4 General Cell Culture Methods
37
Procedure
1. Replace the culture medium with a digesting solution.
2. Allow digestion to proceed for an appropriate period of time. The conditions for digestion are different for different enzymes (see
subprotocols 1-3 in this chapter).
3. Stop digestion and detach the cells by gentle pipetting. When trypsin
is used, observe cells frequently under a microscope during the digestion period, and stop the digestion by adding protein-containing solution (e.g., used culture medium removed at step 1) when substrateattached cells with spread cytoplasmic projections become rounded
by retracting their cytoplasmic projections.
4. The resulting cell suspension is centrifuged at 150 g for 5 min.
5. Discard the supernatant.
6. Suspend the cells in fresh culture medium, and dispense to several
vessels.
7. Add fresh culture medium to each vessel to adjust the cell density to
the desired level.
Results
When a subculture is made, the cell density is reduced. The subcultured
cells will resume multiplication, although some may have been injured
during the subculturing process and may die. If there are many dead
cells or cell debris in the culture, it is better to change the medium after
all cells have settled. In the first several passages following primary culture, cell growth after subculturing is very slow, and the intervals between subcultures are long and variable. However, with advancing passages, the intervals between subcultures become shorter, and cells are
able to be subcultured at regular intervals. This can be used as a criterion for judging the cell line as a continuous cell line. When a new continuous cell line is obtained, it should be reported in an appropriate journal. The report should preferably contain the culture history, and some
characteristics of the cell line, e.g., multiplication rate, karyotype, isozyme
pattern, and sensitivity to viruses.
Subprotocol 5
Hybrid-Cell Technique for Obtaining Growing Cells
In general, cells differentiated to have specific functions scarcely proliferate. In contrast, cells that make up continuous cell lines have the potential to proliferate without limitation, but are in a rather undifferentiated state. Then, the idea of making a hybrid between differentiated cells
and continuous cell lines arises in order to obtain a continuous cell line
with specific function. This strategy can also be applied to obtain prolif-
37
Procedure
1. Replace the culture medium with a digesting solution.
2. Allow digestion to proceed for an appropriate period of time. The conditions for digestion are different for different enzymes (see
subprotocols 1-3 in this chapter).
3. Stop digestion and detach the cells by gentle pipetting. When trypsin
is used, observe cells frequently under a microscope during the digestion period, and stop the digestion by adding protein-containing solution (e.g., used culture medium removed at step 1) when substrateattached cells with spread cytoplasmic projections become rounded
by retracting their cytoplasmic projections.
4. The resulting cell suspension is centrifuged at 150 g for 5 min.
5. Discard the supernatant.
6. Suspend the cells in fresh culture medium, and dispense to several
vessels.
7. Add fresh culture medium to each vessel to adjust the cell density to
the desired level.
Results
When a subculture is made, the cell density is reduced. The subcultured
cells will resume multiplication, although some may have been injured
during the subculturing process and may die. If there are many dead
cells or cell debris in the culture, it is better to change the medium after
all cells have settled. In the first several passages following primary culture, cell growth after subculturing is very slow, and the intervals between subcultures are long and variable. However, with advancing passages, the intervals between subcultures become shorter, and cells are
able to be subcultured at regular intervals. This can be used as a criterion for judging the cell line as a continuous cell line. When a new continuous cell line is obtained, it should be reported in an appropriate journal. The report should preferably contain the culture history, and some
characteristics of the cell line, e.g., multiplication rate, karyotype, isozyme
pattern, and sensitivity to viruses.
Subprotocol 5
Hybrid-Cell Technique for Obtaining Growing Cells
In general, cells differentiated to have specific functions scarcely proliferate. In contrast, cells that make up continuous cell lines have the potential to proliferate without limitation, but are in a rather undifferentiated state. Then, the idea of making a hybrid between differentiated cells
and continuous cell lines arises in order to obtain a continuous cell line
with specific function. This strategy can also be applied to obtain prolif-