The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

Engineering functional tissues of clinically relevant size (in mm-scale) in vitro is still a challenge in tissue engineering due to low oxygen diffusion and lack of vascularization. To address these limitations, a perfusion bioreactor was used to generate contractile engineered muscles of a 3 mm-thickness and a 8 mm-diameter. This study aimed to upscale the process to 50 mm in diameter by combining murine skeletal myoblasts (SkMbs) with human adipose-derived stromal vascular fraction (SVF) cells, providing high neuro-vascular potential in vivo. SkMbs were cultured on a type-I-collagen scaffold with (co-culture) or without (monoculture) SVF. Large-scale muscle-like tissue showed an increase in the maturation index over time (49.18 ± 1.63% and 76.63 ± 1.22%, at 9 and 11 days, respectively) and a similar force of contraction in mono- (43.4 ± 2.28 µN) or co-cultured (47.6 ± 4.7 µN) tissues. Four weeks after implantation in subcutaneous pockets of nude rats, the vessel length density within the constructs was significantly higher in SVF co-cultured tissues (5.03 ± 0.29 mm/mm²) compared to monocultured tissues (3.68 ± 0.32 mm/mm²) (p < 0.005). Although no mature neuromuscular junctions were present, nerve-like structures were predominantly observed in the engineered tissues co-cultured with SVF cells. This study demonstrates that SVF cells can support both in vivo vascularization and innervation of contractile muscle-like tissues, making significant progress towards clinical translation.

Funding Acknowledgements: Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Foundation The development of novel adjuvant angiogenic therapies to restore the low-perfused microvascular network upon myocardial infarction (MI) is crucial to avoid a possible end-stage heart failure. Of the current adult cell-based therapies, human adipose tissue-derived stromal vascular fraction cell (SVF) has vast reparative potential, principally due to: 1) its heterogeneous composition rich in mesenchymal stem cells (MSC), endothelial cells (EC), pericytes and hematopoietic cells, among others. In vitro engineering of SVF-based patches under unidirectional flow, applied by the help of a perfusion-based bioreactor, was found to increase certain cellular SVF subgroups such as pericytes, compared to static culture. In this study, we aimed at studying the potential of SVF-based engineered tissues in a model of chronic MI in nude rats. Human SVF cells were isolated upon liposuction and cultured on 3D collagen sponges (8 mm diameter, 3 mm thickness) either under constant unidirectional perfusion or in static condition for 5 days. Patches were characterized in terms of cellular composition prior to implantation. MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery in male nude rats. Cardiac MRI was performed 4 weeks after MI; prior to the suture of patches and before sacrifice (4 weeks after implantation). Left ventricular ejection fraction (EF) was the surrogate marker and primary end point for cardiac pump function. Controls included untreated MI animals. Following perfusion culture, SVF cells were composed with a statistically superior percentage of pericytes, identified as CD45- CD34- CD146+ compared to static culture (28.06±10.03 and 3.37±2.50, respectively, p<0.0007). The presence of other cell subpopulations was similar in the patches generated in perfusion or static culture. While the percentage of EF at the time of sacrifice resulted to be not statistically different between static and perfusion-based patches, statically generated constructs showed a general trend of decrease in the % EF before and after treatment (rat 1: 61.96 vs 52.90; rat 2: 55.39 vs 53.00; rat 3: 52.34 vs 50.62, respectively). Perfusion-cultured patches, instead, rather improved the cardiac function, measured as % EF (rat 1: 51.82 vs 58.72; rat 2: 51.66 vs 60.45; rat 3: 53.50 vs 52. 36, respectively for 4 weeks following MI and 4 weeks following treatment). When comparing the ratio of the % EF 8 weeks and 4 weeks between static or perfusion-based patches and the untreated controls, rats treated with patches generated under perfusion resulted to show higher levels of % EF, with an almost statistically difference (p=0.0556), compared to the control group. The observed results showed the great potential of human SVF-based patches in the improvement of the heart pump function.