Three-Dimensional Model of Angiogenesis: Coculture of Human Retinal Cells with Bovine Aortic Endothelial Cells in the NASA Bioreactor

Abstract

Ocular angiogenesis is the leading cause of blindness and is associated with diabetic retinopathy and age-related macular degeneration. We describe, in this report, our preliminary studies using a horizontally rotating bioreactor (HRB), developed by the National Aeronautics and Space Administration (NASA), to explore growth and differentiation-associated events in the early phase of ocular angiogenesis. Human retinal (HRet) cells and bovine endothelial cells (ECs) were cocultured on laminin-coated Cytodex-3 microcarrier beads in an HRB for 1-36 days. Endothelial cells grown alone in the HRB remained cuboidal and were well differentiated. However, when HRet cells were cocultured with ECs, cordlike structures formed as early as 18-36 h and were positive for von Willebrand factor. In addition to the formation of cords and capillary-like structures, ECs showed the beginning of sprouts. The HRB seems not only to promote accelerated capillary formation, but also to enhance differentiation of retinal precursor cells. This leads to the formation of rosette-like structures (which may be aggregates of photoreceptors that were positive for rhodopsin). Upregulation of vascular endothelial growth factor and basic fibroblast growth factor was seen in retinal cells grown in the HRB as compared with monolayers and could be one of the factors responsible for accelerated capillary formation. Hence, the HRB promotes three-dimensional assembly and differentiation, possibly through promoting cell-to-cell interaction and/or secretion of growth and differentiation factors.

Engineering Retina from Human Retinal Progenitors (Cell Lines)

“…These progenitors when grown attached to cytodex beads in the NASA-developed rotating culture vessel system and formed large 3D constructs. 50,51 We realized that until well-defined biocompatible substrates could be generated, we will be unable to test these transplants in an animal model.…”

Vascularization Strategies for Tissue Engineering

“…74,75 The horizontally rotating bioreactors, or slowturning lateral vessels, were designed as a rotating cylinder of culture medium, with a gas-permeable membrane for oxygen and carbon dioxide exchange, and fittings for sampling or medium exchange. 75,76 The speed of the rotation could be controlled to match the settling velocity of the tissue constructs, keeping them suspended within the vessel. This approach has been used for engineering of bone, cartilage, and cardiac tissue.…”

“…In coculture, the endothelial cells formed cords and capillary-like structures as well as the beginning of sprouts, indicators of a developing vasculature. 75 …”

Bioreactors Addressing Diabetes Mellitus

“…38 Due to the vast mechanical and pathological difference in the cause of type 1 versus type 2 diabetes mellitus, it is only logical for therapy and treatment of the 2 diseases to also vary significantly. Since type 2 diabetes mellitus is typically tied to other health complications, many tissue engineers have taken to bioreactor technologies to address the diseases' symptoms and characteristics, including diabetic retinopathy 41 and foot wound ulcers. 42,43 Dutt et al applied the horizontally rotating bioreactor developed by NASA to establish a coculture of human retinal cells and bovine endothelial cells incorporated onto laminin-coated Cytodex-3 microcarrier beads over 36 days.…”

“…42,43 Dutt et al applied the horizontally rotating bioreactor developed by NASA to establish a coculture of human retinal cells and bovine endothelial cells incorporated onto laminin-coated Cytodex-3 microcarrier beads over 36 days. 41 The bioreactor was reported to accelerate capillary formation as well as differentiation of retinal precursor cells. With such neovascularization modeling, the bioreactor system could provide an ideal 3-dimensional platform to study retinal diseases, including diabetic retinopathy.…”