Warm trypsin dissociation of tissues
Warm trypsin dissociation of tissues
The tissue was minced and stirred in trypsin for several hours, and the isolated cells were collected every half hour, centrifuged, and added to serum-containing medium.
Operation method
Scheme 12.5 Warm trypsin dissociation of tissues
Principle
The tissue was minced and stirred in trypsin for several hours, and the isolated cells were collected every half hour, centrifuged, and added to serum-containing medium.
Materials and Instruments
Tissue DBSS Crude Trypsin D-PBSA Move 1. Transfer the tissue (1 to 5 g) into a Picasso dish with freshly prepared sterile DBSS (50 ml) and wash. For more product details, please visit Aladdin Scientific website.
Growth medium Petri dishes
Culture flasks Centrifuge tubes Bottles Magnetic stirrers Tweezers Pipettes Hematocrits
2. Transfer to a 2nd Petri dish, excise excess tissue such as fat or necrotic tissue, and transfer to a third Petri dish.
3. Cross-cut with a scalpel into approximately 3 mm3 pieces. 
4. Transfer the tissue into pre-weighed centrifuge tubes or containers (two pre-weighed 50 ml centrifuge tubes or two conventional containers) using curved forceps.
5. Allow the tissue block to settle.
6. Resuspend and wash the block with DBSS to settle the block and remove the supernatant, and repeat this process 2 or more times.
7. Weigh the tube or container again.
8. Transfer all tissue blocks to an empty trypsin (2.5 % crude trypsin in D-PBSA or normal saline) digestion vial and rinse the container or centrifuge tube with D-PBSA (200 ml).
9. Remove excess liquid and add 180 ml of PBSA.
10. Add 20 ml of 2.5% trypsin (other enzymes including collagenase, hyaluronidase, deoxyribonuclease may be added if desired).
11. Add a stirrer (autoclaved in a test tube) to a conical flask (trypsin digestion bottle, 250 ml conical flask or mixing flask).
12. Seal the conical flask, place it on a stirrer and stir at 37°C in a warm box or greenhouse.
13. Stir for 30 min at 37°C at approximately 100 revolutions per minute.
14. After 30 min, collect the dissociated cells:
(a) Allow the tissue mass to settle.
(b) Aspirate the supernatant into a centrifuge tube and place on ice.
(c) Add fresh trypsin solution to the conical flask to digest the remaining tissue mass, continue stirring and incubate for 30 min .
(d) Centrifuge the cell suspension from step 14b at 500 g for 5 min to collect the cells.
(e) Resuspend the cells closed with 10 ml serum-containing medium and store in an ice bath.
15. Repeat the procedure in step 14 until the tissue mass is completely digested or no further digestion is visible (about 3~4 h ).
16. Collect the chilled cell suspension and count the cells with a blood counting plate or an electronic counter to check for cell viability.
17. The cell population is heterogeneous and, due to the difficulty of calibrating the caliber, the initial use of the electronic counter requires confirmation with a hemocyte counting plate.
18. Remove large aggregates by filtration through a sterile cotton cloth or homemade sieve. 
19. Dilute the cell suspension with growth medium (serum-containing growth medium (e.g., DMEM/F 12 plus 10% fetal bovine serum)) to contain 1 x 106 cells per mL of medium. Inoculate in culture flasks at approximately 2 x 105 cells per square centimeter. Cell counts are of little significance when viability is uncertain or unpredictable (e.g. tumor biopsies with a high percentage of dead cells). In such cases, it is recommended to inoculate at a concentration of 5-25 mg of tissue per milliliter.
20. Change the medium at regular intervals (2-4 days, depending on the pH drop). Check the supernatant for viable cells before discarding it, as some cells are slow to adhere or even easy to grow in suspension.