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Isolation and Culture of Primary Mouse Retinal Pigment Epithelial (RPE) Cells with Rho-Kinase and TGFβR-1/ALK5 Inhibitor
摘要： Primary RPE cells could be a reliable model for representing in vivo status of RPE compared with cell lines. We present a protocol for in vitro isolation and culture of primary RPE cells from C57BL mice. We used C57BL mice ages 7 days to 4 months. The RPE layer was separated from the neural retina layer by digestion with 2% Dispase for 45 min and scraped off from the choroid after 25-min incubation in 37°C. Collected RPE sheets were gently pipetted up into smaller sheets. RPE sheets were transferred into well plates and cultured in vitro for 2 weeks. To inhibit epithelial-mesenchymal transition (EMT) of RPE cells, we used Y27632 and Repsox to treat cultured primary RPE cells. RPE cells isolated from C57BL mice maintained pigmented and hexagonal morphology in culture. However, long-term in vitro culture lead to the periphery cells of a RPE sheet becoming mesenchymal-like cells. In contrast to the control group, Y27632 and Repsox, which are inhibitors of Rho-kinase or TGFbR-1/ALK5, promoted primary RPE cells to maintain epithelial-like morphology and eventually become confluent. RPE cells isolated from C57BL mice could be a powerful cell model to study the biological function of RPE. Especially, C57BL mice with different defective genetic background resulting in ocular diseases, would expand the genome type of RPE cells. The method presented here could be an efficient and applicable technique to obtain large numbers of primary RPE cells that maintain some characteristics of in vivo RPE.
关键词： Primary Cell Culture,Mice,Retinal Pigment Epithelium,Inbred C57BL
更新于2025-09-23 15:23:52
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Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells
摘要： Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.
关键词： Developmental Biology,Somitogenesis,Biological Clocks,Primary Cell Culture,Primary Culture,Zebrafish,Oscillator,Fluorescence,In Vitro,Time-lapse Imaging
更新于2025-09-10 09:29:36