Rethinking freshly isolated RPE culture: a steptowards Animal Origin-Free, 3R-aligned, and celltherapy conform conditions

Diplôme Master Rethinking freshly isolated RPE culture: a steptowards Animal Origin-Free, 3R-aligned, and celltherapy conform conditions eng 2026-08-25 author Morgado Alice 401790 director Kropp Martina 0000-0001-6861-5851 793095 Département des neurosciences cliniques (OTONO) 53 In cell culture, Fetal Bovine Serum (FBS) is widely used as a supplement. However, batch-to-batch variability, the emergence of the 3R principles in bioethics, and risks of zoonoses and contamination in clinical cell product manufacturing have raised concerns regarding reproducibility, ethics and safety. Human platelet lysate (hPL) has emerged as a promising alternative to FBS for culturing stem cells and cell lines. However, animal origin-free (AOF) culture of freshly isolated post-mitotic cells, such as Retinal Pigment Epithelial (RPE) cells, remains challenging. We therefore established and optimized AOF culture conditions for primary porcine (p)RPE cells to support future translation to human (h)RPE cell therapy applications. The study was divided into screening (7 days) and validation (21 days) phases. Primary pRPE cells were cultured in DMEM/Ham's F12 supplemented with 6, 8, 10, 15, 20% hPL and 10% FBS and serum-free medium were used as controls. Cells were cultured in 6-well plates (cells from 3 eyes/well) or 48-well plates (400,000 cells from a pool of 6–9 eyes/well). For the screening phase, cell morphology, pigmentation, cell number and viability were evaluated. Based on these results, 8% and 10% hPL were selected for validation and compared with 10% FBS and serum-free conditions. RPE65 and ZO-1 expression were assessed by RT-qPCR and immunostaining, while PEDF secretion and epithelial barrier function were evaluated by ELISA and TEER, respectively. Different coating materials (hCollagen IV, rh-Laminin-521 and rh-Vitronectin-N) were also tested. Finally, TrypLE™ Express was evaluated as an alternative to Trypsin, and primary human RPE cells were cultured under selected AOF conditions. Overall, pRPE cells cultured in 6-well plates showed higher cell counts than those cultured in 48-well plates. After 7 and 21 days, 8% and 10% hPL supported the highest cell numbers, desired cobblestone morphology and pronounced pigmentation. After 21 days, 10% hPL showed a trend towards increased ZO-1 and RPE65 expression compared with 10% FBS, consistent with immunostaining results. Promising PEDF secretion and a steady increase in TEER were also observed. rh-Laminin-521 and rh-Vitronectin-N with 10% hPL showed encouraging results, whereas TrypLE™ Express was less effective than Trypsin. Human RPE cells were more sensitive to medium changes but few survived in medium supplemented with 8% hPL and 1% ITS. This study demonstrates that hPL can support RPE morphology, viability and functionality while reducing reliance on animal-derived FBS. Plate format and coating conditions also influence RPE culture outcomes, highlighting the need for further optimization towards robust and clinically relevant AOF culture conditions. RPE AOF HPL Human Platelet Lysate 3R Post-mitotic cells Primary cells Animal Origin-Free Retinal Pigment Epithelium Dewey 616.8 Dewey 590 44 Département de génétique et évolution 162 61 Département des neurosciences cliniques 226 false 582 Ophtalmologie expérimentale 925 8bf9e40f-2d35-4318-88d3-008a0d2bfeb3 Master thesis Restricted MasterThesis_AliceMorgadodeAlmeida2.pdf 14369591 application/pdf f3bfa871ecea2705343fb911b630d850447a4fd248517bff0cc0d45731e040f5 restrictedAccess Faculté des sciences / Section de biologie / Département de génétique et évolution Faculté de médecine / Section de médecine clinique / Département des neurosciences cliniques

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Rethinking freshly isolated RPE culture: a steptowards Animal Origin-Free, 3R-aligned, and celltherapy conform conditions				2026	1	0

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Morgado, Alice

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