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B.C. Barron, J. Wen, E. Garcia, J.A. Kapp; Isolation and Characterization of Neural Retinal Progenitor Cells from the Murine Ciliary Margin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1693.
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Purpose: To isolate and culture neurospheres from the ciliary margin of green fluorescent protein (GFP) transgenic mice and C57/BL6 mice ciliary margin and characterize the resulting neural retinal progenitor cells (NRPCs). Methods: Whole eyes from Balb/C and C57/BL6 were placed in an artificial cerebral spinal fluid (aCSF) and dissected to remove the ciliary margin tissue. The dissected tissue was place in Dispase and incubated to facilitate removal of the underlying basement membrane. The tissue was further dissected in aCSF containing trypsin, centrifuged and resuspended in DMEM/F12 containing a trypsin inhibitor. The isolated neurospheres were cultured in a serum-free media for 5-7 days. Single sphere colonies were dissociated by enzymes and cultured on poly-lysine/laminin coated plates in DMEM/F12 containing 1% FBS. Confluent cultures were fixed in 4% paraformaldehyde, stained with the primary antibodies Ret-P1 (anti-opsin), MAP2, Nestin, GFAP, and α-internexin and secondary antibodies Alexa Fluor 488 (Ex/Em: 495/519), Alexa Fluor 568 (578/603), and Alexa Fluor 660 (663/690). Cells were counterstained with DAPI. Positive controls were sectioned retina; negative controls were sectioned spleen. Results: Neurospheres isolated from the murine ciliary margin were cultured in a serum-free media. Any contaminating ocular cells did not survive after 5-7 days in this media. The resulting neurospheres were dissociated and cultured in a low-serum media (1% FBS). The cultured cells were identified as neural retinal progenitor cells (NRPCs) by immunocytochemistry using specific markers. NRPCs were cultured and stained from 1-4 passages. Conclusions: Immunocytochemistry showed isolated neurospheres from the ciliary margin of Balb/C and C57/BL6 mice differentiated to neural retinal progenitor cells in vitro.
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