Experiments on the isolation and culture of colonic crypts
Experiments on the isolation and culture of colonic crypts
Although there have been many reports of continuous cell lines for human colon cancer, there are fewer reports of normal intestinal epithelial cultures.Owens et al. were able to isolate cells from fetal intestinal epithelium to establish a limited cell line (FHS74Int). The murine intestinal epithelial cell line IEC-6 has been widely used. This protocol is simplified from the method of Booth and O'Shea. For a detailed description, different methods and applications one should consult their method.
Operation method
Experiments on the isolation and culture of colonic crypts
Principle
Digest the chopped tissue blocks with collagenase and dispase, and then separate the crypts with sorbitol.
Materials and Instruments
HBSS PSG Digestive Mix Growth Medium S-DMEM Move makings The above liquid should be at 37℃. 5. 90 mm Petri dishes For more product details, please visit Aladdin Scientific website.
90 mm Petri dishes Plain containers 24-well plates Cling film Culture flasks Moveue pipettes
non-sterile
1. HBSS/PSG: HBSS with lOOU/ml penicillin, 3ug/ml streptomycin and 25ug/ml gentamicin.
2. Digestion mix: containing 150U/ml type XI collagenase, 40ug/ml dispase and 1% FBS, prepared in DMEM.
3. growth medium: DMEM with 25 mmol/L glucose, 6.8 mmol/L pyruvate, 0.25 U/ml insulin, 50 ng/ml EGF, 2.5% FBS, and antimicrobials in the above HBSS, and pH adjusted to 7.4 with sodium bicarbonate at 7.5% CO2.
4. S-DMEM: growth medium containing 2% sorbitol, filter sterilized
6. Plain container or 30-50 ml centrifuge tube
7. 24-well culture plate, pre-coated with collagen:
(a) Dilute 10X Vitrogen stock solution;
(b ) Add 0.5 ml of Vitrogen to each well and incubate for 3 h. The plates were pre-coated with collagen;
(c) Pipette off the excess Vitrogen and place the plate on a laminar flow ultra-clean bench and air dry.
8. The plates can be wrapped in plastic wrap (Saran Wrap) and stored at 37℃ for use within 1 week.
9. 25 cm2 culture flasks
10. scalpel (#4 handle, #22 blade)
11. Moveue pipette or similar large-bore pipette
II. Procedure
Isolation of human colonic crypts
1. Place the tissue in DMEM as soon as possible after removal of the colon from the patient. Preliminary studies have shown that colon tissue can be stored at 4°C for a short period of time (2 h) as long as it is placed in DMEM as soon as possible, but should not be stored for longer periods of time.
2. Wash the colon tissue twice with 1X HBSS/PSG to remove contaminants.
3. Transfer the colon tissue to a clean Petri dish containing HBSS/PSG.
4. Remove as much smooth muscle as possible and cut the tissue into small pieces using two opposing scalpels.
5. Transfer the tissue blocks into 25 cm2 culture flasks containing 20 ml of HBSS/PSG and rinse the blocks by blowing with a Movette pipette.
6. Allow the block to sink and then aspirate the HBSS/PSG.
7. Repeat rinsing until HBSS/PSG is clear. Repeat rinsing 5 times.
8. Transfer the tissue block to the Petri culture muscle and aspirate off excess HBSS/PSG.
9. Trim the tissue block with a scalpel so that the surface of the block is fairly flat. Use a large-bore pipette to transfer the block into a 25 cm2 culture flask. The inside of the pipette must be wetted with HBSS/PSG in advance to prevent the block from sticking to the pipette.
10. Allow the tissue block to stand and then aspirate off the excess HBSS/PSG.
11. Add 25-30 ml of mixed digestive solution.
12. Incubate for 1 h at 37°C.
13. Replace the mixed digestive solution:
(a) Let the tissue sink.
(b) Gently aspirate the mixed digest from the culture flask without aspirating the undigested tissue pieces.
(c) Add the mixed digestive solution and continue to incubate for 1 to 2 h at 37℃.
(d) Check the tissues every 15-30 min without over-digesting the crypts. The digestion process is completed when about 70% to 80% of the crypts are separated.
Do not wait until all crypts have been separated, as overdigestion will result in fewer crypts being obtained.
Sedimentation of human colonic crypts
Purify the isolated crypt foci by sorbitol sedimentation. Larger agglutinates of fat and mesenchymal tissue are seen at the top of the sedimentation tube. Since the agglutination interferes with the sedimentation, the agglutination should be removed in the first few steps.
14. Dispense the digested tissue suspension from the culture flask into two sterile 30 ml centrifuge tubes labeled "Set1".
15. Add S-DMEM to the top of the tissue suspension to 25 ml and remove any floating fat or mesenchymal tissue.
16. Allow the tissue suspension to stand for 1 min to allow undigested tissue to settle.
17. Remove any lipidic material floating on top and gently transfer the upper suspension to two centrifuge tubes labeled "Set 2".
18. Allow the suspension to stand for 30s.
19. Transfer the upper suspension to two centrifuge tubes labeled "Set 3".
20. Add S-DMEM on top of the suspension.
21. Centrifuge (200-300 g, 4 min)
22. Aspirate the supernatant.
23. Flick the bottom of the centrifuge tube to disperse the aggregated crypts.
24. Repeat the suspension of crypts with S-DMEM and centrifugation. the S-DMEM wash and centrifugation steps must be repeated approximately 5 times until the supernatant is clear.
25. The settled tissue in the Set 1 and Set 2 tubes should be examined under a microscope for crypts. If it contains a large number of crypts, shake the settled tissue well and repeat the sedimentation from step 14. If only undigested tissue is present, it may be discarded.
26. When the supernatant becomes clear, it is ready for implantation of crypts. For good cell growth, a suitable implantation density is required. In general, the best crypt growth occurs when the cells are grown at a density of 800-1000 cells/ml per well (24-well plate). Determine the crypt density as follows.
(a) Suspend the isolated crypts with 10 ml of growth medium and shake well.
(b) Remove 100ul and add 900ul of growth medium.
(c) Take 100ul of suspension from the upper solution and count the crypts. Number of crypts per ml of suspension = Number of crypts counted X 100
27. Incubate at 37°C and 7.5% C02 for 2d to allow the cells to adhere to the wall.
28. Aspirate the culture medium containing unattached cells from the plate and replace it with fresh culture medium.
29. Replenish with 0.5 ml of fresh culture medium every 5 days.