Data Availability StatementThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. freshly isolated lipoaspirate, as well as after attachment onto aligned nanofibrillar scaffolds. Flow cytometry results demonstrated that the BMS-986205 SVF consisted of 33.1 9.6% CD45+ cells, a small fraction of CD45C/CD31+ (4.5 3.1%) and 45.4 20.0% of CD45C/CD31C/CD34+ cells. Although the subpopulations of SVF did not change significantly after attachment to the aligned nanofibrillar scaffolds, protein secretion of vascular endothelial growth factor (VEGF) significantly increased by six-fold, compared to SVF cultured in suspension. Importantly, when SVF-seeded scaffolds were transplanted into immunodeficient mice with induced hindlimb ischemia, the cell-seeded scaffolds induced a significant higher mean perfusion ratio after 14 days, compared to cells delivered using saline. Together, these results show that aligned nanofibrillar scaffolds promoted cellular attachment, enhanced the secretion of VEGF from attached SVF cells, and their implantation with attached SVF cells stimulated blood perfusion recovery. These findings have important therapeutic implications for the treatment of PAD using SVF. expansion, SVF can be derived autologously, extracted in a minimally invasive manner in a clinical setting (Levi et al., 2011), and transplanted back within hours. Consequently, SVF may have greater translational relevance than other stem cells types for treatment of limb ischemia. We have previously shown that collagen scaffolds seeded with human SVF and subcellular populations thereof significantly improved revascularization to dermal wounds (Brett et al., 2017b), which supports the safety of SVF-seeded collagen scaffolds. Regardless of the kind of stem cell used, a major limitation to stem cell therapy is poor survival of the cells when transplanted in saline. As an alternative to saline as a cell delivery vehicle, BMS-986205 biological scaffolds can localize cell delivery to the site of the scaffold, while also providing important extracellular matrix cues that modulate the survival and angiogenic capacity of the transplanted cells. In particular, cues derived from nano-scale anisotropic patterns of fibrillar collagen can modulate cellular organization, growth factor secretion, and upregulation of integrin gene expression (Huang et al., 2013a, b; Nakayama et al., 2015, 2019). We have previously demonstrated that parallel-aligned nanofibrillar scaffolds promote the survival and angiogenic capacity of transplanted primary human endothelial cells or human induced pluripotent stem cell-derived endothelial cells in a mouse model of PAD (Huang et al., 2013b; Nakayama et al., 2015). These studies suggest that nanoscale spatial patterning cues can directly modulate biological functions of therapeutic cells upon transplantation Rabbit Polyclonal to CLM-1 into the ischemic limb. Toward clinical translation, these nanofibrillar scaffolds have been demonstrated to improve angiogenesis (Huang et al., 2013b), arteriogenesis (Nakayama et al., 2015), and lymphangiogenesis (Hadamitzky et al., 2016) = 6) undergoing elective procedures in accordance with the Stanford University Institutional Review Board and kept at 4C until processing. All samples were processed within 24 h from the time of collection. SVF cells were isolated based on established methods (Tevlin et al., 2016). Lipoaspirate was rinsed twice with equal volume of phosphate buffered saline (PBS) to separate fat from blood. Fresh collagenase digestion buffer was prepared using M199 medium containing 2.2 mg/ml type II collagenase (SigmaCAldrich), 1000 U/ml DNAse, 0.5 M calcium chloride, 0.1% bovine serum albumin, 1% polaxamer-188 (SigmaCAldrich), and BMS-986205 2% hydroxyethyl piperazine ethanesulfonic acid (Life Technologies), and filtered using a 0.22-m filter system. Aliquots of the rinsed fat (12.5 ml) were transferred into 50-ml Falcon tubes, and an equal volume of collagenase digestion buffer was added to the fat. The tube caps were sealed with Parafilm (Bemis NA). The fat/collagenase mixture was incubated at 37C in a water bath for 10 min to activate the collagenase. The tubes with fat/collagenase mixture were placed into the orbital shaker set at 220 r/min for 45 min. Collagenase activity was then neutralized by addition of an equal volume of cold buffer consisted of PBS containing 2%.