In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.
Cell Proliferation/*drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelium, Corneal/*cytology ; Epithelium, Corneal/*cytology ; Nerve Growth Factor/*pharmacology

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(6) cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67+/-2.13% and 6.63+/-2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61+/-0.42% and 5.21+/-4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67+/-2.24% and 1.17+/-6.13%, respectively (p<0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.
Trypsin/pharmacology ; Stem Cells/*cytology/drug effects ; Rabbits ; Limbus Corneae/*cytology/drug effects ; Humans ; Epithelium, Corneal/*cytology/drug effects ; Edetic Acid/pharmacology ; Cells, Cultured ; Cell Culture Techniques ; Cell Count ; Animals

To study the possibility of using hydroxypropyl chitosan-based blend membranes as carriers of corneal cells in tissue engineering, we prepared three kinds of blend membranes labeled hydroxypropyl chitosan/chondroitin sulfate, hydroxypropyl chitosan/gelatin/chondroitin sulfate and hydroxypropyl chitosan/oxidized hyaluronic acid/chondroitin sulfate. The transparency, water content and ability of protein adsorption of the blend membranes were measured. To evaluate the cytocompatibility of the blend membranes with corneal epithelial cells, rabbit corneal epithelial cells were cultured on the surface of the carrier membranes. The morphological characteristics, cell adhesion, cell proliferation and the activity of lactate dehydrogenase (LDH) in the media were investigated. Three kinds of blend membranes had good optical transmittance, suitable water content and ability of protein adsorption. The results showed that the less injury was made to corneal epithelial cells by the hydroxypropyl chitosan/gelatin/chondroitin sulfate blend membrane than the others. This kind of membrane was favor of the growth and adhesion of corneal epithelial cells. The hydroxypropyl chitosan/gelatin/chondroitin sulfate blend membrane is a promising carrier of corneal cells and can be used in reconstruction of tissue engineered cornea.
Animals ; Biocompatible Materials ; chemistry ; pharmacology ; Cell Culture Techniques ; methods ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chitosan ; chemistry ; Chondroitin Sulfates ; chemistry ; Epithelium, Corneal ; cytology ; Gelatin ; chemistry ; Membranes, Artificial ; Rabbits ; Tissue Engineering ; methods

This study investigated the toxicity of commercial non-steroid anti-inflammatory drug (NSAID) eye solutions against corneal epithelial cells in vitro. The biologic effects of 1/100-, 1/50-, and 1/10-diluted bromfenac sodium, pranoprofen, diclofenac sodium, and the fluorometholone on corneal epithelial cells were evaluated after 1-, 4-, 12-, and 24-hr of exposure compared to corneal epithelial cell treated with balanced salt solution as control. Cellular metabolic activity, cellular damage, and morphology were assessed. Corneal epithelial cell migration was quantified by the scratch-wound assay. Compared to bromfenac and pranoprofen, the cellular metabolic activity of diclofenac and fluorometholone significantly decreased after 12-hr exposure, which was maintained for 24-hr compared to control. Especially, at 1/10-diluted eye solution for 24-hr exposure, the LDH titers of fluorometholone and diclofenac sodium markedly increased more than those of bromfenac and pranoprofen. In diclofenac sodium, the Na+ concentration was lower and amount of preservatives was higher than other NSAIDs eye solutions tested. However, the K+ and Cl- concentration, pH, and osmolarity were similar for all NSAIDs eye solutions. Bromfenac and pranoprofen significantly promoted cell migration, and restored wound gap after 48-hr exposure, compared with that of diclofenac or fluorometholone. At 1/50-diluted eye solution for 48-hr exposure, the corneal epithelial cellular morphology of diclofenac and fluorometholone induced more damage than that of bromfenac or pranoprofen. Overall, the corneal epithelial cells in bromfenac and pranoprofen NSAID eye solutions are less damaged compared to those in diclofenac, included fluorometholone as steroid eye solution.
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage/*toxicity ; Benzophenones/administration & dosage/toxicity ; Benzopyrans/administration & dosage/toxicity ; Bromobenzenes/administration & dosage/toxicity ; Cell Movement/drug effects ; Cells, Cultured ; Diclofenac/administration & dosage/toxicity ; Epithelial Cells/drug effects/metabolism/ultrastructure ; Epithelium, Corneal/cytology/*drug effects/metabolism ; Fluorometholone/administration & dosage/toxicity ; Humans ; L-Lactate Dehydrogenase/metabolism ; Microscopy, Electron, Transmission ; Ophthalmic Solutions ; Propionates/administration & dosage/toxicity

The role of nitric oxide (NO) in the ocular surface remains unknown. We investigated the conditions leading to an increase of NO generation in tear and the main sources of NO in ocular surface tissue. We evaluated the dual action (cell survival or cell death) of NO depending on its amount. We measured the concentration of nitrite plus nitrate in the tears of ocular surface diseases and examined the main source of nitric oxide synthase (NOS). When cultured human corneal fibroblast were treated with NO producing donor with or without serum, the viabilities of cells was studied. We found that the main sources of NO in ocular surface tissue were corneal epithelium, fibroblast, endothelium, and inflammatory cells. Three forms of NOS (eNOS, bNOS, and iNOS) were expressed in experimentally induced inflammation. In the fibroblast culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblast cells caused by serum deprivation in a dose dependent manner up to 500 micrometer SNAP, but a higher dose decreased cell viability. This study suggested that NO might act as a doubleedged sword in ocular surface diseases depending on the degree of inflammation related with NO concentration.
Animals ; Apoptosis/drug effects/physiology ; Aqueous Humor/metabolism ; Blood Proteins/pharmacology ; Cell Survival/drug effects/physiology ; Cells, Cultured ; Epithelium, Corneal/*cytology/*enzymology ; Fibroblasts/cytology/enzymology ; Humans ; Nitric Oxide/biosynthesis/*physiology ; Nitric Oxide Donors/pharmacology ; Nitric Oxide Synthase/metabolism ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Penicillamine/*analogs & derivatives/pharmacology ; Peroxynitrous Acid/biosynthesis ; Rabbits ; Tears/metabolism ; Uveitis/metabolism