Engineering transferrable microvascular meshes for subcutaneous islet transplantation

Abstract

The success of engineered cell or tissue implants is dependent on vascular regeneration to meet adequate metabolic requirements. However, development of a broadly applicable strategy for stable and functional vascularization has remained challenging. We report here highly organized and resilient microvascular meshes fabricated through a controllable anchored self-assembly method. The microvascular meshes are scalable to centimeters, almost free of defects and transferrable to diverse substrates, ready for transplantation. They promote formation of functional blood vessels, with a density as high as ~220 vessels mm^-2, in the poorly vascularized subcutaneous space of SCID-Beige mice. We further demonstrate the feasibility of fabricating microvascular meshes from human induced pluripotent stem cell-derived endothelial cells, opening a way to engineer patient-specific microvasculature. As a proof-of-concept for type 1 diabetes treatment, we combine microvascular meshes and subcutaneously transplanted rat islets and achieve correction of chemically induced diabetes in SCID-Beige mice for 3 months.

Fig. 1

Fabrication and function of transferrable microvascular meshes via anchored self-assembly. a Schematic illustration of organization of vascular endothelial cells into a microvascular mesh, which is transferred and attached to a cellular device. In a poorly vascularized subcutaneous space, microvascular mesh can enhance vascularization and anastomoses with host vasculature to provide oxygen/nutrients to donor cells. b Design of micropillar arrangement (blue) and ASA-enabled cell organization (purple): the key to the ASA is that inner micropillars (yellow) provide a geometric template for cell self-assembly to form square mesh and boundary micropillars (green) serve as anchoring points for cell attachment to prevent assembled mesh structure from shrinking. c A fluorescent image of HUVECs expressing GFP in fibrin matrix organized into a square mesh (1 × 1 cm) on a micropillar substrate via ASA after 2 days of culture. d After 2 days of culture, a HUVEC square mesh (5 × 5 cm) is lifted from the micropillar substrate for transfer. The insert is a magnified image of HUVEC mesh and scale bar is 1 mm. e HUVEC microvascular mesh generates angiogenic sprouts (white arrows) when embedded in fibrin matrix during 2 weeks of culture in vitro. f Microscopic images of retrieved HUVEC microvascular mesh device show a high degree of vascularization after 2 weeks of subcutaneous implantation in SCID-Beige mice (n = 4)

Fig. 2

Simulation and characterization of the ASA-enabled microvascular meshes. a, b The contraction simulation shows an in-plane displacement contour plot of organized cellular mesh structure (a) and the normal stress distribution in the X (Cauchy stress component 11) direction (b) on a 4 × 4 micropillar substrate. The initial shape of cells and fibrin matrix is displayed in light gray. The micropillar diameter is 400 μm and micropillar-to-micropillar interval is 200 μm. The contracted region is marked as dotted purple ellipse and the junction region is purple circle. The displacement unit is μm and the unit of stress is mN μm⁻². c Cross-sectional images showing a HUVEC mesh suspended between micropillars. The micropillars are pseudo-colored as blue and HUVECs are pseudo-colored as purple. d SEM images of a HUVEC mesh (purple) at the inner and boundary regions on the micropillar substrate (blue). e Confocal images of a HUVEC mesh at the contracted and junction regions on the micropillar substrate showing the tubular structures. Human CD31 antibody is green, F-actin is red, and nucleus is blue. f Screenshots of a glass pipette poking a HUVEC mesh showing high resilience of the mesh

Fig. 3

Enhancement of vascularization and anastomoses in subcutaneous space of SCID-Beige mice. a Schematics and a digital photo of a Mesh device, which is a diffusion chamber with HUVEC meshes (~25 μm thick, purple) in the fibrin gel (gray) on the top and bottom. The diffusion chamber is a cylindrical cell container with a PDMS ring (blue) as the wall and two nylon grids (green) as the top and bottom. The dimensions of each component are labeled in the schematic. b Fluorescent images of randomly mixed cells (top) and microvascular mesh (bottom) placed on diffusion chambers after 2 days of culture in EGM-2 medium. HUVEC:NHDF = 9:1, Human CD31 antibody is green to show HUVEC, α-smooth muscle actin (α-SMA) antibody is red to show NHDF, and nylon grid is blue. c Cross-sectional hematoxylin/eosin staining images of retrieved devices after 14 days of implantation. Yellow arrowheads point to blood vessels with erythrocytes inside. d Density and area percentage of blood vessels at the interface between the device and panniculus carnosus muscle. n = 6 in the No cell and Random, and n = 8 in Mesh groups. Data are mean ± SEM; **P < 0.01, ***P < 0.001, NS (P > 0.05) no significant difference. One-way analysis of variance. e Cross-sectional immunostaining images of human (red) and mouse (green) CD31 antibodies showing the human and mouse blood vessels at the interface between the device and panniculus carnosus muscle. f Confocal images of perfused lectins bound to human (UEA-I, green) and mouse (GSL-I, red) endothelial cells, confirming the anastomoses between human and mouse vessels. g Blood-perfused human vasculatures anastomosed with mouse vascular system in Mesh device after 10 days of subcutaneous implantation. HUVEC-GFP is green and perfused dye DiI in vessels is red. h Representative immunostaining images of mature human vasculatures (human CD31 antibody is green) covered with perivascular cells (α-SMA antibody is red) in retrieved Random and Mesh device after 10 days of subcutaneous implantation. i The percentage of perivascular cell (PVC) coverage is 19 ± 9% (n = 3; mean ± SEM) and 65 ± 6% (n = 6) for the Random and Mesh devices, respectively. **P < 0.01. Unpaired two-tailed t test

Fig. 4

Improvement of re-vascularization of rat islets and diabetes correction in SCID-Beige mice. a Schematics and a microscopic image of rat islets (green) in a Mesh device. Microvascular mesh is red and nylon grid is blue. b Non-fasting blood glucose (BG) concentration of the mice after transplantation. Grafts were retrieved after different time points. Most of the mice were kept alive for 1 week after retrieval while 3 mice from the Mesh group were used for perfusion studies prior to retrievals. During 42 days of transplantation (n = 9 in No cell, n = 11 in Random, and n = 14 in Mesh): *P < 0.05 and NS (P > 0.05) no significant difference. ANCOVA, time was treated as continuous covariate. c BG concentrations during intraperitoneal glucose tolerance tests (IPGTT) after 30 and 90 days of transplantation. IPGTT at Day 30 (n = 6 in Normal mice and No cell, n = 8 in Random and Mesh). Data are mean ± SEM. *P < 0.05 and NS (P > 0.05) no significant difference. ANCOVA, time was treated as continuous covariate. d Hematoxylin/eosin staining of rat islets and blood vessels in retrieved devices (Day 42) and the number of blood vessels around a rat islet (within a 200 μm distance). Yellow arrowheads point to blood vessels with erythrocytes inside. White arrows point to rat islets. The round material in the cross-sections is nylon grid. No cell group consists of 11 islets pooled from 3 mice; Random group consists of 13 islets pooled from 3 mice; Mesh group consists of 12 islets pooled from 3 mice. *P < 0.05, ***P < 0.001, and NS (P > 0.05) no significant difference. One-way analysis of variance. e Cross-sectional immunostaining images of rat insulin (red) and mouse blood vessels (CD31, green) in a retrieved Mesh device. f Immunostaining images (parallel section) using human (red) and mouse (green) CD31 antibodies indicate the anastomoses between human and mouse blood vessels. g Confocal image of perfused blood vessels in re-vascularized rat islets in the Mesh group after 42 days of transplantation. h Fluorescent images of re-vascularized rat islets after 91 and 112 days of transplantation from the Mesh group. The rat islets expressing GFP are green and perfused blood vessels are red

Fig. 5

Human iPSC-EC-derived microvascular meshes enhance vascularization. a Fluorescent images of iPSC-EC meshes with different geometries (square, pentagon, hexagon, and octagon). b iPSC-EC meshes with more complex patterns (spider web and capillary bed). c Confocal images of an iPSC-EC mesh at the contracted and junction regions. iPSC-EC expressing GFP is green, human CD31 antibody is red, and nucleus is blue. All samples were imaged after 2 days of culture. d Hematoxylin/eosin and immunostaining images of retrieved devices after 2 weeks of subcutaneous implantation in SCID-Beige mice. In all in vivo experiments, NHDFs were mixed with iPSC-ECs (iPSC-EC:NHDF = 9:1). The yellow arrowheads point to blood vessels with erythrocytes inside. The white dash lines mark the interface between the device and panniculus carnosus muscle. Mouse CD31 antibody is green and α-smooth muscle actin (α-SMA) is red. e The density and area percentage of blood vessels at the interface. n = 5 in all groups. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and NS (P > 0.05) no significant difference. One-way analysis of variance. f Immunostaining images of human (red) and mouse CD31 (green) antibodies showing the anastomoses between iPSC-EC derived vessels and mouse vessels

Fig. 6

Improved diabetes correction by the iPSC-EC microvascular meshes in SCID-Beige mice. a A microscopic image of rat islets in an iPSC-EC mesh device. The gray color aggregates are rat islets. The white arrow points to iPSC-EC mesh. In all in vivo experiments, NHDFs were mixed with iPSC-ECs (iPSC-EC:NHDF = 9:1). b Non-fasting BG concentration of the mice during 91 days of subcutaneous transplantation (n = 5 in No iPSC-EC, n = 6 in Random iPSC-EC and Mesh iPSC-EC). *P < 0.05. ANCOVA, time was treated as continuous covariate. c BG concentration during intraperitoneal glucose tolerance test (IPGTT) after 30 and 90 days of transplantation. IPGTT at Day 30 (n = 6 in Normal mice, Random iPSC-EC, and Mesh iPSC-EC, and n = 4 in No iPSC-EC). Data are mean ± SEM. *P < 0.05. NS (P > 0.05) no significant difference. IPGTT at Day 90 (n = 6 in Normal mice, n = 3 in No iPSC-EC, n = 4 in Random iPSC-EC, and n = 6 in Mesh iPSC-EC): *P < 0.05. ANCOVA, time was treated as continuous covariate. d Immunostaining images (parallel section) of human (red) and mouse CD31 (green) antibodies indicating the anastomoses between human and mouse blood vessels. e Confocal image of perfused blood vessels in a re-vascularized rat islet retrieved from the Mesh group at Day 91. f Hematoxylin/eosin staining image of a retrieved iPSC-EC mesh device at Day 91. Black arrow points to a rat islet and yellow arrowheads point to blood vessels containing erythrocytes. g Immunostaining image of insulin and blood vessels surrounding rat islets in a retrieved iPSC-EC mesh device at Day 91. Rat insulin antibody is red and mouse CD31 antibody is green