School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4000, Australia.
Queensland Eye Institute, South Brisbane, QLD 4101, Australia.
Molecules. 2021 Nov 11;26(22):6810. doi: 10.3390/molecules26226810.
A recombinant formulation of silk fibroin containing the arginine-glycine-aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal-epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.
一种含有精氨酸-甘氨酸-天冬氨酸（RGD）细胞结合基序的丝素蛋白重组制剂（RGD-丝素蛋白）为角膜细胞的培养提供了潜在的优势。因此，我们研究了 RGD-丝素蛋白表面上角膜基质细胞和上皮细胞的生长情况，并与天然丝素蛋白进行了比较。在存在或不存在血清的情况下，比较了细胞在 90 分钟内的附着情况，并通过定量双链 DNA（dsDNA）含量进行了分析。通过使用低分子量聚乙二醇（PEG；300 Da）作为造孔剂、辣根过氧化物酶（HRP）作为交联剂以及在对面的膜表面上生长基质细胞，通过共聚焦荧光显微镜检查了上皮细胞在独立膜上的分层情况，并通过优化进行了检查。RGD-丝素蛋白减少了基质细胞培养物形成团块的趋势，并促进了上皮细胞的分层。与 HRP 一起使用的 PEG 支持更具渗透性的独立 RGD-丝素蛋白膜的制造，为基质-上皮共培养提供了有效的支架。我们的研究鼓励使用 RGD-丝素蛋白进行角膜细胞培养。需要进一步的研究来确认这种配方的益处是否归因于整合素表达的变化、细胞外基质的组成部分或转录水平的其他事件。
