Protokoll

We invite you to take a look at a collection our optimalised and working methods on the field of cell breeding, microscopy, histology and sample analysis.

  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 cm2 culture 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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We have use two types of isolation protocols:

The first protocol was developed for the isolation of tumor tissue. After the enucleation the tumor was removed from the bulbus, half of it was taken for histological examination, the other half of the tumor was used for cell isolation. The tumor was disinfected once with 70% ethanol and four times with Betadine solution. For each step new equipment was used. After the disinfection the tissue was minced to 2x2 mm pieces with 20 gauge hypodermic needle, and digested in 400 U/ml collagenase IV and 0,25% trypsin/EDTA in Ham’s F12 media. The digestion lasted 60 minutes. After the 60 minutes the cell suspension was centrifuged on 500G, 5 min, then the cell viability was examined directly with a Bürker chamber using trypan blue stain. The cells were moved into a 25cm2 cell culture flask into Ham’s F12 medium supplemented by 10% foetal bovine serum (FBS) and 1% penicillin neomycin streptomycin solution (PSN). After 24 hours the media were remowed, and only the adherent cells were cultivated.

The second isolation protocol was developed for fine needle aspiration biopsy. The cell suspension was centrifuged and after viability assay, the cells were cultured in a 25 cm2 cell culture flask in RPMI-1640 medium supplemented by 10% foetal bovine serum (FBS) and 1% penicillin streptonycin neomycin (PSN) solution. After 24 hours the media was removed and only the adherent cells were cultured.

 

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