Integration of Self-Assembled Microvascular Networks with Microfabricated PEG-Based Hydrogels

Abstract

Despite tremendous efforts, tissue engineered constructs are restricted to thin, simple tissues sustained only by diffusion. The most significant barrier in tissue engineering is insufficient vascularization to deliver nutrients and metabolites during development in vitro and to facilitate rapid vascular integration in vivo. Tissue engineered constructs can be greatly improved by developing perfusable microvascular networks in vitro in order to provide transport that mimics native vascular organization and function. Here a microfluidic hydrogel is integrated with a self-assembling pro-vasculogenic co-culture in a strategy to perfuse microvascular networks in vitro. This approach allows for control over microvascular network self-assembly and employs an anastomotic interface for integration of self-assembled micro-vascular networks with fabricated microchannels. As a result, transport within the system shifts from simple diffusion to vessel supported convective transport and extra-vessel diffusion, thus improving overall mass transport properties. This work impacts the development of perfusable prevascularized tissues in vitro and ultimately tissue engineering applications in vivo.

Figure 1

Microfabrication system design schematic. PDMS is replica molded to fabricate an external perfusion housing (Step 1). The interior of the housing is coated with photoinitiator (Step 2), and then photopolymerizable PEG precursors are injected into the housing and mask directed photolithography is used to fabricate hydrogel microchannels within the external PDMS housing (Step 3). The PDMS–PEG multilayer device is conformally sealed to coverglass and perfused with media and buffer (Step 4). The final schematic shows the spatial relationship of the perfused media (red) and buffer (blue) microchannels to PEG hydrogel (cyan) regions imaged for analysis in this study.

Figure 2

Microvascular network formation. In this representative immunohistochemistry (IHC) image of the microvasculature at 96 h in the region 0–300 μm from the media microchannel, HUVECs (green; anti-PECAM) are shown forming connected networks with 10T1/2 cells (red; anti-smooth muscle α-actin) acting as pericytes to wrap and therefore stabilize vessel networks (inset; arrow). Cell nuclei appear blue with DAPI counterstaining. Dimensions of the field of view (FOV) for the large image are 318 μm × 318 μm with an inset FOV of 76 μm × 57 μm.

Figure 3

Spatiotemporal microvascular network morphology. (a) Immunohistochemistry fluorescent micrographs of HUVECs (green; anti-PECAM), 10T1/2 (red; anti-smooth muscle α-actin) and DAPI (blue) as a function of culture time (0, 48, and 96 h) and distance (0–3000 μm) from the perfused media microchannel. Co-cultures begin as homogeneously dispersed 3D suspensions (0 h) and self organize to form tubules over 96 h. The distance between dotted lines is 318 μm. (b) Enlarged images of the 0-300 (left) 1200–1500 (center) and 2700–3000 μm (right) regions at 96 h.

Figure 4

Spatiotemporal self-assembled microvascular network morphology. Total tubule number (a) and total tubule length (b) in a Media-Buffer configuration (solid bars) and a Media-Media configuration (hashed bars) as a function of distance from the microchannel (0–3000 μm) and perfusion time (48 h and 96 h). Robust microvascular network formation in the Media-Buffer system was confined to regions closest to the media microchannel (0–600 μm) with the interior regions (1200–2100 μm) not forming networks at 96 h. The Media-Media configuration permitted microvascular network formation farther from the microchannel but with an overall lower number of total tubules and a decrease in total tubule length at each time and location when compared to the Media-Buffer configuration. p < 0.05, n = 3, paired t-test, (+) indicates 48 h vs. 96 h temporal significance, (*) indicates spatial significance compared to the 0–300 μm region, and ‡ indicates significance between Media-Media and Media-Buffer at 96 h.

Figure 5

Spatiotemporal apoptotic activity. Spatiotemporal apoptotic activity was assessed using the TUNEL marker for damaged DNA. After 96 h of culture, the region nearest the media microchannel (0–300 μm) had a significantly lower fraction of TUNEL positive cells. Furthermore the TUNEL positive fraction was shown to increase from 48 h to 96 h in the same regions where total tubule length and number were shown to decrease.

Figure 6

Mass transport regimes in vascularized and non-vascularized PEG scaffolds. (a) Time-lapse high molecular weight dextran intensity maps of regions directly adjacent to the microchannel wall. Non-vascularized transport profiles behave according to Fickian diffusion through a polymer matrix. (b) Time-lapse high molecular weight dextran intensity maps of regions directly adjacent to the microchannel wall in vascularized scaffolds at 96 h of culture. Rapid convective like transport through the matrix in regions coinciding with vessel structures suggests a shift in transport from diffusional transport to convection through vessel structures. (c) Enlarged high molecular weight dextran intensity map at 120 min post perfusion. Dotted arrows indicate newly perfused vessels, dashed and solid arrows indicate progressively broadening intensity profiles suggesting extra-vessel diffusion. (d) Confocal z-stack and orthogonal projection of fixed high molecular weight dextran (red) and anti-PECAM IHC (green) in vascularized scaffolds at 96 h of culture. Dextran co-localization within PECAM labeled vessel lumen suggests transport via vessel structures. Scale bars in (a) and (c) are 50 μm, FOV for the primary image in (d) is 318 μm.

Figure 7

Vascularization effects on mass transport. (a) High molecular weight dextran intensity profiles in non-vascularized (black trace) and vascularized (red trace) hydrogels. Non-vascularized hydrogels rely on diffusional transport with construct characteristic diffusion lengths (λ_D) limited to large distances between perfused microchannels. Vascularized hydrogels exhibit convective transport that rapidly penetrates hundreds of micrometers into the hydrogel with characteristic diffusion lengths (λ_V) limited to the small distance between vessels. (b) At early time points when convection dominates, high molecular weight dextran accumulates at a significantly greater rate in the vascularized system (red trace) when compared to the nonvascularized system (black trace) (p < 0.01, n = 5, paired t-test). The difference in vascularized and non-vascularized transport or the % convective transport shown as a percentage of total intensity (blue dash trace) is greatest at early time points and decreases as transport becomes diffusion limited. (c) Time-lapse (0-120 min) high molecular weight dextran intensity profiles in the vascularized constructs show diffusion out of vessel structures as indicated by (d) peak width broadening and a decrease in peak aspect ratio over time. FOVs for the inset images are 318 μm.