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Title: Cryopreservation of retinal organoids and applications of RPE cells for disease modelling in retinitis pigmentosa
Authors: Georgiou, Maria
Issue Date: 2021
Publisher: Newcastle University
Abstract: Generation of retinal cells from human induced pluripotent stem cells (hiPSCs) provides great opportunities for research and therapeutics. In this study, we generated hiPSC-derived retinal cells to evaluate their ability to be cryopreserved, shipped at room temperature (RT), and assess their application in disease modelling by studying the mechanisms causing Retinitis Pigmentosa (RP). To develop an effective cryopreservation protocol for the long-term storage of retinal organoids (ROs), previously published cryopreservation methods were used. The structure and presence of retinal cells in cryopreserved ROs were assessed by immunofluorescence analysis. The ‘Master Liver Supercooling’ protocol was the most promising among all in preserving the overall retinal structure. However, success was limited, and only PRs survived. To assess whether hiPSC-derived ROs can be shipped at RT, ROs were kept at RT for 5 days or shipped internationally for 3 days using a commercial container. The structure, morphology and function of ROs were assessed by immunofluorescence, transmission electron microscopy and electrophysiology, indicating no differences between control, RT incubated and shipped organoids. This study provides an effective shipping method to facilitate the transportation of ROs at RT. To identify the disease mechanisms of RP associated with PRPF31 mutations (known as RP11), proteomic analyses of hiPSCs-retinal pigmented epithelium (RPE) cells from control and RP11- patients were conducted. These showed that RNA splicing, retinoid metabolism and visual perception, and protein folding pathways were affected. RP11-RPE cells were characterised by reduced functional PRPF31 protein and the presence of insoluble aggregates containing mutant PRPF31, misfolded and ubiquitin-conjugated proteins. The waste disposal mechanisms were impaired exacerbating aggregate formation which was associated with cell death activation. Treatment of RP11-RPE cells with rapamycin (autophagy activator) reduced cytoplasmic aggregates and improved cell survival. This thesis highlights the applications of hiPSC-derived retinal cells and provides more insights in cryopreservation, transportation and potential therapeutics for RP.
Description: PhD Thesis
Appears in Collections:Biosciences Institute

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