IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Carlos III National Institute of Health, Spain.
Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Carlos III National Institute of Health, Spain; Biomaterials for Regenerative Therapies Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Barcelona, Spain; Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC), Barcelona, Spain.
Colloids Surf B Biointerfaces. 2019 May 1;177:121-129. doi: 10.1016/j.colsurfb.2019.01.054. Epub 2019 Jan 28.
Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.
角膜缘上皮干细胞（LESCs）负责角膜上皮的更新。培养的角膜缘上皮移植术是目前恢复 LESCs 缺失或功能障碍的首选治疗方法。为了进行此操作，需要一种基质来体外培养角膜缘上皮细胞，并将其随后移植到眼表面。在这项工作中，我们评估了经 IV 型胶原蛋白（col IV）功能化的聚 L/DL-乳酸 70:30（PLA）薄膜作为 LESCs 的潜在体外载体基质。我们首先证明 PLA-col IV 薄膜具有生物相容性，适合人角膜上皮细胞的增殖。随后，从人角膜缘环中分离出角膜缘上皮细胞悬液，使用不含动物成分的培养基进行培养。与组织培养塑料（TCP）相比，细胞在 PLA-col IV 薄膜上的附着速度明显更快。在 PLA-col IV 薄膜上培养的角膜缘上皮细胞中，LESC 特异性标志物 K15、P63α 和 ABCG2 的 mRNA 表达水平相似或更高（在 K15 的情况下显著更高）。在这两种基质上培养的细胞表达角膜（K3、K12）和 LESC（P63α、ABCG2）特异性标志物的百分比相似。这些结果表明，PLA-col IV 薄膜促进了 LESC 的附着，并有助于维持其未分化的干细胞表型。因此，这些基质为角膜缘细胞移植到眼表面提供了替代方案。
