Department of Biomedical Engineering, Tufts University, Medford, MA, USA.
Department of Chemical Engineering, Tufts University, Medford, MA, USA.
J Tissue Eng Regen Med. 2018 Jan;12(1):285-295. doi: 10.1002/term.2499. Epub 2017 Sep 28.
With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro. Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in coculture on the optimized silk scaffolds.
由于满足角膜移植临床需求的选择有限，一个新兴的重点领域是角膜组织工程。在本研究中，目的是将角膜基质和上皮组合成一个共培养系统，以监测人角膜基质干细胞（hCSSC）和人角膜上皮细胞（hCE）在这些三维组织系统中向角膜基质细胞和成纤维细胞分化，并在体外分化为上皮细胞。首先优化了共培养条件，包括培养基、气液界面培养以及基于丝蛋白的生物材料支架膜的表面形貌和化学性质。丝蛋白被用作基质和上皮组织层的支架，因为它具有细胞相容性、可进行表面图案化，并且光学透明。接下来，在该体外系统中研究了增殖和分化的 hCE 和 hCSSC 的影响，包括对细胞增殖、免疫化学基质形成和定量逆转录聚合酶链反应基因表达的影响。与单独的角膜基质细胞和角膜上皮细胞相比，将两种细胞类型纳入共培养系统显示出两种细胞类型的更完全分化和生长。优化了用于角膜上皮细胞培养的丝蛋白膜，将 4.0-μm 尺度的表面图案与大量负载的 IV 型胶原相结合。在优化的丝支架上共培养 6 周后，每种细胞类型的分化都表现为角膜基质和上皮蛋白的表达增加，以及转录水平增加。
