Figure 1.
In vitro vascularization of islet multicultures.
10-day-old multi-cellular cultures of mouse pancreatic islets, human ECs and HFFs, grown on 3D PLLA/PLGA polymer scaffolds were analyzed. (a) Whole scaffold, bright field photo; scale bar- 2mm. (b) A paraffin-embedded section stained with H&E; scale bar-200 µm. (c–f) Stereomicroscope confocal images of engineered vascularized pancreatic islet scaffolds stained for insulin (red) and HUVEC-GFP (green). (g–h) Laser scanning confocal image of a cell-embedded scaffold stained for insulin (green), vWF (red) and nuclear content (blue); scale bar-100 µm. (i–j) 3D reconstruction of images collected in (g–h) using the Imaris software.
Figure 2.
In vitro vascularization of engineered pancreatic tissue: 2D versus 3D growth environments.
20 isolated mouse islets seeded alone, with HFF or with HFF and HUVEC were either grown on 3D polymer scaffolds or on 2D ECM-coated plates for up to 28 days. Scaffolds and plates were triple-stained with anti-vWF (red), anti-insulin (green) antibodies and DAPI (blue). (a) 10-day old, immunostained sections; scale bar-200 µm. (b) Staining of 9-, 21- and 42-day-old sections. Scale bar-200 µm (c) Islet survival in 2D and 3D cultures was determined by quantification of the number of viable islets under light microscope using trypan blue, insulin and DAPI staining for the 2D-plated islets and immunofluorescently via insulin- and DAPI-stained islets for 3D scaffolds.
Figure 3.
Addition of endothelial cells to islet cultures promotes islet function in vitro.
Real-Time PCR analyses of (a) insulin and (b) glucagon were performed on islets cultured in 2D or 3D microenvironments for 8 days, in the absence or presence of HFF and/or HUVEC. Values were normalized to mouse 18s and are presented as the recorded levels versus those of free islets cultured under the same conditions. Differential expression analyses of (c) insulin and (d) glucagon were performed by comparing their levels in 3D versus 2D islet culture on days 8 and 21. Values were normalized to mouse 18s RNA and are presented as the ratio of levels expressed in 3D versus 2D islet culture environments. (e) Real-Time PCR analyses of the mature islet genes PDX1, NKX6.1, GLUT2, MAFA, PC1/3 and glucokinase were performed for islets cultured in 2D and 3D microenvironments, in the absence or presence of HFF and/or HUVEC. Values were normalized to mouse 18s and are presented as the ratio of the recorded levels versus those of free islets cultured under the same conditions.
Figure 4.
Expression profiles of EC morphogenesis-related genes.
Quantitative real-time PCR analyses, using human specific primers were performed on islet-EC settings following their culture (a) with HFF in a 3D co-culture system or (b) with HFF in a 2D co-culture system. (c) Relative gene expression between 3D/2D multicellular culture systems. (d) Relative gene expression between 3D/2D co-culture systems. Values were normalized to human GAPDH.
Figure 5.
Endothelial cells promote upregulation of ECM-associated genes in islet cultures.
Quantitative real-time PCR analyses of the ECM-associated (a) ITGB1 and (b) VEGF genes were performed on islets cultured in 2D or 3D microenvironments in the absence or presence of HFF and/or HUVEC for 8 days. Values were normalized to mouse 18s and are presented as the fold increase in recorded expression levels versus those of free islets cultured under the same conditions. Expression analyses of (c) ITGB1and (d) VEGF-A were performed by comparing 3D to 2D settings of various cell cultures. Values are presented as the fold increase in expression levels of 2D versus 3D cultures. (e) c-peptide concentrations in KRB media were measured from 8-day-old cultures of 3D scaffolds seeded with islets, islets + HFF or islets + HFF + HUVEC after challenge with low glucose doses (2.5 mM) and high glucose doses (17.5 mM).
Figure 6.
In vivo biofunctioning of 3D vascularized islet constructs.
5-day old vascularized or non-vascularized 3D islet constructs were implanted into the peritoneal subcutaneous area of streptozotocin-induced diabetic mice. (a, b) 3D pre-vascularized constructs were removed 2 weeks after implantation, sectioned and immunostained for insulin (green) and vWF (red) to identify human ECs. Scale bar-200 µm. (c, d) Histological examination of H&E-stained, implanted vascularized islet constructs, retrieved 1 week following restoration of blood glucose levels (200 mg/dl) in each animal. S = scaffold. Scale bar-200 µm. (e, f) Vascularized islet implants were retrieved from mice 14 days post-implantation and following injection of FITC-dextran to the tail vein. Samples were then immunofluorescently stained with anti-insulin antibodies (red) and viewed with a laser scanning confocal stereomicroscope (Leica). Scale bar-100 µm. (g) Functional, FITC-dextran-perfused vessels in the engineered vascularized islet implant, viewed with a laser scanning confocal stereomicroscope on day 14 post-implantation. Scale bar-200 µm. (h) Blood glucose levels and (i) Survival of streptozotocin-induced diabetic mice after implantation of vascularized or non-vascularized islet-bearing scaffolds (50 islets per scaffold) implanted after a 5-day culturing period (vascularized implant, n = 6, non- vascularized, n = 4).