The cut tissues were placed in trypsin at 4℃ for 16-18 h. After removing the trypsin, the cells were warmed and dispersed in warm medium.

Operation method

Program 12.6 Cold Trypsin Dissociation of Tissues

Principle

The cut tissues were placed in trypsin at 4°C for 16-18 h. After removing the trypsin, the cells were warmed and dispersed in warm medium.

Materials and Instruments

Tissue DBSS Crude Trypsin
Growth medium Petri dish
Culture flasks Tweezers Pipettes Conical flasks Scissors Ice baths

Move

1. Transfer the tissue (1 to 5 g, pre-weighed) into a Picasso Petri dish with freshly prepared sterile DBSS and wash.

2. Transfer to another Petri dish and excise excess tissue such as fat and necrotic tissue.

3. Transfer to a third Petri dish and finely chop into small pieces of approximately 3 mm³ using a crossed scalpel. If the organs of the embryo are smaller than 3 mm³, all of them may be retained.

4. Transfer the blocks of tissue with curved forceps into a pre-weighed 15 ml or 50 ml sterile centrifuge tube.

5. Allow the block to settle.

6. Resuspend and wash the block with DBSS, remove the supernatant after settling, and repeat this step more than twice.

7. Carefully remove remaining liquid and pre-weigh.

8. Add 10 ml of 0.25 % trypsin dissolved in RPMI1640 or S-MEM (0.25 crude trypsin dissolved in serum-free RPMI1640 or MEM/Stirrer salt (S~MEM )) per gram of tissue and place at 4 °C.

9. Place at 4°C for 6 to 18 h.

10. Carefully aspirate the trypsin and leave the tissue and trypsin residue (if necessary, 1-2 ml of other enzymes such as collagenase, hyaluronidase, deoxyribonuclease, etc. can be added at this time).

11. Place at 37°C for 20~30 min.

12. Add warm medium, approximately 1 ml for every 100 mg of initial tissue, and gently pipette the mixture up and down until the tissue is completely dispersed.

13. If some of the tissue is not dispersed, the cell suspension can be filtered through sterile plain gauze or a stainless steel sieve (100-200 μm), or a disposable plastic sieve, or large pieces of tissue can be allowed to settle. When more tissue is present, an additional 20 ml of medium per gram of tissue can be added to facilitate sedimentation and subsequent collection of the suspension. 2-3 min is usually sufficient to remove most of the large pieces of tissue.

14. Determine the cell density of the suspension using a blood counting plate or an electronic counter to check the viable cell ratio.

15. Cell populations are heterogeneous and are difficult to calibrate due to the caliber, thus requiring confirmation with a hemocytometer when using an electronic counter for the first time.

16. Dilute the cell suspension growth medium (growth medium (e.g., DMEM/F12 with 10% fetal bovine serum)) to contain 1 x ¹⁰⁶ cells per milliliter of medium. Inoculate as required in multiple culture flasks (25 ^cm2, 75 ^cm2 flasks) at approximately 2 x ¹⁰⁵ cells per square centimeter. Cell counting is of no value when viability is unclear 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 to 25 mg of tissue per ml.

17. 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.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/