Analysis of Immunomodulatory Properties of Spheroids from Adipose-derived Stem Cells

Purpose: Adipose Stem Cells (ASCs) represent promising cell therapies for regenerative medicine and immunomodulation. The recently isolated spheroids from adipose-derived stem cells (S-ASCs) have shown being superior stemness and enhanced regenerative capability than the classical ASCs. The aims of this study were (1) to investigate the effects of adipose tissue donor-site, harvesting technique and patients’ characteristics on the yield of S-ASCs and (2) to investigate the immunomodulatory properties of S-ASCs comparing them with the adherent ASCs. Material and Methods: The study was divided into two phases. Phase I (prospective clinical study) between July 2016 and August 2019 was conducted to evaluate the effects of adipose tissue donor-site, harvesting technique and patients’ characteristics on the yield of S-ASCs. Seventy-six subcutaneous fat tissues samples from flanks (n=13), abdomen (=30), thigh (n=21) and breast (n=12), were harvested from healthy patients using syringe-assisted Coleman technique with (n=34) or without centrifugation (n=42). Differences in S-ASC yields were assessed with one-way analysis of variance. Phase II (Immunological in vitro study) was designed to assess and to compare the immunomodulatory properties of human S-ASCs and ASCs toward γδ T cells (subpopulations of PBMCs) using a CFSE-mediated Mixed lymphocyte reaction (MLR) assay and their cytokine secretion profile by Cytofluorimetric analysis. Results: In Phase I, the total number of S-ASCs in fat harvested from the thigh was significantly higher than flanks or breast (P < 0.05). Comparing the technique used for fat harvesting, samples from thigh and abdomen with centrifugation yielded significantly more S- ASCs thank those without centrifugation. In Phase II, both ASCs and S-ASCs effectively suppressed mitogen-induced γδ T cells proliferation. Levels of interferon (IFN)- γ secreted from activated γδ T cells increased over time, but these levels were significantly reduced when co-cultured with both type of ASCs. A statistical significant stronger immunosuppressive effect of S-ASCs was observed in the downregulation of the pro-inflammatory cytokines and in upregulation of the anti-inflammatory cytokines (P