Institute for Science and Technology in Medicine, School of Medicine, Keele University , Stoke-on-Trent, United Kingdom .
Tissue Eng Part A. 2014 Jan;20(1-2):225-38. doi: 10.1089/ten.TEA.2013.0167. Epub 2013 Sep 9.
In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-β1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial-stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-β1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-β1 media supplementation. It was then revealed that epithelial-stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal-epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital.
在体内，上皮细胞在解剖和功能上都与基质角膜细胞相连。共培养旨在通过将两种或多种细胞类型聚集在同一培养环境中，重新获得这种细胞解剖和功能。角膜基质细胞被激活为损伤表型（成纤维细胞）并扩增，然后在 I 型胶原水凝胶构建体中包封。然后检查了三种不同的上皮-基质共培养方法：上皮外植体；transwell；和使用条件培养基。目的是确定是否可以在体外恢复天然的、失活的角膜细胞表型。然后用转化生长因子β-1 (TGF-β1) 补充培养基，以确定失活的基质细胞在体外是否保持其可塑性，并可被重新激活为成纤维细胞表型。最后，用wortmannin 补充培养基以抑制上皮-基质细胞相互作用。两种不同的非破坏性技术，球形压痕和光相干断层扫描，分别用于揭示 TGF-β1 与上皮-基质共培养以及 wortmannin 培养基补充如何影响基质细胞行为和分化，以及构建体收缩和弹性模量测量。细胞活力、表型、形态和蛋白质表达进行了研究，以证实我们的力学发现。结果表明，通过共培养可以将激活的基质细胞失活到角膜细胞表型，并且它们在体外保持其可塑性。激活的具有成纤维细胞表型的角膜基质细胞在行为和生物学特性方面成功地逆转为非激活的角膜细胞谱系；然后通过 TGF-β1 培养基补充再次转回。然后表明，上皮-基质相互作用可以通过wortmannin 抑制来阻断。对基质-上皮相互作用及其介导机制的更深入了解，为调节角膜伤口愈合提供了巨大的药理学潜力，有可能通过这种相互作用治疗角膜疾病和损伤。
