Hypoxic Retinal Precursor Cells Co-Cultured with Neural Progenitor Cells

Immortalized R28 retinal precursor cells were cultured as previously described [9 (link)]. H9 human ESCs (WiCell Research Institute, Madison, WI, USA) were routinely maintained on Matrigel-coated culture dishes (BD Biosciences, San Jose, CA, USA) in TeSR™-E8™ medium (STEMCELL Technologies, Vancouver, BC, Canada). The culture process for NPCs is detailed in a recent publication [8 (link)]. A hypoxic environment was created in R28 cells using cobalt chloride (CoCl₂). R28 cells were seeded at a density of 2 × 10⁵ and then treated with 300 µM CoCl₂ for 3 h. Following this, NPCs were added to the damaged R28 cells. After 24 h, the cells were harvested and prepared for analysis.