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Master
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Establishment of a FBS- and xeno-free culture system for low numbers of freshly isolated (and transfected) Iris Pigment Epithelial (IPE) and induced Pluripotent Stem cell (iPS)-derived Retinal Pigment Epithelial (RPE) cells

ContributorsBreuillard, Nathan
Defense date2022-02-11
Abstract

Current in vitro cell culture systems frequently use animal-derived components to support cell growth. Iris Pigment Epithelial (IPE) cells proliferate in vitro, if seeded in sufficient cell number with Fetal Bovine Serum (FBS) as supplement. However, in the pre-clinical context, a xeno-free system is highly recommended and furthermore, FBS is subject to major ethical and scientific controversies. We are developing a gene therapy approach, in which autologous IPE cells will be genetically modified to overexpress the anti-angiogenic pigment epithelium-derived factor (PEDF) to treat neovascular Age-Related Macular Degeneration (nvAMD), a major cause of blindness in elderly people. To prove both, safety and efficacy of the cell product, 5’000 to 50’000 IPE cells need to be cultured in physiological, xeno-free conditions. For this purpose, the present project aimed to establish FBS-free culture conditions using human Platelet Lysate (hPL) in different concentrations as medium supplement alternative for low numbers of freshly isolated human and porcine IPE (hIPE, pIPE) cells cultured for 2 to 4 weeks. The culture system was additionally enhanced by the co-culture of pIPE with mitotically arrested ARPE-19 feeder cells, the use of commercial growth factors, and a commercially available matrix. Microscopical analyses, staining and biochemical assays were performed to assess cell morphology, cell number and viability. The project could identify the best hPL concentration of 3% for IPE cell proliferation, though the culture system remained less efficient than the classical FBS-based system. The secondly performed implementation of a xeno-free and economic culture system for iPS cells could be successfully finalized allowing the broadening of the gene therapy approach to iPS-derived RPE cells.

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Citation (ISO format)
BREUILLARD, Nathan. Establishment of a FBS- and xeno-free culture system for low numbers of freshly isolated (and transfected) Iris Pigment Epithelial (IPE) and induced Pluripotent Stem cell (iPS)-derived Retinal Pigment Epithelial (RPE) cells. 2022.
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Master thesis
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  • PID : unige:159634
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Creation02/24/2022 12:32:00 PM
First validation02/24/2022 12:32:00 PM
Update time03/16/2023 2:53:13 AM
Status update03/16/2023 2:53:12 AM
Last indexation02/01/2024 7:56:53 AM
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