Cultivation of Human Oral Mucosal Explants on Contact Lenses

<i>Purpose/Aim</i>: Autologous cultivated oral mucosal (OM) epithelial transplantation has been successfully used as corneal epithelial replacement in bilateral limbal stem cell deficiency. Recently, lotrafilcon A contact lens (CL) surface was described as a suitable carrier for cultured stem cells in corneal epithelial transplantation. Our aim was to establish explant cultures from human OM on CL carriers that are free of animal-derived materials and feeder cells. <i>Materials and methods</i>: Human cadaveric 2 mm OM explants were sutured onto CL surfaces and cultivated with fetal calf serum (FCS) or human serum (HS) supplemented culture medium without feeder cells. Confluent cultures were harvested and evaluated morphologically with hematoxylin and eosin stain and with immunofluorescence microscopy for the presence of p63, vimentin and cytokeratins (CK) 3, 4, 13 and 14. <i>Results</i>: Confluent cell sheets covering the whole CL surface were produced from OM explants after 2 weeks of culture with HS and after 3 weeks with FCS. A basal layer consisted of small, vimentin, p63 and CK14 positive putative stem/progenitor cells, which were present in the whole cell sheet. Large, CK3, CK4 and CK13 positive, differentiated cells appeared to spread above this confluent layer. <i>Conclusions</i>: We have established an animal-free culture system from human OM explants on CL surface. The cultured OM sheets contain large numbers of putative stem cells including limbal-like CK3 and CK14 positive cells. This method can be adapted to good manufacturing practice (GMP) conditions and has, therefore, great potential for clinical use.

<i>研究目的</i>：自体培养口腔黏膜（oral mucosal，OM）上皮移植已成功应用于双侧角膜缘干细胞缺乏症的角膜上皮替代治疗。近期研究表明，洛特拉菲康A（lotrafilcon A）接触镜（contact lens，CL）表面可作为角膜上皮移植中培养干细胞的适宜载体。本研究旨在建立以无动物源材料及饲养层细胞的接触镜（CL）为载体的人口腔黏膜（OM）外植体培养体系。<i>材料与方法</i>：将人尸源性2mm口腔黏膜（OM）外植体缝合于接触镜（CL）表面，使用添加胎牛血清（fetal calf serum，FCS）或人血清（human serum，HS）且不含饲养层细胞的培养基进行培养。收集汇合后的培养物，通过苏木精-伊红染色进行形态学评估，并通过免疫荧光显微镜检测p63、波形蛋白（vimentin）以及细胞角蛋白（cytokeratins，CK）3、4、13、14的表达。<i>研究结果</i>：使用人血清（HS）培养2周、胎牛血清（FCS）培养3周后，可从口腔黏膜（OM）外植体获得覆盖整个接触镜（CL）表面的汇合细胞片层。细胞片层的基底层由小型、呈波形蛋白、p63及CK14阳性的潜在干细胞/祖细胞构成，此类细胞遍布整个细胞片层；大型、呈CK3、CK4及CK13阳性的分化细胞则分布于该汇合层上方。<i>研究结论</i>：本研究成功建立了以接触镜（CL）为载体的人口腔黏膜（OM）外植体无动物源培养体系。所培养的口腔黏膜（OM）细胞片层含有大量潜在干细胞，其中包括类似角膜缘干细胞的CK3及CK14阳性细胞。该方法可适配药品生产质量管理规范（good manufacturing practice，GMP）标准，因此具备巨大的临床应用潜力。