(165y) A Physiologically Relevant 3D in-Vitro Model of Retinal Degenerative Diseases

Statement of Purpose: Age-related macular degeneration (AMD) is one of the incurable retinal diseases and the leading cause of central vision blindness in people over the age of 50 worldwide ¹. AMD can present in two forms of ‘dry’ or ‘wet’ (Neovascular form) that ultimately lead to photoreceptor cell death¹. Retinal organoids (ROs) derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are advancing to potentially replace animal models for drug development and be used to model retinal diseases such as AMD ². Current retinal organoids’ protocols are mostly focused on the enrichment of specific cells, such as photoreceptors. Retinal organoid disease models also generally lack vasculature. Yet studying interactions between vasculature and retinal tissue may be highly important in modeling certain diseases such as AMD. Of particular interest is the close association of photoreceptors and retinal pigment epithelium (RPE) in the presence of vasculature which mimics the outer blood-retinal barrier, which is important in modeling wet AMD ^3-5. In this study, we investigated the interaction of retinal organoids derived from mouse iPSCs (miPSCs) and human umbilical vein endothelial cells (HUVECs) in three experimental approaches, namely, 1. culture of mature organoids along with HUVECs in a dish at early and late stages of RO maturation, 2. co-culture of miPSCs and HUVECs, and 3. use of a microfluidic system to model angiogenesis.

Methods: Retinal organoids were cultured in Matrigel as a control and an alginate-based gel system containing hyaluronic acid (HA) in 96 U-well plates until 8 days, and subsequently transferred to 6-well plates to further mature in media customized for retinal maturation (Optic cup (OC) medium). GFP-labeled HUVECs were mixed with organoids at two stages of organoid maturation and plated on 24-well plates for a week of culture in 1:1 serum‐free basal media (Vasculife®): OC media. Growth and interaction with the organoids were monitored using fluorescence microscopy and immunostaining of organoids for PECAM-1 (endothelial cell marker).

Co-culture of miPSCs and HUVECs was done in 1:1 media composition and resulted in organoid-like structures that were grown for 25 days using the same protocol stated above. Organoids were fixed and immunostained for rhodopsin (photoreceptor cell marker) and CD31/PECAM-1. In an assay to test if endothelial cells could develop perfusable vessels in 1:1 media composition without any organoids involved, individual cells were grown and allowed to interconnect and form into a vessel network over 72 h, followed by lumen formation⁶.

Results: In the mixed culture of mature organoids and HUVECs, endothelial cells grew well in 1:1 media and seemed to grow around and within the mature organoids (Fig. 1). To confirm whether HUVEC cells penetrated the organoids, ROs were fixed after one week and stained with CD31/PECAM-1. The immunofluorescent imaging showed clear expression of CD31/PECAM-1 in the retinal organoids matured from day 9 to day 16 (Fig. 2).

Co-culture of miPSCs with endothelial cells generated self-organized retinal organoids, with immunostaining showing the expression of endothelial cells in the outer layer of organoids. However, neural retina cellular organization was lost, and the co-culture system did not promote photoreceptor organization or maturation (Fig. 3).

To model angiogenesis with retinal organoids, the 1:1 media composition was first examined for ability to support microvessel formation in the fluidic device. Based on these analyses, OC media composition was adjusted to allow maintenance of healthy endothelial cells and perfusable vessels. Eliminating lipid factors from RO media aided the formation of perfusable vasculature, and microvessels were developed over the course of approximately one week (Fig. 4).

Conclusion: In modeling the wet form of AMD, a more physiologically relevant model of neural retina, incorporating RPE and choroid capillary layers can better capture in vivo structure and give insight into disease stages. The preliminary results herein support the feasibility of developing a vascularized retinal organoid as a first step in creating a useful model for understanding neovascular macular degeneration.

References

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