Advantages of nanofibrous membranes for culturing of primary RPE cells compared to commercial scaffolds

Abstract

Purpose: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. Methods:We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm.Results: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehydebinding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na + /K + ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of a-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. Conclusions:Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

The Effects of the Coating and Aging of Biodegradable Polylactic Acid Membranes on In Vitro Primary Human Retinal Pigment Epithelium Cells

“…Before performing the experiments, the cultures were carefully rinsed with DPBS and the cells were harvested by incubating the cultures on TrypLE TM (Gibco ® , Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at 37 • C in a 5% CO 2 atmosphere. The cells were then seeded on either commercially available inserts (Corning Transwell polycarbonate (PC) membrane cell culture inserts, 6.5 mm diameter, 0.4 µm pore) or coated or uncoated electrospun polylactide-based (PLA) membranes (PLA membranes characterized elsewhere, cell culture insert wit 10 mm diameter, PLA membrane with 0.4 µm pore size, porosity of 72%, membrane thickness of 3.7 µm, and fiber diameter of 380 nm) [2,10] at a density of 1.5 × 10 5 −1.8 × 10 5 cell/cm 3 . For coating, Matrigel ® was used at a concentration of 8.7 µg/cm 3 , which was deposited on target PLA membranes, cultured for 1 h at 37 • C, and subsequently rinsed with DPBS.…”

“…Several promising transplantation studies have already been performed on rodents and pigs [8,9]. Electrospun PLA-based membranes have especially exhibited excellent properties in comparison to commercial polyester inserts [10]. In general, one of the most important advantages of ultrathin nanofibrous PLA-based membranes in comparison to commercial membranes is that their thickness corresponds very well to the thickness of healthy Bruch's membrane to which RPE cells adhere in the eye.…”

“…For that reason, porous ultrathin PLA-based nanofibrous membranes can offer better support for growing cells, allowing for the physiological flow of nutrients and thus preserving the function and anatomy of the neuroretina. In our previous work, it was proven that a porcine primary RPE cell can be successfully cultivated even on a xenofree PLA-based membrane without using a biomimetic coating [10]. However, although PLA-based membranes appear to be a good starting point as carriers, they usually need to be coated with biomimetic components such as laminin or vitronectin [7,8] in order to ensure a better initial adhesion of cells and avoid the possible detachment of the cell layer during mechanical manipulation in subretinal implantation.…”

“…Here, we investigated how the prolonged storage of the PLA membrane influenced the maturation of primary human retinal pigment epithelial cells (hRPE). We evaluated Matrigel-uncoated and coated PLA membranes [8][9][10][11] to determine their influence on hRPE morphology, pigmentation, epithelial barrier function, genomic integrity, and the expression of maturity markers.…”

Progress in Stem Cells-Based Replacement Therapy for Retinal Pigment Epithelium: In Vitro Differentiation to In Vivo Delivery

“…Ultrathin nanofibrous scaffolds based on degradable polylactide (PDLLA) facilitated the physiological flow of nutrients through porosity over 70% and thus preserved the functioning of RPE cells. 88 Ultrathin PDLLA nanofibrous membrane with thickness of 3-4 µm corresponds with thickness of Bruch’s membrane of 2-6 µm, however, the wet ultrathin membrane is difficult to handle during biological and surgical manipulations. Popelka et al reported embedding of the peripheral frame into the nanofibrous PDLLA membrane that enabled the ultrathin membrane to be handled without irreversible folding and allowed the membrane to regain its flat shape when transplanted subretinally.…”