Department of Pharmaceutical, Sciences and Technology, Institute of Chemical Technology, Mumbai, India.
Department of Chemical Engineering, Indian Institute of Technology, Mumbai, India.
Biotechnol J. 2022 Sep;17(9):e2100530. doi: 10.1002/biot.202100530. Epub 2022 Jun 9.
The investigation is focused on the development of a compartmentalized microfluidic device for coculturing the cells of crucial retinal cellular layers and assessing cell-to-cell interactions. A perfusion-based microfluidic co-culture device was employed and computationally validated for determining the pressure drop and fluid flow rate within the device microchannels. Fabrication was performed using PDMS polymer and coating of fibronectin and collagen facilitated adherence of the cells over the glass surface. Microfluidic device successfully supported cell proliferation, under continuous perfusion of 1 μl min flow rate. The barrier integrity of this coculture was confirmed by evaluating the permeability of fluorescently labeled molecules. The coculture expressed characteristic phenotypic protein markers like recoverin, PAX6, for retinal precursor cells, and RPE65 for retinal epithelial cells. The coculture also exhibited basal expression of TNF-α under normal conditions. Differentiated photoreceptor cells positively expressed rhod inherently possess sensitivity toward violet/blue light, which was validated in R28 cells by exposure to light having a wavelength of 405 nm, which significantly decreased cell viability via increased TNF-α production and reduced rhodopsin expression. This proof-of-concept investigation proved the functionality of the retinal coculture, which may be used as an appropriate perfusion-based, preclinical tool for the evaluation of novel retinal drugs and delivery systems.
本研究专注于开发一种分隔式微流控装置，用于共培养关键视网膜细胞层的细胞，并评估细胞间的相互作用。采用基于灌注的微流控共培养装置，并对其进行了计算验证，以确定装置微通道内的压降和流体流速。使用 PDMS 聚合物进行制造，并涂覆纤维连接蛋白和胶原蛋白，以促进细胞在玻璃表面上的黏附。微流控装置在 1 μl/min 的连续灌注流速下成功支持细胞增殖。通过评估荧光标记分子的通透性，证实了这种共培养的屏障完整性。该共培养表达了特征性的表型蛋白标志物，如视网膜前体细胞的 recoverin 和 PAX6，以及视网膜上皮细胞的 RPE65。在正常条件下，共培养还表现出 TNF-α 的基础表达。分化的光感受器细胞阳性表达 rhod，对紫光/蓝光具有固有敏感性，这在 R28 细胞中通过暴露于波长为 405nm 的光得到了验证，该光通过增加 TNF-α 的产生和减少 rhodopsin 的表达，显著降低了细胞活力。这项概念验证研究证明了视网膜共培养的功能，它可以作为一种合适的基于灌注的、临床前工具，用于评估新型视网膜药物和输送系统。
