The high demand for donated organs vastly outnumbers the supply, leading to the suffering of millions of patients in need. Tissue and organ engineering can be a possible solution to overcome this problem. However, prevascularization strategies to ensure irrigation of complex engineered tissues after transplantation continues to be challenging. The stromal vascular fraction (SVF) of adipose tissue possesses a strongly angiogenic character due to its cellular composition. Previously, we have developed cell sheets of human SVF capable of undergoing spontaneous vasculogenesis in vitro in the absence of added growth factors, combining the cellular variety of SVF with the extracellular matrix (ECM) richness of cell sheets. In the present work, we aimed at defining optimal culture conditions of SVF cell sheets for downstream extraction and characterization of angiogenic ECM. SVF cells were cultured up to 32 days in the presence of different concentrations of ascorbic acid (AA) to boost ECM production. In addition, since cell sheet detachment is a common outcome of prolonged in vitro culture, a Rho-associated kinase (ROCKi) inhibitor, which modulates the cytoskeleton and stress fiber formation, was also added to the culture. As expected, the presence of AA resulted in higher collagen levels after 25 days of culture, which was also true for fibronectin. Furthermore, the addition of ROCKi did not inhibit protein levels and, in fact, increased the detected levels of laminin. Additional proteomic characterization is underway. This work is the first step towards defining a protocol for producing ECM-enriched SVF cell sheets for downstream ECM extraction. 

Acknowledgements: EU Horizon 2020 research and innovation program under the ERC grant CapBed (805411), IF/00347/2015, PD/BD/135252/2017