Selective culture of primary tumor cells: confluent feeder layer tumor cell screening assay

Summary

The feeder layer technique depends on the prevention of fibroblast overgrowth through other contacts that inhibit cell formation in the monolayer. The feeder layer technique does not selectively inhibit normal epithelial cells, W as both normal epidermal cells and normal mammary epithelium can form clones on confluent feeder layers. However, the results of the glioma study suggest that it is possible to selectively culture equivalent normal cells on the same feeder layer.

Operation method

Scheme 24.1 Confluent feeder layer tumor cell screening assay

Principle

At mid-exponential growth, feeder layer cells were treated with mitomycin C, and cells were cultured to form confluent monolayers. Tumor cells are isolated from biopsy specimens by collagenase digestion or obtained from primary cultures by trypsin digestion and then inoculated onto a confluent monolayer (Fig. 24.1). Epithelial tumor cells can form clones within 3 weeks to 3 months. Fibrosarcomas and gliomas do not always form clones but can invade the feeder layer and grow in a gradual transition.

Materials and Instruments

3T3 cells STO cells 10T1 2 cells
Growth medium Collagenase Trypsin
Scalpel Petri dish

Move

I. Materials

aseptic

1. feeder cells (e.g. 3T3, STO, 10T1/2 or FHS74Int)

2. 1 mg/ml mitomycin C (Sigma)

Caution:

When using feeder layer cells for the first time, it is advisable to perform a dose-response curve for mitomycin C to confirm that this dose will allow feeder layer cells to survive for 2 to 3 weeks, but will cause the cells to undergo a maximum of two multiplications before they continue to proliferate. In this case, proceed as follows:
(1) Cells in 25 ^cm2 culture flasks were treated with 1~100 μg/ml mitomycin C overnight (18 h);
(2) After digesting the cells with trypsin, all the cells in the culture flasks were re-inoculated into 75 ^cm2 culture flasks with 20 ml of fresh culture medium;
(3) The cells were cultured for 3 weeks, and the solution was changed twice a week;
(4) Stain the cells and examine the surviving clones.

2. Growth medium

3. 2000 U/ml collagenase, CLS grade (Worthington) or equivalent

4. 0.25% trypsin, prepared in PBSA

5. tumor biopsy specimens or primary cultures

6. scalpel with 22-gauge blade

7. Petri dish for dissection, same as Petri dish for primary culture

II. Procedure

1. In six 75 ^cm2 culture flasks, feeder cells are cultured to 80% confluence.

2. Add mitomycin C to a suitable final concentration, typically around 5 μg/ml.

3. Incubate cells with mitomycin C overnight (about 18 h).

4. Remove the culture medium containing mitomycin C and wash the cell monolayer with fresh culture medium.

5. Incubate the cells further for 24~48 h.

6. Digest the cells with trypsin and re-inoculate the cells into 25 ^cm2 culture flasks at a density of 5X105 cells/ml (1X105 ^cells/cm2 ). The cells were incubated for 24 h.

7. If using a biopsy specimen, cut the specimen and place it in collagenase in step 2.

8. Inoculate the cell suspension obtained from the specimen at 20-100 mg/vial into two 25 ^cm2 culture flasks, each containing 6 ml of cell suspension. Remove 1 ml of suspension from each culture flask and put it into two other culture flasks and add culture solution to 4 ml. The third pair of culture flasks was used as a control to check the viability of cells in the feeder layer after treatment with mitomycin C. The third pair of culture flasks was used as a control to check the viability of cells in the feeder layer after mitomycin C treatment.

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