Following passaging culture, cells assume growth forms characterized by delayed, exponential or logarithmic, and stationary (plateau) phases. The logarithmic and plateau phases provide important information about the cell lineage, while the exponential growth phase gives their PDT and the plateau phase gives the maximum cell density (i.e., saturation density).Measurement of the PDT can be used for quantitative studies of the responsiveness of the cells to different inhibitory factors or stimulating culture conditions, such as varying nutrient concentrations, hormonal effects, or toxic drugs. It is also a good monitor for continuous passaging. It counts the cell yield, as well as the dilution required for passaging.

Operation method

Scheme 21.7 Experiments for plotting growth curves of cells cultured in monolayers in culture flasks

Principle

Start a series of cultures, counting cells at certain times each day until they reach plateau.

Move

makings

non-sterile
Monolayer cell culture, A549, Vem or HeLa-S3, 75 ^cm2 culture flask, 1 bottle in late log phase

0.25% crude trypsin mixed with lOmmol/L EDTA 5 ml

Growth solution with 2 mmol/L _NaHC03 200 ml

D-PBSA (for washing and cell counting) 50 ml

25 ^cm2 culture flasks 24 pcs.

Procedure

1. For normal passaging, first digest the cells with trypsin (see protocol 13.2).

2 . Dilute the cell suspension to ^2X104 cells/ml, 150 ml medium, respectively.

3 . Inoculate in 24 25 ^cm2 culture flasks.

4. Seal the flasks and place them in a 37°C incubator.

The use of low carbonate (4 mmol/L) medium is convenient for this protocol, but if the carbonate concentration is too high (e.g., 23 mmol/L), pass through 5% C02 or place in a C02 incubator with a gas permeable lid.

5. After 24 h, remove the first 2 bottles of cells from the incubator and count the number of cells:

(a) Completely aspirate each vial of culture fluid;
(b) Add 2 ml of trypsin/EDTA to each bottle;
(c) Incubate the bottles for 15 min;
(d) Cells were detached with trypsin/EDTA and 0.4 ml of cell suspension was taken into 19.6 ml of D-PBSA for counting;
(e) Count the cells with an electronic cell counter.

6 . Repeat sampling at 4, 8 and 72 h as in step 5.

7 . Change the culture solution at 72 h or earlier, depending on the decrease in PH (see Scheme 13.1; Fig. 22b).

8. For fast-growing cells (i.e., cells with a PDT of 12-24 h), sampling can be done continuously every day, but for slow-growing cells (i.e., cells with a PDT > 24 h), sampling should be done every two days until the cells reach a plateau.

9 . Change the medium every 1, 2, or 3 days, depending on the pH change.

10. Stain cells in a culture flask on days 2, 5, 7, and 10 (see section 16.4.2).

It is also possible to count the cells using a hemocytometer plate, but this is difficult at low cell concentrations, especially at the beginning of the growth curve. If a hemocytometer plate is to be used, reduce the volume of trypsin to 0.5 ml, carefully dispense the cells with a micropipette so that no air bubbles are generated, and then transfer the cells to the hemocytometer plate.

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