ABSTRACT
The shortage of donor corneal tissue worldwide has led to extensive research for alternate corneal equivalents utilizing tissue engineering methods. We conducted experiments using Poly D, L lactic acid polymer along with a copolymer (Eudragit) in varying concentrations to create a biodegradable scaffold suitable for in vitro growth of corneal epithelial stem cells. It was found that stable, spherical, and porous microparticles can be prepared by combining PDLLA and Eudragit RL100 polymers in the ratio of 90:10 and 70:30. The microparticles can then be fused to form scaffold membranes with porous architecture and good water retention capacity at room temperature using methanol, which can withstand handling during transplantation procedures. The scaffolds made using a 70:30 ratio were found to be suitable for the promotion of growth of laboratory corneal epithelial stem cell lines (SIRC cell lines). This innovation can pave way for further developments in corneal stem cell research and growth, thus providing for viable laboratory-derived corneal substitutes.
ABSTRACT
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.
Subject(s)
Rabbits , Humans , Animals , Trypsin/pharmacology , Stem Cells/cytology , Limbus Corneae/cytology , Epithelium, Corneal/cytology , Edetic Acid/pharmacology , Cells, Cultured , Cell Culture Techniques , Cell CountABSTRACT
PURPOSE: To investigate the minimal requirements of the limbal epithelium for successful limbal stem cell transplantation and the healing process. METHODS: Nine rabbits were divided into 4, 6, and 8 clock-hour transplantation groups. Limbal autografts from the healthy fellow eye were transplanted to the iatrogenic damaged eye. The amniotic membrane served as a stem cell niche. Experimental corneas were evaluated by slit lamp examination and immunohistochemistry. RESULTS: In the over 9 hours transplantation group, the healing process of the epithelium from the limbal stem cell was revealed and cornea-specific keratin k3, transcription factor p63, and connexin 43 were detected by immunohistochemistry. The normal corneal epithelium was regenerated after 60 days postoperatively in the fellow donor eye. CONCLUSIONS: Limbal cell transplantation of over 9 hours seems to be a safe and effective method in the treatment of severe ocular surface disorders. In addition, the donation of limbal epithelium for up to 8 hours did not affect the normal corneal regenerating capability.