Abstract
Purpose :
To create a structurally relevant model of human retinal pigment epithelium (RPE) and choroidal endothelial cells (CECs) in order to investigate diseases involving Bruch’s membrane.
Methods :
Human fetal RPE was dissected and cultured as reported previously. Dissected choroidal tissue was dissociated and expanded separately. Human CECs were enriched and selected as CD144+CD45-EpCam- populations via FACS sorting, followed by expansion and passaging. CECs and RPE were co-cultured in a modified transwell system and an engineered microvascular system. The modified transwell system consisted of CECs plated onto transwell filter inserts on or suspended in collagen for 2-7 days, and RPE cultured on top of collagen for an additional week. The microvascular system consisted of CEC-lined engineered collagen lumen, and CECs were cultured under flow. Both systems were fixed for either TEM or immunofluorescent staining for confocal 3D imaging.
Results :
CECs, identified as VeCad+, EpCam- and CD45- were 1.6% in the total single cell population of dissected choroidal tissue. In the modified transwell system, we found that RPE cultured on collagen I (3mg/mL) alone was least favorable for RPE survival and morphology, while RPE cultured on collagen mixed with CECs demonstrated accelerated maturation with the development of apical microvilli and robust RPE pigmentation by 7 days in culture. CECs were observed to migrate through the collagen matrix (nearly 100 mm thick) and form a layer underlying polarized RPE. A mean thickness of 0.752 micrometers (SE 0.075) collagen separated the CECs from the basal aspect of the RPE. In the engineered microvascular system, our preliminary studies showed the successful co-culture and maintenance of RPE on collagen scaffolds supported with engineered perfusable, 3D microvessel networks in which the lumen were lined with CECs or human umbilical vein endothelial cells.
Conclusions :
In co-culture with CECs, human RPE appear to achieve maturation more quickly as determined by ultrastructure formation based on TEM imaging. CECs migrate through collagen to form a monolayer of cells separated by a thin layer of collagen from the overlying basal aspect of the RPE. RPE and CECs can also be co-cultured in a 3D microvascular network.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.