Corneal epithelial cell culture experiment
Corneal epithelial cell culture experiment
There has been a great deal of interest in forming corneas in culture, as attempts have been made to replace the rabbit eye experiments (Draze's experiments) with cultured corneal epithelium. The following description and protocol 23.2 describe the culture of normal human corneal epithelial cells in serum-free culture medium. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition.
Operation method
Scheme 23.2 Corneal epithelial cell culture assay
Principle
Corneal tissue was placed on collagen for attachment. Then, the culture was expanded in Petri dishes coated with fibronectin and collagen.
Materials and Instruments
D-PBSA Serum-free Keratinocyte Culture Solution Trypsin EDTA Biocoat 6-well Plate Move I. Materials Sterilization Primary culture Expansion culture 7. After removing the tissue slices, change the fluid twice a week. 8 When the cell confluence reaches 70%~80%, the cells are washed with D-PBSA. Then, the cells were treated with a mixture of trypsin and EDTA at 37°C for 4 min. 9. Terminate the digestion with D-PBSA containing 10% FBS. 10 Centrifuge the cells and suspend with KGM. Cells were counted and inoculated into FNC-coated petri dishes at a density of 1X104cells/cm2. 11 Incubate at 37°C, 95% air and 5% CO2. 12. Trypsin treatment and planting for 1d followed by a change of culture medium. For more product details, please visit Aladdin Scientific website.
Serum-free keratinocyteserum-freemedium (KGM; Clonetics): containing 0.15 mmol/LCa2-, 0.1 ng/ml human epithelial growth factor, 5ug/ml insulin, 0.5ug/ml hydrocortisone and 30ug/ml bovine pituitary gland extract.
EMEM (Eagle's minimalessentialmedium).
D-PBSA: Ca2+ and Mg2+ free DulbeccoPBSA liquid FBS
Trypsin and EDTA mixture: 0.05% trypsin, 0.5 mmol/L EDTA
Fibronectin/collagen (FNC) mixture: 10ug/ml fibronectin, 35ug/ml collagen and lOOug/ml BSA. BSA acts as a stabilizer.
Biocoat 6-well plates: coated with mouse tail collagen type I (B-D Biosciences)
Steps
1. Place the donor cornea on a sterilized object with the epithelial side facing up and cut into 12 triangular tissue slices with a scalpel. This should be done in a single cut, avoiding sawing. Such careful handling of the cornea reduces damage to the collagen matrix and loss of fibroblasts.
2. Turn the epithelial side of the corneal tissue slice down and place it into a 6-well culture plate precoated with collagen type I from rat tail, placing 4 tissue blocks per well.
3. Gently press the slice with forceps so that the slice is in full contact with the surface of the Petri dish. Leave the tissue piece for 20 min to dry.
4. A drop of KGM was gently placed on the tissue slice and incubated overnight at 37°C and 5% CO2. Although donor corneas from the eye bank are preserved in culture medium containing antimicrobials (McCarey-Kauffman or Dexsol), all operations should be performed under sterile conditions.
5. On day 2, lml of culture medium was added to the culture dish. Early in the culture, cells were observed to migrate only from the corneal rim, but not from the central cornea or sclera. The serum-free culture medium contains a low concentration of calcium (0.15 mmol/L), which reduces the degradation of the collagen matrix, and therefore this medium reduces fibroblast outgrowth.
6 On day 5 after implantation of the corneal tissue slice, the slice was removed with forceps and 3 ml of culture medium was added. After removal of the tissue sheet, the adherent cells continued to proliferate. At the second week of culture, the cells grew to form a monolayer, showing the typical pebble-like arrangement of the epithelium. About 1X106~5X106 epithelial cells were obtained from each cornea.
Shortly after passaging, the epithelial cells were long shuttle-shaped, refractive, and had a strong migratory ability. In the first week after passaging, when the cell confluence reached 70%~80%, the cells were arranged in a pebble shape. If the monolayer is formed and the culture is continued, the cells can grow into scattered complex structures.
Although corneal epithelial cells can multiply about 9 to 10 times up to 5 generations, most cell proliferation occurs in the 1st to 3rd generation. Each cornea acquires approximately 1X106 to 2X106 epithelial cells. Cell senescence often begins in the 5th generation.