1. The limbal tissues are disinfected once with 70% ethanol and four times using Betadine solution. For round of disinfectant new sterile tools should be used.

  2. After the disinfection process the tissues are washed with complete medium( DMEM/F12(Sigma Aldrich D8437)) supplemented by 10% FBS(PAAA15101) and 1% PSN (Sigma Aldrich P4083).

  3. Following the wash we cut the tissue into 2x2 mm pieces and digest it in 800 U/ml collagenase IV (Sigma Aldrich C1889) for 60 minutes on 37 °C .

  4. When the digestion is done we centrifuge the solution containing the isolated cells on 1000 rpm for 5 minutes and culture the cells in a 25 cmculture falsk (PAA70025X) using complete medium.

With this method we succeeded in isolating two cell types:

               endo-and epithelial like cells (passage number 11)

               fibroblast like cells ( passage number 5)


The isolated cells are kept in a stable environment, on 37°C, 5% CO2,in complete medium.

During subculturing we use trypsin-EDTA solution to pause the adherence of cells to the flasque surface.

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