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.

Cellularized Microcarriers as Adhesive Building Blocks for Fabrication of Tubular Tissue Constructs

“…By contrast, little to no inter-microcarrier bead cellular bridging was apparent when Cultisphers containing only endothelial cells (HUVECs) were used ( data not shown ). This observation is similar to other findings showing that co-culture of endothelial cells with retinal cells on microcarrier beads was required to stimulate endothelial cells to form endothelial cords that interconnect microcarrier beads 35 .…”

Cellularized Microcarriers as Adhesive Building Blocks for Fabrication of Tubular Tissue Constructs

“…By contrast, little to no inter-microcarrier bead cellular bridging was apparent when Cultisphers containing only endothelial cells (HUVECs) were used ( data not shown ). This observation is similar to other findings showing that co-culture of endothelial cells with retinal cells on microcarrier beads was required to stimulate endothelial cells to form endothelial cords that interconnect microcarrier beads 35 .…”

Simulated microgravity promotes nitric oxide‐supported angiogenesis via the iNOS–cGMP–PKG pathway in macrovascular endothelial cells

“…Dutt et al [12] first demonstrated that simulation of microgravity by horizontally rotating bioreactor upregulates growth factors and promote cellular differentiation and three‐dimensional assembly, a prerequisite for blood vessel formation [12]. Recently, Infanger et al [13] showed that after 72 h of clinorotation, EC assemble to three‐dimensional tubular structures [13].…”

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.…”

Cellularized Microcarriers as Adhesive Building Blocks for Fabrication of Tubular Tissue Constructs

“…By contrast, little to no inter-microcarrier bead cellular bridging was apparent when Cultisphers containing only endothelial cells (HUVECs) were used ( data not shown ). This observation is similar to other findings showing that co-culture of endothelial cells with retinal cells on microcarrier beads was required to stimulate endothelial cells to form endothelial cords that interconnect microcarrier beads 35 .…”

Simulated microgravity promotes nitric oxide‐supported angiogenesis via the iNOS–cGMP–PKG pathway in macrovascular endothelial cells

“…Dutt et al [12] first demonstrated that simulation of microgravity by horizontally rotating bioreactor upregulates growth factors and promote cellular differentiation and three‐dimensional assembly, a prerequisite for blood vessel formation [12]. Recently, Infanger et al [13] showed that after 72 h of clinorotation, EC assemble to three‐dimensional tubular structures [13].…”

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.…”

Cellularized Microcarriers as Adhesive Building Blocks for Fabrication of Tubular Tissue Constructs

“…By contrast, little to no inter-microcarrier bead cellular bridging was apparent when Cultisphers containing only endothelial cells (HUVECs) were used ( data not shown ). This observation is similar to other findings showing that co-culture of endothelial cells with retinal cells on microcarrier beads was required to stimulate endothelial cells to form endothelial cords that interconnect microcarrier beads 35 .…”

Simulated microgravity promotes nitric oxide‐supported angiogenesis via the iNOS–cGMP–PKG pathway in macrovascular endothelial cells

“…Dutt et al [12] first demonstrated that simulation of microgravity by horizontally rotating bioreactor upregulates growth factors and promote cellular differentiation and three‐dimensional assembly, a prerequisite for blood vessel formation [12]. Recently, Infanger et al [13] showed that after 72 h of clinorotation, EC assemble to three‐dimensional tubular structures [13].…”

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.…”