I never tried it. My suggestion: 1. remove the outer surface (conjunctival, Tenon´s and episcleral tissue and inner surface tissue /retina, retinal pigment epithelium and choroid. Then clean the inner and outer surface of the sclera by ablating tissue with an excimer laser. Finally pouch a scleral button with a 10 to 15 mm trephine . This button should be cultured. Hopefully scleral will spread out of the button. Good luck!

Thank you for your suggestion, Dr. Gerding. This procedure looks like complicated. Our lab don't have excimer laser for use. I nerve do trephine before, and don't know which machine should I use to do it on sclera.

Kindly use the method described by Cui et al.

Extract from the article:

Method:

Eyeballs washes in HBSS containing penicillin (200 µg/ml) and gentamicin sulfate (400 µg/ml). The retinas and choroids were removed from the sclera. The sclera was trimmed into pieces approximately 1×1 mm in size, placed in 75 mm2 plastic culture bottles in DMEM with 1X antibiotic/antimycotic and 10% FBS, and incubated at 37 °C in a humidified incubator containing 5% CO2. The growth medium was changed twice a week. When a heavy primary monolayer was achieved, cells were trypsinized in 0.25% trypsin/EDTA solution in PBS at room temperature for 2 min and subcultured at a split ratio of 1:3 in a 75 mm2 plastic bottle. The third passage of fibroblasts was used for this experiment. The fibroblasts were grown on cover glasses in six well plates until 70%–80% confluence occurred. The cells were washed with PBS three times, fixed with cool acetone for 15 min, air-dried, and kept frozen at −20 °C until use.

The purity of fibroblast cell cultures was confirmed by staining for vimentin and stain resistance for cytokeratin, desmin, and S-100 in the indirect immunofluorescence procedure described below. The morphology of HSF was observed with light microscopy.

Indirect immunofluorescence

The slices were washed three times with PBS, covered with 10% normal goat serum diluted in PBS, and incubated for 20 min at 37 °C. The slices were then incubated at 4 °C overnight with the primary antibodies (anti-vimentin, anti-cytokeratin, anti-desmin, anti-S-100, anti-ADORA1, anti-ADORA2A, anti-ADORA2B, and anti-ADORA3) diluted at 1:500 in PBS. A negative control was run in which the cells were incubated in PBS without the primary antibodies. The slices of both the antibody-treated and the negative control samples were washed with PBS and exposed to fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit IgG antibodies diluted at 1:50 in PBS at 37 °C for 30 min. The slices were again washed with PBS three times and added to propidium iodide (PI) for 5 min to stain the cell nuclei red. Immunofluorescent images were taken using a confocal microscope (LSM 510 META, Carl Zeiss, Jena, Germany).

Western blot analysis

HSF were cultured in DMEM/F12 for 48 h and harvested; human sclera were shivered into pieces and centrifuged (15,000x g) at 4 °C for 1 h. HSF lysates were prepared in a lysis buffer and boiled in a sample buffer for 5 min. The protein concentration was detected by BAC kits. Protein (40 μg) was loaded in each lane of 10% SDS-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes for electrophoresis, blocked in TBST (5% fat-free dry milk, 0.1% Tween 20, 150mM NaCl, and 50 mM Tris at pH 7.5) for 1 h. The membranes were exposed to 1 µg/ml of anti-ADORA1, anti-ADORA2A, anti-ADORA2B, and anti-ADORA3 polyclonal antibody and incubated overnight at 4 °C. They were then incubated with a secondary horseradish peroxidase-labeled antibody for 1 h. Protein bands were visualized with the use of a chemiluminescence Phototope(R)-HRP Western Blot detection system (Cell Signaling Technology, Inc., Danvers, MA) and exposed onto a negative film, developed, and fixed. The film was scanned and analyzed with Gel-Pro Analyzer software.

I hope this helps you. The technique is described below in more detail. All rights are attributed to the authors.

Distribution of adenosine receptors in human sclera fibroblasts

Dongmei Cui, Klaus Trier, Xiang Chen, Junwen Zeng, Xiao Yang, Jianmin Hu, Jian Ge Mol Vis. 2008; 14: 523–529. Published online 2008 Mar 14. PMCID: PMC2268861

Regards,

Sahil

Thanks, Sahil. I should give it a try.