We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.
Total time
~3–5 days (dissection ~1 hour; isolation ~2 hours; culture to confluence ~3–4 days)
Model organism
Human fetal retinal pigment epithelial cells (hfRPE)
Steps
1
Understand retinal pigment epithelium biology and function
Review anatomical and physiological background on RPE tissue, its role in the eye, and the rationale for in vitro modeling of eye diseases.
▶ 01:08
2
Dissect human fetal eye tissue
Perform careful dissection of the human fetal eye to isolate the retinal pigment epithelium layer for downstream cell culture.
▶ 03:26
3
Isolate and dissociate hfRPE cells
Enzymatically digest and mechanically dissociate the isolated RPE tissue to obtain single-cell suspension suitable for monolayer culture.
▶ 05:57