Generation of a functional liver tissue mimic using adipose stromal vascular fraction cell-derived vasculatures

Abstract

One of the major challenges in cell implantation therapies is to promote integration of the microcirculation between the implanted cells and the host. We used adipose-derived stromal vascular fraction (SVF) cells to vascularize a human liver cell (HepG2) implant. We hypothesized that the SVF cells would form a functional microcirculation via vascular assembly and inosculation with the host vasculature. Initially, we assessed the extent and character of neovasculatures formed by freshly isolated and cultured SVF cells and found that freshly isolated cells have a higher vascularization potential. Generation of a 3D implant containing fresh SVF and HepG2 cells formed a tissue in which HepG2 cells were entwined with a network of microvessels. Implanted HepG2 cells sequestered labeled LDL delivered by systemic intravascular injection only in SVF-vascularized implants demonstrating that SVF cell-derived vasculatures can effectively integrate with host vessels and interface with parenchymal cells to form a functional tissue mimic.

Figure 1. Adipose stromal vascular fraction cells form perfused microvasculatures in vivo.

Fresh (fSVF) and cultured (cSVF) isolated from GFP rats were seeded in 3-dimensional collagen type I gels and implanted subcutaneously into immunocompromised mice. After 4 weeks, host mice were perfused with dextran-TRITC through jugular injection. Representative images of vasculatures formed 4 weeks post-implantation are shown. Vessel density (number of vessels/field of view), percentage of vessels perfused (*p = 0.001) and average vessel diameter (*p = 0.02) are shown. Values are reported as mean ± s.e.m.; n = 3/condition.

Figure 2. Adipose stromal vascular fraction cells contribute to angiogenesis.

Freshly isolated and cultured SVFs significantly differ in their ability to incorporate into sites of neovascularization. fSVF and cSVF isolated from GFP rats were co-implanted with microvessel fragments derived form non-GFP rats into immunocompromised mice for 14 or 28 days, when implants were removed and vessels stained with GSI-TRITC. Representative images of vasculatures formed 14 and 28 days post-implantation. Black arrow shows SVF in endothelial cell position. White arrows show SVF incorporated in perivascular position. Quantification of SVF incorporation into neovessels 28 days post-implantation (percentage of total vessel volume) Values are reported as mean ± s.d.; *p = 0.01, n = 4 (fSVF); n = 8 (cSVF).

Figure 3. Expression of cell surface markers in freshly isolated (black bars) and cultured (white bars) SVF cells.

Cells were stained for the different molecules and analyzed by fluorescent flow cytometry. Percentage of cells positive for a specific molecule above isotype control is shown. Values are reported as mean ± s.d.; *p = 0.009 (CD31) and 0.02 (c-kit); n = 3/condition. At the 95% confidence level, α = 0.929.

Figure 4. Human freshly isolated adipose SVF cells vascularize implanted parenchymal cells.

(A–C) Freshly isolated human SVF seeded in collagen type I gels and implanted subcutaneously into immunocompromised mice. After four weeks, implants were stained with UEA-TRITC. (D–F) Human SVF and HepG2 beads constructs implanted for 6 weeks.

Figure 5. Freshly isolated adipose SVF cells form a functional interface with implanted parenchymal cells that allows for DiI-LDL uptake.

(A) HepG2-GFP⁺ coated Cytodex-3 microcarrier beads. (B) DiI-LDL within construct. (C) GS1-Cy5⁺ staining of murine endothelium, demonstrating formation of a vascular bed around beads. (D) HepG2-GFP⁺ and DiI-LDL overlay showing co-localization. (E) HepG2-GFP⁺ coated Cytodex-3 microcarrier beads implanted without SVF cells do not form a GS1-Cy5⁺ vascular network. No DiI-LDL uptake was observed. (F) DiI-LDL uptake within host liver confirming adequate DiI-LDL delivery to host circulation. (G) Percentage overlap of HepG2-GFP⁺ clusters and GS1-Cy5⁺ vasculature and DiI-LDL in implants containing SVFs and HepG2-GFP⁺. No HepG2 clusters lacking associated GS1-Cy5⁺ and DiI-LDL signal were identified (+). *p = 0.03; †p = 0.008 with n = 5/condition. At the 95% confidence level, α = 0.569. Values are reported as mean ± s.e.m. (H) Scatter plot of implants (n = 5/condition) with HepG2 clusters comparing DiI-LDL with GS1-Cy5⁺ vasculature. Pearson correlation coefficient of r = 0.909 was calculated.