Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface
Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface
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DOI：
10.1167/iovs.04-0813
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发表时间：
2005-05-01
影响因子：
4.4
通讯作者：
Tano, Y
中科院分区：
文献类型：
--
作者：
Hayashida, Y;Nishida, K;Tano, Y
目的.自体角膜缘干细胞移植治疗角膜缘干细胞缺乏症在免疫学上是可取的，以避免同种异体移植排斥反应。本研究旨在探讨一种新的组织工程方法用于眼表重建的可能性，使用自体口腔粘膜上皮干细胞在温度响应性细胞培养表面上离体扩增。兔口腔粘膜上皮细胞培养的温度响应培养表面与丝裂霉素C处理的3 T3饲养细胞2周产生汇合的上皮细胞片。通过集落形成试验估计推定的祖细胞群体。将这些细胞片自体移植到手术操作的眼睛中，并检查眼表面重建和细胞表型调节。所有培养的口腔上皮细胞通过将培养温度降低至20 ℃而非酶促收获为可移植的完整细胞片。口腔上皮细胞分层为3 ~ 5层，与角膜上皮相似，而非口腔粘膜上皮。p63、β 1-整联蛋白和连接蛋白43的集落形成测定和免疫荧光表明细胞片中保留了活的干细胞和/或祖细胞群。自体角膜移植成功地重建了兔角膜表面，恢复了透明。移植后4周，上皮分层与角膜上皮相似，但角蛋白表达谱保留了口腔粘膜上皮的特征。细胞片收获技术能够制造活的、可移植的、组织工程化的上皮细胞片，其保留来自自体口腔粘膜上皮细胞的推定祖细胞。自体组织工程上皮细胞片用于眼表重建的临床能力是有前途的。
PURPOSE. Autologous stem cell transplantation for total limbal stem cell deficiency is immunologically preferable, to avoid allograft rejection. This study was undertaken to investigate the possibility of a novel tissue engineering approach for ocular surface reconstruction, using autologous oral mucosal epithelial stem cells expanded ex vivo on temperature-responsive cell culture surfaces.METHODS. Rabbit oral mucosal epithelial cells cultured on temperature-responsive culture surfaces with mitomycin-C-treated 3T3 feeder cells for 2 weeks produced confluent epithelial cell sheets. Putative progenitor cell populations were estimated by colony-forming assays. Autologous transplantation of these cell sheets to surgically manipulated eyes was performed, and ocular surface reconstruction and cell phenotypic modulation were examined.RESULTS. All cultured oral epithelial cells were nonenzymatically harvested as transplantable intact cell sheets by reducing culture temperature to 20 degrees C. Oral epithelial cells were stratified in three to five cell layers more similar to corneal epithelium than to oral mucosal epithelium. Colony-forming assays and immunofluorescence for p63, beta 1-integrin, and connexin 43 indicated retention of viable stem and/or progenitor cell populations in cell sheets. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface, with restoration of transparency. Four weeks after transplantation, epithelial stratification was similar to that in the corneal epithelium, although the keratin expression profile retained characteristics of the oral mucosal epithelium.CONCLUSIONS. Cell sheet harvest technology enables fabrication of viable, transplantable, tissue-engineered epithelial cell sheets that retain putative progenitor cells from autologous oral mucosal epithelial cells. Promising clinical capabilities for autologous tissue-engineered epithelial cell sheets for ocular surface reconstruction are indicated.