Human retinal pigment epithelial (RPE) cells in culture secrete and accummulate drusen-like material that forms visible microscopic debris within weeks in vitro rather than decades in vivo (PMID: 28146236). To develop a 3D model that mimics the retinal/choroidal interface, we tested various endothelial cells frequently used in tube formation assays to ask if such a co-culture system can be maintained for an extended period of time. We set out to establish a long-term co-culture system using RPE (primary and human embryonic stem cell-derived) and endothelial (primary retinal microvascular endothelal [HRMVEC]) and immortalized hTERT-HRMVEC) cells. RPE differentiation medium alone, or mixed with spent medium collected from the basal chamber of RPE cells were used for endothelial cell growth. The effects of these culture conditions on endothelial cells were compared to the commercially recommended one. The morphology of the vascular network was analyzed by tube formation assay (Angiogenesis Analyzer tool of ImageJ) and LDH release was measured for cell toxicity. Primary RPE and HRMVEC were also characterized in co-culture using immunochemistry for pMEL17 (for RPE pigmentation), CD31 and UEA-1 (for endothelial cell morphology). Only the primary HRMVEC and immortalized hTERT-HRMVEC cells were able to keep their vascular characteristics (>4 weeks) when kept in RPE medium. The total meshwork area was significantly lower (p<0.0001) for both cell lines, without any increase in number of isolated branches. LDH release was reduced after 21 days in culture (24.2±1.1%, 31.6±1.8% for primary and immortalized cells in RPE differentiation medium, respectively). Besides, the addition of conditioned medium of RPE cells led to further decrease of LDH release (23.1±0.9% and 21.4±2%). When RPE and endothelial cells were grown is co-culture, endothelial cells retained their vascular characters. Simultaneously, RPE cells also developed extensive pigmentation (pMEL17-positivity), and showed a characteristic cobblestone morphology. RPE differentiation media is compatible with long-term endothelial cell mono- and co-culture. The total meshwork of microvascular endothelial cells remained stable without increasing cell toxicity. These data strongly suggest that it is possible to develop 3D model system mimicking the choroidal/RPE interface in long-term cultures. This is a 2020 ARVO Annual Meeting abstract.
|Journal||Investigative ophthalmology & visual science|
|Publication status||Published - 2020|