Chopped tissues were placed in complete medium containing collagenase for incubation, and after the tissues were broken down, collagenase was removed by centrifugation, inoculated and cultured at high concentrations.

Operation method

Scheme 12.8 Collagenase dissociation of tissues

Principle

Chopped tissues were placed in complete medium containing collagenase for incubation, and after the tissues were broken down, collagenase was removed by centrifugation, inoculated and cultured at high concentrations.

Materials and Instruments

Collagenase DBSS
Culture media Pipettes Pipettes Culture flasks Centrifuge tubes Scalpels Centrifuges

Move

1. Transfer the tissue to fresh, sterile DBSS and wash.

2. Transfer to another Petri dish (9 cm Picasso dish, non-tissue culture grade) and excise excess tissue, such as fat or necrotic tissue.

3. Transfer the tissue to a third Petri dish and finely chop it to approximately 1 mm³ with a crossed scalpel.



4. Use a 10-20 ml wide-mouth pipette to transfer the tissue into a 15 ml or 25 ml sterile centrifuge tube or a commonly used container (wet the pipette with DBSS first, otherwise the tissue mass will stick to the wall).

5. Allow the tissue mass to settle.

6. Resuspend the washed tissue in DBSS, remove the supernatant after the mass has settled, and repeat this procedure more than twice.

7. Inoculate 20-30 tissue blocks in a 25 ^cm2 flask and 100-200 blocks in another flask.

8. Aspirate the DBSS and add 4.5 ml of serum-containing medium (e.g. DMEM/F12 with 10 % fetal bovine serum) to each bottle.

9. Add 0.5 ml of crude collagenase (2000 U/ml, Worthington CLS or Sigma 1A) to a final concentration of 200 U/ml.

10. Incubate at 37°C for 4-48 h without shaking. Tumor tissues (e.g., sclerocarcinoma of the breast or colon) may act for 5 days or more due to slow decomposition. In some cases, due to excessive pH drop (pH < 6.5) over time, the tissue is centrifuged and resuspended with fresh medium and collagenase.

11. Gently shake the culture flask to check the separation: the tissue mass is distributed in spots at the bottom of the flask and disperses into single cells or small tissue masses when properly aspirated.



12. Small clumps of epithelial cells from some tissues (lung, kidney, colon, or breast) are collagenase-resistant and can be separated from the rest of the tissue after 2 min of precipitation. If they are resuspended in DBSS and the precipitate is inoculated into the culture medium after precipitation, islands of actively growing epithelial cells will form. Epithelial cells tend to survive better when not completely dissociated.

13. After complete dissociation or sedimentation of the tissue mass, collect the cells in the supernatant and centrifuge at 50~100g for 3 min (centrifuge tubes or conventional containers, 15~50 ml, depending on the amount of tissue treated).

14. Remove DBSS or medium supernatant, resuspend, add 5 ml of medium and inoculate into 25 ^cm2 culture flasks. If the pH drops during collagenase action (pH < 6.5 for 48 h), remove collagenase and dilute the cell suspension with 2x or 3x medium.

15. Replace the culture medium after 48 hours.

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