Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

Abstract

Microvascular endothelial cells (MVEC) are a preferred cell source for autologous revascularization strategies, since they can be harvested and propagated from small tissue biopsies. Biomaterials-based strategies for therapeutic delivery of cells are aimed at tailoring the cellular microenvironment to enhance the delivery, engraftment, and tissue-specific function of transplanted cells. In the present study, we investigated a modular tissue engineering approach to therapeutic revascularization using fibrin-based microtissues containing embedded human MVEC and human fibroblasts (FB). Microtissues were formed using a water-in-oil emulsion process that produced populations of spheroidal tissue modules with a diameter of 100-200 µm. The formation of MVEC sprouts within a fibrin matrix over 7 days in culture was dependent on the presence of FB, with the most robust sprouting occurring at a 1:3 MVEC:FB ratio. Cell viability in microtissues was high (>90%) and significant FB cell proliferation was observed over time in culture. Robust sprouting from microtissues was evident, with larger vessels developing over time and FB acting as pericyte-like cells by enveloping endothelial tubes. These neovessels were shown to form an interconnected vascular plexus over 14 days of culture when microtissues were embedded in a surrounding fibrin hydrogel. Vessel networks exhibited branching and inosculation of sprouts from adjacent microtissues, resulting in MVEC-lined capillaries with hollow lumens. Microtissues maintained in suspension culture aggregated to form larger tissue masses (1-2 mm in diameter) over 7 days. Vessels formed within microtissue aggregates at a 1:1 MVEC:FB ratio were small and diffuse, whereas the 1:3 MVEC:FB ratio produced large and highly interconnected vessels by day 14. This study highlights the utility of human MVEC as a cell source for revascularization strategies, and suggests that the ratio of endothelial to support cells can be used to tailor vessel characteristics. The modular microtissue format may allow minimally invasive delivery of populations of prevascularized microtissues for therapeutic applications.

Keywords: Modular tissue engineering; fibrin; fibrinogen; injectable scaffolds; microtissues; microvascular endothelial cells; minimally invasive delivery; vascularization.

Figure 1

Schematic of a strategy to treat ischemic tissue with modular microtissues containing embedded endothelial cells and supporting perivascular cells.

Figure 2

Schematic of microtissue fabrication process.

Figure 3

Endothelial sprouting in bulk fibrin hydrogels. A) Fluorescent images of embedded MVEC stained red with UEA-1 in gels made with different MVEC:FB ratios and cultured in different medium volumes. B) Quantitation of the average sprout length in bulk gels (n=3). C) Quantitation of the number of sprouts >100 µm in length in bulk gels (n=3). Error bars represent standard deviation of the mean. * indicates statistical significance (*p<0.05, **p<0.01 and ***p<0.001).

Figure 4

Characterization of modular microtissues. A) Single MVEC:FB microtissues (1:1 and 1:3) under phase contrast showing microtissue morphology and embedded cells immediately after microbead fabrication. B) Single MVEC:FB microtissues (1:1 and 1:3) under fluorescence showing cell viability (green = live cells, red = dead cells). C) Quantitation of cell viability for a population of microtissues. D) Quantitation of total cells per unit volume for a population of microtissues. E) Total DNA in microtissues as a function of time. Best viewed in color. Error bars represent standard deviation of the mean. * indicates statistical significance (*p<0.05).

Figure 5

Flow cytometry analysis of MVEC and FB cell population in fibrin co-cultures over time. A,B) Mono-culture of MVEC and FB. C- H) MVEC and FB cell population in co-cultures over time. Error bars represent standard deviation of the mean. * indicates statistical significance (*p<0.01).

Figure 6

Maximum intensity projections of confocal stacks showing FB enveloping endothelial vessel surface (Day 14).

Figure 7

Sprouting of neovessels from modular microtissues. A) Fluorescence images (green = fibrin, red = MVEC) at day 7 and 14 with different MVEC:FB ratios. B) Quantification of vessel area normalized to microtissue area. C) Box plot of vessel diameter. The solid center line in the box plot represents the median, the dotted center line in the box represents the mean, and the lower and upper boundaries of the box represent the 25^th and 75^th percentiles, respectively. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. Large dots represent outliers.

Figure 8

Fluorescence images showing vessel network (red) formation in between microtissues (green) embedded in a surrounding acellular fibrin hydrogel. Best viewed in color.

Figure 9

Inosculation of MVEC neovessels. A) MVEC (red) networks (i) sprouted from microtissues (green) and formed branches (ii) with hollow lumens (iii, iv). B) Serial histological sections showed inosculation of adjacent MVEC vessels.

Figure 10

Microtissues cultured in suspension aggregated to form larger tissue structures. A, B) By day 7, neovessels were evident in tissue masses. C, D) By day 14, networks of vessels had formed in tissue masses made with 1:3 MVEC:FB microtissues, but were less evident in those made at 1:1. E) Fluorescence staining and F) PECAM/CD-31 IHC confirmed that vessels within tissue structures were created by MVEC.