Abstract
Abstract: :
Purpose:To evaluate the feasibility of replacing fetal bovine serum (FBS) with human serum (HS) for the culturing and expansion of limbal progenitor cells on amniotic membrane Methods:The limbal rim of human donor corneolimbal buttons was cut into 1 x 1 mm2 explants, which were then placed over cryopreserved amniotic membrane for culturing in a SHEM medium with either 5% FBS or 5% HS. Before epithelial cultures were confluent, BrdU labeling was done for 24 hours to identify rapid-cycling cells. Xenotransplantation to NIH-bg-nu-xidBR mice was also performed to induce epithelial differentiation and stratification. Results:A monolayer composed of small and compact epithelial cells with a similar morphology was noted in both culture conditions. The rate of explant outgrowth was similar in both cultures (P=0.006). The 24 hr BrdU labeling index was 4.4 ± 0.9% for the SHEM with 5% FBS group and 12.2 ± 9.4% for the SHEM with 5% HS group (P=0.001). The resultant stratified epithelium following xenotransplantation revealed limbal phenotype characteristics as the basal epithelial cells showed negative staining to both K3 keratin and connexin 43 in either culture condition. BrdU labeled cells were identified in the basal layer of the stratified epithelium five days after transplantation to the nude mice in both groups. media Conclusion:Collectively, the above findings support that it is feasible to replace FBS with HS in ex vivo preservation and expansion of limbal epithelial progenitor cells on amniotic membrane cultures. The use of autologous HS will avoid the risks of transmitting diseases from bovine species to human patients when this new procedure is contemplated.
Keywords: 372 cornea: epithelium • 607 transplantation • 356 clinical (human) or epidemiologic studies: systems/equipment/techniques