall phenotype of the culture will change with time, as each passage will preferentially select for the more tightly adherent cells.
2. Wash 1 x with 5 ml of PBS.
3. Add 2"C3 ml trypsin. Allow the trypsin to cover the plate. Tilt plate and remove excess trypsin.
4. Incubate at 37jaC. The time will vary depending on the cell type and whether it is a primary culture or an established cell line, but generally it will take 2"C3 min. It is a good idea to remove the plate from the incubator and look to see if the cells have rounded up after about 2 min and check every minute thereafter. If the cells slough off the plate when its side is gently tapped against the bench, they are ready. Do not trypsinize beyond the time required to detach cells to this degree, since this will damage the cells and may reduce plating efficiency.
5. When the cells have rounded up and are coming off the plate, resuspend in 5 ml of serumcontaining medium and wash cells by centrifugation at 800 rpm. Resuspend in 5 ml
medium. [This wash step may be omitted if the cells are to be split at a high split ratio (> 1:50) in serum containing medium.] If using serum-free media, the trypsin should be neutralized with 1 ml of a 1 mg/ml solution of soybean trypsin inhibitor (STI), diluted to 10 ml with medium, and centrifuged in a clinical centrifuge for 3"C4 min at 900 rpm. You must wash the cells if culturing serum free. This wash removes any residual enzyme and removes the STI, which can prevent attachment of some cells. The supernatant is then aspirated and the pellet resuspended by repeated pipetting in 5 ml of growth medium.
6. If the cells are primary or secondary cultures or the cell line is one with which you have had no experience, a high seeding density is recommended, i.e., 2.5 ml of cell suspension to 7.5 ml of growth medium (a 1:4 split ratio). If precise cell counts are needed or the required seeding density is known, an aliquot of the cell suspension should be counted at this time. It is a good idea to seed replicate stock plates at several densities, for example, 1x, 1/2, 1/5x, and 1/10x the density chosen (or in the example above, 1:4, 1:8, 1:20, and 1:40 split ratio).
Subculturing Suspension Cultures
Cultures of cells that grow in suspension in flasks or spinners can be maintained by diluting an aliquot of the suspension into fresh growth medium. See Chapter 11 for information on adapting attached cells to suspension culture and growing cells in spinners.
Materials (All Materials Are Sterile)
1. Flasks (75 cm2)
2. Growth medium
3. Hemacytometer or electronic particle counter Procedure
1. Hold the flask upright and pipette the cell suspension up and down two or three times to disperse any clumps.
2. Either remove an aliquot for counting or, if precise counts are not required, transfer 200 ^l to 1 ml of the suspension to a fresh flask containing 10 ml of growth medium. If a split ratio is less than 1:10, the appropriate volume of cell suspension should be placed in a 15 ml conical tube, diluted to 10 ml with medium, and centrifuged at 900 rpm for 3"C4 min. Resuspend the resulting cell pellet into fresh growth medium and aliquot the appropriate number of cells into the number of flasks needed. In this way, one can avoid diluting fresh medium with exhausted medium components or carrying over toxic cell metabolites or proteases. Note: While many published methods suggest washing or diluting cells in PBS, we prefer to use serum-free medium (outdated medium can be used here). This seems to improve viability, especially for cells carried continuously in serum-free medium and when handling "delicate" or "picky" cells. Using serum to wash cells also prevents any rapid changes in osmolarity that may occur when switching from medium to PBS and back and maintains an energy source for the cells. Basically, the cells are less stressed during an already difficult period.
Growth Curves and Measuring Cell Growth
In order to analyze the growth characteristics of a particular cell type or cell line, a growth curve can be established from which one can obtain a population doubling time, a lag time, and a saturation density.
A growth curve generally will show the cell population's lag phase, that is, the time it takes for the cell to recover from subculture, attach, and spread; the log phase, in which the cell number begins to increase exponentially; and a plateau phase, in which the culture becomes confluent and the growth rate slows or stops (Fig. 5.1). An increase in cell number is also a frequently used method of assessing the effect of hormones, nutrients, and so forth on a specific cell type. Growth, or increase in total cell number over time, is a good measure of a biological response because it is so broadly defined and influenced by many different factors, including mitogens, changes in nutrient level, transport, membrane integrity, attachment factors, and so forth. However, merely seeing a difference in cell number or some secondary endpoint such as [3H]thymidine incorporation, dye uptake, and so on gives only a limited amount of information. A factor can affect cell number by changing attachment, shortening or lengthening the lag phase, changing the plating efficiency (or survival at subculture), changing the death rate, changing the rate of progression through the cell cycle, or changing the plateau density. A growth curve will help differentiate between these effects.