Cells are inoculated at low density, cultured until colony formation, stained (for clone formation rate and survival assays ) or used for screening when cells are isolated and then expanded into cell lines.

Operation method

Scheme 14.1 Clonal culture by dilution method

Principle

Cells are inoculated at low density, cultured until colonies form, and cells are stained (for clone formation rate and survival assays ) or used for screening when cells are isolated and then expanded into cell lines.

Materials and Instruments

CHO Cells Trypsin
Culture medium Straws Petri dishes Hematocrit

Move

1. Cells are digested with trypsin (trypsin, 0.25 %, 1 : 250 or equivalent activity) to prepare single cell suspensions. Insufficient digestion results in the formation of cell clusters; excessive digestion reduces cell activity. For clonal culture the cells in single suspension are the basis. If this is the first time a new cell line is cloned, it is necessary to feel out the digestion time and concentration to ensure that the cells are digested into single suspensions for optimal clone formation.



2. During cell digestion, the dishes are numbered (written on the bottom of the dish), the medium for each dilution step is assigned (medium: Ham F12, 5 % _CO2 equilibrium, 10 % FBS), and four dilutions may be necessary to dilute the original monolayer of cells to a concentration suitable for cloning.



3. When the cells become round and begin to detach, terminate the digestion by adding medium containing serum or trypsin inhibitors to disperse the cells.

4. For cell counting, dilute the cell suspension to ^1×105 /ml and then to a concentration that produces 50 colonies per dish. For CHO cells (25 ^cm2 CHO cells, late logarithmic growth), this is 10 cells per ml and the dilution step is:



(a) Dilute the digested cells to ^1×105 /ml (approximately 1 : 10 or 1:20, depending on the number of cells in the culture flask).

(b) Take 200 μl of ^1×105 cells/ml and add to 20 ml (1:100) to make ^1×103 cells/ml.

(c) Take ^1×103 cells /ml 200 μl and add to 20 ml (1:100) to become 1×10 cells/ml.

If it is desired to vary the clonal culture conditions, such as a range of serum concentrations, different sera or growth factors, prepare a series of tubes at this time and add 20 μl of ^1×103 cells /ml to each tube separately.

If this is the first cloning culture to determine the rate of clone formation, choose to experiment with 10, 50, 100, 200, and 2000 cells per milliliter.

5. Inoculate 3 dishes with 5 ml of medium per dish containing the final dilution concentration of cells. It is also recommended to inoculate dishes with 2000 cells per ml as a control to confirm that the cells have been added in case clones do not form at a lower concentration.

6. Place the dishes in a clear plastic box.

7. Place the plastic box in a humidified _CO2 incubator or in a vented airtight container (2 % ~10 % _CO2 ).

8. Leave to incubate for one week and if colonies form, do the following:

(a) Clone formation rate analysis; stain and count colonies.

(b) Clone screening; isolation of individual colonies.

If there are no visible colonies, change the medium and incubate for another week, if there is still no colony formation, also change the medium and incubate for a third week, if no more colonies appear, there is no longer a possibility of colonization.

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