Maturation and Protection Effect of Retinal Tissue-Derived Bioink for 3D Cell Printing Technology

Abstract

Retinal degeneration is a leading cause of incurable vision loss and blindness. The increasing incidence of retinal degeneration has triggered research into the development of in vitro retinal models for drug development and retinal alternatives for transplantation. However, the complex retinal structure and the retinal microenvironment pose serious challenges. Although 3D cell printing technology has been widely used in tissue engineering, including in vitro model development and regeneration medicine, currently available bioinks are insufficient to recapitulate the complex extracellular matrix environment of the retina. Therefore, in this study, we developed a retinal decellularized extracellular matrix (RdECM) from the porcine retina and evaluated its characteristics. The RdECM conserved the ECM components from the native retina without cellular components. Then, we mixed the RdECM with collagen to form a bioink and confirmed its suitability for 3D cell printing. We further studied the effect of the RdECM bioink on the differentiation of Muller cells. The retinal protective effect of the RdECM bioink was confirmed through a retinal degeneration animal model. Thus, we believe that the RdECM bioink is a promising candidate for retinal tissue engineering.

Keywords: 3D cell printing; bioink; retina; tissue engineering; vision.

Figure 1

Characterization of retinal decellularized extracellular matrix (RdECM). (a) Biochemical analysis of collagen and glycosaminoglycans (GAGs) in the native retina (Native) and RdECM. (b) Immunohistochemical analysis of native retina (Native) and RdECM. Scale bar: 200 µm. The error bars represent the standard deviation.

Figure 2

Proteomic analysis of retinal decellularized extracellular matrix (RdECM). (a) Molecular function. (b) Biological process. (c) Cellular component. (d) Matrisome (Core-Col: Core matrisome-Collagens, Core-Pro: Core matrisome-Proteoglycans, Core-ECM: Core matrisome-ECM Glycoproteins, Mat-Sec: Matrisome-associated secreted factors, Mat-ECM: Matrisome-associated ECM-affiliated proteins).

Figure 3

Rheological analysis and printability test of retinal decellularized extracellular matrix (RdECM). (a) Viscosity. (b) Complex modulus. (c) Sol-gel transition test. (d) Printability test (30-gauge nozzle). The error bars represent the standard deviation.

Figure 4

Proliferation and viability of printed MIO-M1 cells. (a) Proliferation and (b) Live/Dead assay of MIO-M1 cells encapsulated in retinal decellularized extracellular matrix (RdECM) bioink or collagen. The error bars represent the standard deviation. Scale bar: 100 µm. The data were compared using two-way ANOVA and differences were considered significant when * for p < 0.05 and *** for p < 0.001.

Figure 5

Effect of retinal decellularized extracellular matrix (RdECM) bioink for Muller cell differentiation (rhodopsin: Rho, blue opsin: BOp, red-green opsin: RGOp, glial fibrillary acidic protein: GFAP, glutamine synthetase: GS). Scale bar: 20 µm.

Figure 6

Retinal protective effect of retinal decellularized extracellular matrix (RdECM). (a) FITC-dextran image of the retina from Laser-CNV model mice. Red: LASER irradiated lesion. Yellow: vascular structure. (b) Remaining vascular area in the Laser-CNV model. (c) H&E staining images of the retina from NMU-RD model mice. (d) Total retinal thickness in the NMU-RD model. The error bars represent the standard deviation. The data were compared using the Student’s t-test and differences were considered significant when *** for p < 0.001. Scale bar: (a) 100 µm, (c) 50 µm.