One-stop assembly of adherent 3D retinal organoids from hiPSCs based on 3D-printed derived PDMS microwell platform

Abstract

The three-dimensional (3D) retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs), mimicking the growth and development of the human retina, is a promising model for investigating inherited retinal diseases (IRD) in vitro. However, the efficient generation of homogenous ROs remains a challenge. Here we introduce a novel Polydimethylsiloxane (PDMS) microwell platform containing 62 V-bottom micro-cavities for the ROs differentiation from hiPSCs. The uniform adherent 3D ROs could spontaneously form using neural retina (NR) induction Our results showed that the complex of NR (expressing VSX2), ciliary margin (CM) (expressing RDH10), and retinal pigment epithelium (RPE) (expressing ZO-1, MITF, and RPE65) developed in the PDMS microwell after the differentiation. It is important to note that ROs in PDMS microwell platforms not only enable one-stop assembly but also maintain homogeneity and mature differentiation over a period of more than 25 weeks without the use of BMP4 and Matrigel. Retinal ganglion cells (expressing BRN3a), amacrine cells (expressing AP2a), horizontal cells (expressing PROX1 and AP2α), photoreceptor cells for cone (expressing S-opsin L/M-opsin) and rod (expressing Rod opsin), bipolar cells (expressing VSX2 and PKCα), and Müller glial cells (expressing GS and Sox9) gradually emerged. Furthermore, we replaced fetal bovine serum (FBS) with human platelet lysate (HPL) and established a xeno-free culture workflow that facilitates clinical application. Thus, our PDMS microwell platform for one-stop assembly and long-term culture of ROs using a xeno-free workflow is favorable for retinal disease modeling, drug screening, and manufacturing ROs for clinical translation.

TGF-β Isoforms and Local Environments Greatly Modulate Biological Nature of Human Retinal Pigment Epithelium Cells

“…As a limitation of this study, it was not determined why the TGF-β isoforms involved in EMT of the RPE are also modulated by oxygen levels (normoxia vs. hypoxia) and local environments (2D vs. 3D). In terms of a 3D culture approach related to the RPE, the aims of most studies were focused on future transplantation therapy against various retinal diseases using transplantation of human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs) [ 68 , 69 , 70 ]. However, our study approach using both 2D and 3D experiments in the RPE to determine various factors such as TGF-β, oxygen conditions, and other factors is quite unique and original.…”

Application of patient-derived induced pluripotent stem cells and organoids in inherited retinal diseases

“…Recently, Slembrouck-Brec et al described a defined xeno-free and feeder-free culture condition for the generation of human iPSC-derived ROs and RPE cells [ 102 ]. In our previous study, fetal bovine serum was replaced with human platelet lysates to establish a xeno-free ROs culture workflow that facilitates clinical application [ 103 ].…”

“…For example, ROs often vary in size and quality, contain some off-target tissues and their development may be inconsistent [ 82 ]. We previously used 3D-printed polydimethylsiloxane (PDMS) microwell platform for adherent ROs cultivation [ 103 ]. Unlike suspended ROs on ultralow adhesion microwell plates, iPSC-derived ROs on PDMS molds were confined to their respective microcavities but shared the same medium and microenvironment, which could not only avoid the fusion of multiple ROs but also ensure the long-term culture and survival of ROs, resulting in efficient and homogeneous ROs with fewer apoptotic cells (Fig.…”