Automated production of patient derived iPSCs for autologous photoreceptor cell replacement.

Abstract

Purpose : In this study we describe the use of custom designed robotics and ISO Class 5 cGMP cell culture atmospheric isolators for the high throughput production of clinical grade patient specific iPSCs and retinal organoids containing transplantable photoreceptor cells.

Methods : Patient derived fibroblast were isolated from 27 individuals, ranging from 6 to 92 years of age, with molecularly confirmed inherited retinal degenerative blindness. Cells were used for iPSC generation using Sendai virus. Oxygen tension was dropped from 20-10% and cultures are subsequently fed daily with E8 media and passaged onto laminin 521 coated 6 well culture plates. Over the next 21 days cultures were fed daily, imaged every 3-5 days, picked, and clonally expanded using the CellX robot. IPSCs were subjected to karyotyping and score card analysis prior to being differentiated using a cGMP compliant 3D differentiation protocol. Differentiated organoids were subjected to immunohistochemistry and single cell RNA sequencing.

Results : Robot generated iPSCs were determined to be pluripotent via score card analysis and no significant difference in the rate of karyotypic abnormalities was detected between iPSCs generated under manual or automated conditions (P>0.05). Robot generated iPSCs gave rise to retinal organoids that were indistinguishable from their manually generated counterparts as determined via immunostaining and confocal microscopy using antibodies targeted against NRL, rhodopsin, cone opsin, cone arrestin and recoverin. At 120 days, retinal organoids were dissociated and subjected to single cell RNA sequencing and were compared to cells generated in the Reh laboratory (scRNAseq comparison dataset PMID 32023475). Clustering analysis demonstrated that cells generated on the robotic platform were comparable to those generated under manual conditions in a separate laboratory. Specifically, each of the expected retinal cell types were present in roughly the same proportions between those generated in both groups.

Conclusions : We successfully developed a high throughput robot iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.