Oral keratinocytes
Oral keratinocytes
This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)
Operation method
Program 23.10 Oral keratin-forming cells
Materials and Instruments
D-PBSA 0.17% trypsin PET Move I. Primary culture For more product details, please visit Aladdin Scientific website.
Transfer medium Growth medium Growth medium with antimicrobials Scalpel and No. 11 blade Ophthalmic scissors Ophthalmic forceps 2 50mm or 100mm culture-grade Petri dishes 35mm and 100mm non-culture-grade Petri dishes 15 ml conical centrifuge tubes Microfuge Microfillers 50mm Petri dishes coated with FN C BSA
1. Take surgically excised or early biopsy tissues, put the tissues into transfer culture (Leibowitz L-15 culture with 100 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml amphotericin B), and send them to the laboratory as soon as possible.
2. Transfer the tissues into 100 mm Petri dishes and wash with P-PBSA (Dulbecco PBSA without Ca2+ and Mg2+ ).
3. Remove as much connective tissue and blood as possible. If the surface area of the tissue specimen is ≥1 cm2, cut it into small pieces.
4. Place the tissue block into a 35 mm dish and add enough 0.17 % trypsin solution (prepared with P-PBSA) to cover the block. Incubate at 4°C overnight.
5. Fix the block with one forceps and use the other forceps to strip the epithelium into the trypsin solution.
6. Gently grind the tissue suspension several times to further disperse the cells. Then, transfer the cell suspension into a centrifuge tube.
7. Soak the petri dish with D-PBSA and add the soaking solution to the cell suspension. Use the same amount of trypsin solution as in Step 4 Tissue Digestion.
8. Aspirate one aliquot with a micropipette to determine cell yield.
9. Centrifuge (125 g) at 4°C, preferably in a refrigerated centrifuge. Suspend the cells in growth medium.
10. Dilute the cell suspension with antimicrobial-containing growth medium (100 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml amphotericin B in the growth medium) as required, and inoculate the cells into FN/C/BSA-coated 50-mm dishes at a density of 5 × 103cells/cm2.
11. The culture medium was changed after 24 h to remove cell debris and erythrocytes.
12. Change the culture medium every other day.
Passage culture
13. Wash the cells with D-PBSA.
14. Add PET solution (containing 1 % polyvinylpyrrolidone (PVP, 40 KDa), 0.5 mmol/L EGTA and 0.025 % trypsin, prepared in D-PBSA), completely covering the cells, e.g., 3 ml in a 100 mm dish.
15. Incubate at room temperature until the cells become rounded or de-attached from the dishes. Observe cell de-adhesion under a microscope, which typically takes 1.5 min. The efficiency of de-adhesion can be improved by adding high concentrations of trypsin or by digesting at 37°C.
16. When most of the cells have de-attached, flick the dish and blow the cells with trypsin solution to mechanically enhance de-attachment.
17. After the cells are de-attached, terminate trypsin digestion by adding 5-10 ml of D-PBSA to the culture dish.
18. The cell suspension was injected into a centrifuge tube and gently ground to mix the cells well.
19. Take the cell suspension with a 1 ml pipette, add it to a blood cell counting plate and count the cells.
20. Calculate the cell density in the suspension.
21. Centrifuge at 4°C (125 g for 5 min).
22. Aspirate the supernatant and flick the tube with your finger until the sunken cells are dispersed.
23. Add an appropriate amount of growth medium and gently mix the cells with a pipette, then inject the cell suspension into a petri dish.
24. Place all petri dishes into a shallow dish and shake the dish by moving the dish by hand and changing the direction of movement. Care should be taken to ensure that the density of cells in each dish is uniform, i.e., concentrated in the center of the dish when shaking it. In addition, care should be taken to ensure that the rack on which the dishes are placed is flat and fixed to the incubator, and that the incubator is not vibrated to avoid uneven distribution of cells during wall attachment.
25. Leave the cells to incubate for 4~24 h, and then change the culture medium.