National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Mol Vis. 2022 Dec 12;28:412-431. eCollection 2022.
The development of biomaterials provides potent promise for the regeneration of neuroretinal cells in degenerative eye diseases and retinal tissue engineering. Biomimetic three-dimensional (3D) microenvironments and specific growth factors motivate the differentiation of human retinal pigment epithelial (hRPE) cells toward a retinal neural lineage. In this study, we evaluated alginate/gelatin (A/G) as a substrate for the culture of hRPE cells.
hRPE cells were isolated from neonatal human cadaver globes and cultivated on A/G substrate under different culture conditions, including 30% human amniotic fluid (HAF), 10% fetal bovine serum (FBS), and serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12). The proliferation of cells in different culture conditions was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and a cell proliferation assay. Immunocytochemistry and real-time PCR were performed to evaluate the effect of the substrate on hRPE cell differentiation.
A significant increase in the cell proliferation rate was observed in hRPE cells cultivated on an A/G substrate. Continuous observations demonstrated that hRPE cells formed densely packed, suspended spheroids in DMEM/F12 culture conditions, with dominant transdifferentiation into amacrine cells. Small adherent clusters of hRPE cells in HAF- and FBS-treated cultures represented dedifferentiation toward retinal progenitor cells. These cultures generated amacrine, rod photoreceptors, and bipolar cells.
These findings indicated that A/G substrate induced neural retinal cell propagation in cultures and would therefore be promising for RPE-based tissue engineering studies.
生物材料的发展为退行性眼病和视网膜组织工程中的神经视网膜细胞再生提供了有力的前景。仿生三维（3D）微环境和特定的生长因子促使人视网膜色素上皮（hRPE）细胞向视网膜神经谱系分化。在这项研究中，我们评估了藻酸盐/明胶（A/G）作为 hRPE 细胞培养的基底。
从新生儿人尸眼球中分离 hRPE 细胞，并在不同的培养条件下（包括 30%人羊水（HAF）、10%胎牛血清（FBS）和无血清的 Dulbecco 改良 Eagle 培养基/营养混合物 F-12（DMEM/F12））在 A/G 基底上培养。使用 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐和细胞增殖测定法测定不同培养条件下细胞的增殖。进行免疫细胞化学和实时 PCR 以评估基底对 hRPE 细胞分化的影响。
在 A/G 基底上培养的 hRPE 细胞的细胞增殖率显著增加。连续观察表明，hRPE 细胞在 DMEM/F12 培养条件下形成密集的悬浮球体，向无长突细胞的转分化占主导地位。在 HAF 和 FBS 处理的培养物中，小的 hRPE 细胞附着簇代表向视网膜祖细胞的去分化。这些培养物产生无长突细胞、杆状光感受器和双极细胞。
这些发现表明 A/G 基底在培养物中诱导神经视网膜细胞增殖，因此有望用于基于 RPE 的组织工程研究。
