Abstract
Purpose :
Stem cell-derived retinal organoids (RtOgs) offer a valuable platform for studying human retinal tissue in vitro and hold promise for in vivo transplantation. Addressing challenges of heterogeneous organoid manufacturing methods, we seek to optimize current RtOg protocols through chemical and physical manipulations. Additionally, we demonstrate ongoing tissue development in a microfluidic bioreactor system, providing a controlled environment with continuous media supply.
Methods :
Organoids were derived from a genetically modified human embryonic stem cell line expressing CRX-GFP (Collin et al., 2016). Two manipulations were tested: Neural Induction Medium with N2 supplement (NIM-N2) and NIM with B27 without retinoic acid supplement (NIM-B27 w/o RA) [Days 0 – 20], and whether 3D-suspended cultures with Matrigel dissolved in media (ultra-low attachment plates) yield more retinal organoids compared to 2D Matrigel-coated cultures. Embryonic body (EB) diameter was assessed on Days 1, 5, and 7 of differentiation. 3D tissue structures from low-attachment plates were categorized on Day 25 and 26 as: retinal, mostly retinal, mostly non-retinal, and non-retinal. Nine retinal organoids were transferred to a polydimethylsiloxane (PDMS) microfluidic bioreactor for long-term culture, with subsequent brightfield and fluorescence imaging taken every week.
Results :
Less variability in average EB diameter was observed across all nine EBs over three timepoints under NIM-N2 compared to NIM-B27 w/o RA, indicating a higher contribution of CRX-GFP cells to the EB structure in the NIM-N2 treatment. In low-attachment plates, significantly more retinal-labeled tissue was observed in NIM-B27 w/o RA treatment compared to NIM-N2 treatment. However, around Day 50, majority of the organoids disintegrated, reducing to nine RtOgs to use for our microfluidic bioreactor system. Utilizing a 3D-printed microfluidic chip plate holder, we captured brightfield and fluorescence images.
Conclusions :
Initial results suggest potential omission of NIM-N2 supplementation, and replacement with NIM-B27 w/o RA for the first 20 days. Additionally, there is promise for proper retinal organoid development and maturation in suspended culture, eliminating the need for dissections used in 2D-Matrigel cultures. Further experiments are required to validate these findings and establish an optimized RtOg protocol.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.