One-Stop Assembly of Adherent 3D Retinal Organoids from Human Induced Pluripotent Stem Cells based on PDMS Microwell Chip

Abstract

Purpose : The three-dimensional (3D) retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs) 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 chip for the one-stop culture of ROs.

Methods : We fabricated a positive mold using 3D printing. Then, a complementary PDMS mold for the microwell was generated by replicating the 3D printing mold. The adherent 3D ROs could be assembed in PDMS microwell chip. We further optimized the PDMS chip by adding microfluidics, In addition, We have also established a disease model of ROs for a retinitis pigmentosa patient with the PRPF8 mutation.

Results : The microwell chip was placed in a 6-well plate for the culture of ROs (Fig.1A). The aggregated differentiated from hiPSCs were semi-attached in the PDMS microwell chip (Fig.1B), and fewer apoptotic cells in the PDMS microwell platform compared with that in 96-well V-bottomed plate (Fig.1C). Notably, the adherent 3D ROs contained the NR-CM-RPE three domains without requiring BMP4 and Matrigel (Fig.1D). Moreover A one-stop long-term culture of adherent 3D ROs was achieved in PDMS microwell chip (Fig.2A). Retinal ganglion cells, amacrine cells, horizontal cells, cone, and rod photoreceptor cells, bipolar cells, and Müller glial cells differentiated gradually (Fig.2B). Furthermore, the application of human platelet lysate (HPL) was enabled to establish a xeno-free workflow. In the perfusion culture mode, RO has better activity than static culture. finaly we found the disrupted photoreceptors was occurred in ROs from a patient carrying the PRPF8 mutation, under this work flow.

Conclusions : The PDMS microwell chip could achieve a one-stop xeno-free workflow for long-term ROs culture, which also can provide vasculature-like perfusion. This chip would contribute to establishing the next generation of ROs, model retinal disease, and facilitate clinical translation and drug discovery research.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1 hiPSCs differentiate into ROs in PDMS microwell chip.

 

Figure 2 One-stop assembly of adherent 3D ROs in PDMS microwell chip for long-term culture.