Epidermal keratin-forming cell model construction experiment
Epidermal keratin-forming cell model construction experiment
Cultured epidermal keratinocytes are used as (1) models of differentiation, ideal tissue constructs for organotypic cultures, and studies of cell-cell interactions, and (2) grafts for burn or trauma repair.
Operation method
Epidermal keratin-forming cell model construction experiment
Principle
The epidermis was separated from the dermis by enzymatic digestion, and then keratin-forming cells were isolated and cultured in serum-free culture medium or on feeder layer cells with arrested growth.
Materials and Instruments
Skin Tissue Move I. Materials Caveat 1. Sampling: Keratinocytes isolated from the foreskin of newborns and young children are superior to those isolated from adult trunk skin in terms of adherence and proliferation. 2. Cryopreservation is often required to preserve large numbers of cells isolated from the same tissue. For more product details, please visit Aladdin Scientific website.
Thermolysin Trypsin SKDM Keratinocyte growth medium D-PBSA EDTA Analytical grade glycerol dispase
Cryopreservation tubes Nylon mesh Centrifuge tubes Cell filters Scalpels Curved forceps
aseptic
Prepare the feeder layer 3 days before the experiment (see Scheme 23.4). Irradiate 3T3 cells (30 Gy) and human fibroblasts (70 Gy), preferably with a suspension containing more cells.
FAD: A high-calcium culture medium developed by Wu et al. [1982] for feeder layer cultures containing Ham's F12, DMEM, and additives. 1.8Xl0-4mol/L adenine, 10-10 mol/L cholera toxin, 1-10 ng/L EGF, 0.4ug/ml hydrocortisone, 5%-10% hydroxycortisone, and 1.4ug/ml hydroxycortisone are added to a 1.8Xl0-4mol/L culture mixture (1:3). cortisone, 5%-10% FCS, 100 U/ml penicillin and 50ug/ml streptomycin.
Keratinocytegrowth medium (KGM ): Serum-free medium based on the MCDB153 medium developed by Boyce and Ham [1983] (see Table 10.1 and Appendix II), supplemented with low-calcium (0.03-0.5 mmol/L) bovine pituitary extract. CaCl2 may be added to elevate calcium ion concentration
Keratinocyte-defined medium (KDM): a serum-free medium based on the MCDB153 medium developed by Boyce and Ham in 1983 (see Appendix 1), which is a chemically defined medium that has been tried in organotypic cultures by Stark et al [1999].
SKDM (supplemented KDM) has been tried in organotypic cultures by Stark et al [1999]:
(a) KDM without pituitary extract (Promocell, Clonetics);
(b) Addition of 5ug/ml insulin, 0.5ug/ml hydrocortisone, 0.1ng/ml rhEGF, 20ug/ml rhEGF, 0.5ug/ml hydrocortisone, 0.1ng/ml rhEGF, 20ug/ml rhEGF.
(b) Add 5ug/ml insulin, 0.5ug/ml hydrocortisone, 0.1ng/ml rhEGF, 20ug/ml transferrin, 0.1% high purity serum albumin (endotoxin and fatty acid free, e.g., A-8806, Sigma) and 50ug/ml ascorbic acid;
(c) Calcium ion concentration was adjusted to 1.3 mmol/L .
(d ) Primary keratinocytes can be cultured in SKDM in uncoated plastic dishes, however, the proliferative activity of the cells is low. The growth of keratinocytes was improved when they were inoculated on dishes coated with type I collagen.
When cultured in organoids on collagen gels containing fibroblasts, the effects of SKDM on epidermal growth and differentiation were indistinguishable from those of FAD.
D-PBSA
0.5% EDTA (~15 mmol/L)
Analytical grade glycerol
FAD culture solution with 20% FCS and 10 % glycerol
Enzymes:
(a) Thermolysin (T-7902, Sigma);
(b) 0.2% and 0.6% trypsin (1:250);
(c) 2.4 U/ml type II dispase (Boehringer Mannheim).
Povidone iodine solution (10% iodine)
Cryopreservation tubes
50 ml centrifuge tube
Nylon mesh
Cell filters (BectonDickinson)
100 mm bacterial culture grade Petri dishes
Scalpel and curved forceps
II. Procedure
Sampling
1. Human skin is usually obtained from the foreskin of newborns and young children, but may also be obtained from trunk skin at the time of surgery or after autopsy (within 48 h of death). Keratinocytes isolated from the foreskin of newborns and young children are superior to those isolated from adult trunk skin in terms of adherence and proliferation.
2. Skin tissue blocks were incubated in povidone-iodine solution (10% iodine) for 30 min to prevent infection. Povidone-iodine solution did not significantly decrease the viability of keratinocytes.
3. The skin was washed with D-PBSA for 10 min twice.
Separation of the epidermis is best done in full-thickness skin.
4. Cut the full-thickness skin with a Castroviejo scalpel (Storz Instrument) to a thickness of 0.1-0.2 mm. The subcutaneous tissue and part of the dermis can be removed with curved scissors.
5. The skin is cut into 1 cmX2 cm pieces with a scalpel.
6. Wash the tissue block with D-PBSA 2 to 5 times.
7. Incubate the blocks with protease by one of the following steps.
(a) Trypsin: float the skin blocks in D-PBSA (pH 7.4) containing 0.6% trypsin for 20-30 min at 37°C. This procedure is also effective for full-thickness skin digestion.
(b) Cold trypsin: Place the tissue block in pre-cooled 0.2% trypsin at 4°C and float for 15 to 24 h. This procedure is also effective for full-thickness skin digestion. The pH of the tryptic solution should be indicated by phenol red to avoid changes in enzyme activity and cell viability due to pH changes.
(c) Type II dispase: incubate with 2.4U/ml dispase for 30 min at 37℃.
(d) Pyrolysin: incubate with 0.5 mg/ml pyrolysin for 12-16 h at 4℃.
8. Observe carefully the separation of epidermis. When the epidermis begins to separate at the edge of the skin block, place the block in a 100 mm plastic Petri dish with the dermis side down. Then, add 5 ml of complete culture medium containing serum.
9. The epidermis is peeled off with two fine curved forceps and collected into a 50 ml centrifuge tube containing 20 ml of complete culture solution.
(a) For isolation by trypsin, viable keratinocytes are isolated from the epidermis by gentle blowing with a pipette and filtration through nylon mesh with a pore size of 100 um (cell strainer, B-DBiosciences). .
(b) To obtain cells from epidermis isolated by dispase or thermolysin digestion, the epidermis was incubated and digested with trypsin and 1.3 mmol/L EDTA (0.2% and 0.05%, respectively) for 10 min at 37°C. The cells were then incubated with trypsin and 1.3 mmol/L EDTA (0.2% and 0.05%, respectively) for 10 min.
10. After separating the skin with trypsin, the poorly adherent epidermal cells were gently scraped from the dermis, and the scraped cells were then added to the epidermal cell suspension. This method yields a higher number of epidermal cells if the purity of the keratin-forming cells is less important. However, there is a lot of mesenchymal cell contamination in keratin-forming cell cultures.
11. Isolated epidermal cells were washed twice with culture medium by centrifugation (100 g, 10 min). Count the total number of cells and the number of viable cells that do not absorb Taipan blue (see Scheme 21.1).
Primary culture
12. Adjust the cell density. Inoculate the cells with FAD or KGM at 37 °C.
(a) Inoculate mitotic 3T3 cells [Rheinwald and Green, 1975] (see Scheme 14.3) or skin fibroblasts [Limat et al, 1989] in Petri dishes 3 days before. Keratinocytes were inoculated on feeder cells at a density of 2X104 to 5X104 cells/cm2 and cultured with FAD.
(b) If fibroblast culture is not used, inoculate primary keratinocytes at high density (1X105 to 5X105 cells/cm2 ) in FAD and maintain high cell density during passaging culture.
(c) Cells were inoculated at low density (1X103 cells/cm2) or high density (5X104 cells/cm2) in KGM containing a low concentration of calcium ions (0.03 to 0.1 mmol/L). The same culture medium was used for passaging culture.
(d) Cell attachment and growth are slower in SKDM, so SKDM is best used for experiments requiring low proliferative activity.
13. After 1 to 3 d, the cells are wall-adhered. After the cells are attached to the wall, the cells are washed well with culture medium to remove the unattached dead cells and differentiated cells. Then/add FAD or KGM and continue the culture: keratinization can be promoted and growth slowed down by adjusting the calcium ion concentration of KGM.
Passage culture
14. The culture method is as follows.
(a) Culture with FAD
(i) Incubate the cells with 1.3-2.7 mmol/L (0.05% 0.1%) EDTA for 5-15 min to de-attach the cells. At this time, the cell gap was enlarged.
(ii) Incubate the cells with 0.1% trypsin and 1.3 mmol/L (0.05%) EDTA for 5-10 min at 37 ℃, and then blow gently with a pipette to fully de-attach the cells.
(b) Incubation with KGM
(i) Due to the low calcium ion concentration of KGM, pretreatment with EDTA is not required.
(ii) If FAD culture method is used, incubate the cells with 0.1% trypsin and 1.3 mmol/L EDTA solution.
Cryopreservation
15. Cryopreservation is often required to preserve large numbers of cells isolated from the same tissue. Cryopreservation is most effective when primary cultured cells are taken before confluence.
(a) Digest the cells with trypsin as described above and centrifuge (100 g, 10 min).
(b) Suspend the cells with complete culture medium containing serum and count.
(c) Adjust the cell density to 2X106 cells/ml with FAD containing 20% FCS and 10 % glycerol.
The cells are then dispensed into cryopreservation tubes.
(d) The tubes were left at 4°C for 1-2 h. The cells were stored at 4°C for 2 hours.
(e) Freeze the cells with a programmable freezer (Planer) at a cooling rate of 1°C/min. Alternatively, the cryopreserved cells can be stored in
Alternatively, the tubes can be inserted into isopropyl alcohol-filled cell freezers (e.g., 1°C freezer containers, No. 5100-0001, NalgeNunc). This allows for proper cooling when placing the cassette in a -80°C freezer.
(d) Cell recovery:
(i) Thaw the cryotubes rapidly in a 37°C water bath.
(ii) Dilute the cells 10 times with culture medium.
(iii) Centrifuge the cells and suspend the cells to a suitable density with the desired culture medium. The cells are then inoculated in Petri dishes.