Keratocyte isolation (corneal fibroblasts)        

Corneal keratocytes (corneal fibroblasts) that this corneal layer is specialized fibroblasts residing in the stroma, representing about 85-90% of corneal thickness, are built up from highly regular collagenous lamellae and extracellular matrix components. Keratocytes play the major role in keeping it transparent, healing its wounds, and synthesizing its components.  In the unperturbed cornea keratocytes stay dormant, coming into action after any kind of injury or inflammation.  Some keratocytes underlying the site of injury, even a light one, undergo apoptosis immediately after the injury.  The corneal stroma matrix consists primarily of collagen I, V, VI, and XII; the proteoglycan decorin, which contains a single dermatan sulfate side chain; and the proteoglycans lumican, keratocan, and osteoglycin/mimecan.  Keratocytes may play a role in different corneal disorders. According to comparative research, their functions drastically diverge from the norm in keratoconus, the most frequent form of corneal dystrophy. In keratoconic corneas they have been shown to commit apoptosis far away from any epithelial injury; a hypothesis exists that presents excessive keratocyte apoptosis as a major pathological event in keratoconus. According to one study, patient's keratocytes have decreased levels of one of the alcohol dehydrogenase subforms, they secrete significantly less superoxide dismutase 3, according to another.

Keratocyte Isolation
1. Bovine keratocytes were isolated using a modification of a sequential primary cell digestion method for embryonic chick corneas.

2. Fourteen freshly harvested, adult bovine eyes were obtained from Pel-Freeze Biologicals by overnight shipment on wet ice.

3. The corneas with a scleral rim were removed using a scalpel and placed epithelium side up on the round end of a rubber centrifuge adapter.

4. Three disks were removed from the central region of the cornea using an 8-mm disposable biopsy punch.

5. The disks were placed in primary cell culture medium containing antibiotics and cut into quarters, using two single-edged razor blades drawn across each other on a rubber stopper.

6. The quartered disks were divided in two groups and each placed in a 50-ml conical centrifuge tube with 21 ml primary cell culture solution containing 3.3 mg/ml collagenase and incubated at 37°C with shaking (126 rpm) for 30 minutes.

7. The tubes were then vortexed for 30 seconds and the tissue pieces separated from the collagenase solution using a cell strainer.

8. Digestion was continued with a second 21-ml aliquot of collagenase for 60 minutes and with a third 21-ml aliquot of collagenase for 180 minutes, followed by a repeat of the separation procedure.

9. The cells in each of the three collagenase digestions were collected by low-speed centrifugation (1400 rpm × 10 minutes) at the end of each digestion period and the cells resuspended in 10 ml primary cell culture solution.

10. The viability and appearance of the cells was determined by trypan blue exclusion, using an inverted microscope and counting the number of the cells by hemocytometer.

11. In initial experiments, cells from the second and third collagenase digest were resuspended in primary cell culture solution containing 10% fetal bovine serum (FBS), plated in tissue culture dishes and visually inspected for attachment and spreading.

References

1. Wilson SE, Chaurasia SS, Medeiros FW. Apoptosis in the initiation, modulation and termination of the corneal wound healing response". Exp. Eye Res. 2007; 85: 305–311.

2. Marianne P. Beales, James L. Funderburgh, James V. Jester and John R. Hassell. Proteoglycan Synthesis by Bovine Keratocytes and Corneal Fibroblasts: Maintenance of the Keratocyte Phenotype in Culture. Invest. Ophthalmol. Vis. Sci. 1999; 40: 1658-1663.