Adipose-tissue-derived therapeutic cells in their natural environment as an autologous cell therapy strategy: the microtissue-stromal vascular fraction

Abstract

The prerequisite for a successful clinical use of autologous adipose-tissue-derived cells is the highest possible regenerative potential of the applied cell population, the stromal vascular fraction (SVF). Current isolation methods depend on high enzyme concentration, lysis buffer, long incubation steps and mechanical stress, resulting in single cell dissociation. The aim of the study was to limit cell manipulation and obtain a derivative comprising therapeutic cells (microtissue-SVF) without dissociation from their natural extracellular matrix, by employing a gentle good manufacturing practice (GMP)-grade isolation. The microtissue-SVF yielded larger numbers of viable cells as compared to the improved standard-SVF, both with low enzyme concentration and minimal dead cell content. It comprised stromal tissue compounds (collagen, glycosaminoglycans, fibroblasts), capillaries and vessel structures (CD31 + , smooth muscle actin + ). A broad range of cell types was identified by surface-marker characterisation, including mesenchymal, haematopoietic, pericytic, blood and lymphatic vascular and epithelial cells. Subpopulations such as supra-adventitial adipose-derived stromal/stem cells and endothelial progenitor cells were significantly more abundant in the microtissue-SVF, corroborated by significantly higher potency for angiogenic tube-like structure formation in vitro. The microtissue-SVF showed the characteristic phenotype and tri-lineage mesenchymal differentiation potential in vitro and an immunomodulatory and pro-angiogenic secretome. In vivo implantation of the microtissue-SVF combined with fat demonstrated successful graft integration in nude mice. The present study demonstrated a fast and gentle isolation by minor manipulation of liposuction material, achieving a therapeutically relevant cell population with high vascularisation potential and immunomodulatory properties still embedded in a fraction of its original matrix.

Isolation of adipose tissue derived regenerative cells from human subcutaneous tissue with or without the use of an enzymatic reagent

“…Nevertheless, Tables 6 and 7 summarize the relative amount of ADRCs expressing the surface markers CD13, CD29, CD34, CD44, CD73, CD90, CD31 and CD45 as reported in all studies describing enzymatic and non-enzymatic methods for isolating ADRCs listed in Tables 1 and 2 (note that in some studies surface markers were investigated but relative amounts of ADRCs expressing a certain surface marker or a combination of surface markers were not provided). The data summarized in Tables 6 and 7 demonstrate that (i) for only very few methods [49,53,54,58] the relative amount of CD34 + ADRCs was determined, with substantial variation among methods (range, 35% -81%); (ii) for most methods CD34 was determined together with at least one other surface marker, resulting in a range of published data between 0.8% (CD34 + /CD90 -/CD31 -/CD45 -/CD105 -/CD146 + cells; [51]) and 44% (CD34 + /CD31 -/CD45cells; [41]); (iii) the relative amount of CD45 + ADRCs varied between 6% [42] and 50% [54] for enzymatic methods, and between 8% [42] and 82% [58] for non-enzymatic methods; (iv) for only few methods the relative amounts of CD13 + cells, CD29 + cells, CD44 + cells, CD73 + cells, CD90 + cells and CD31 + cells were determined; and (v) for no any method the relative amount of CD31 -/CD34 + /CD45 -/CD235acells (as proposed in [73]) was determined.…”

Isolation of adipose tissue derived regenerative cells from human subcutaneous tissue with or without the use of an enzymatic reagent

“…Nevertheless, Tables 6 and 7 summarize the relative amount of ADRCs expressing the surface markers CD13, CD29, CD34, CD44, CD73, CD90, CD31 and CD45 as reported in all studies describing enzymatic and nonenzymatic methods for isolating ADRCs listed in Tables 1 and 2 (note that in some studies surface markers were investigated but relative amounts of ADRCs expressing a certain surface marker or a combination of surface markers were not provided). The data summarized in Tables 6 and 7 demonstrate that (i) for only very few methods [46,50,51,56] the relative amount of CD34+ ADRCs was determined, with substantial variation among methods (range, 35% -81%); (ii) for most methods CD34 was determined together with at least one other surface marker, resulting in a range of published data between 0.8% (CD34 + /CD90 -/CD31 -/CD45 -/CD105 -/CD146 + cells; [48]) and 44% (CD34 + /CD31 -/CD45cells; [40]); (iii) the relative amount of CD45 + ADRCs varied between 6% [57] and 50% [51] for enzymatic methods, and between 8% [57] and 82% [56] for non-enzymatic methods; (iv) for only few methods the relative amounts of CD13 + cells, CD29 + cells, CD44 + cells, CD73 + cells, CD90 + cells and CD31 + cells were determined; and (v) for no any method the relative amount of CD31 -/CD34 + /CD45 -/CD235acells (as proposed in [68]) was determined.…”

Immunomodulatory Effect of Adipose-Derived Stem Cells: The Cutting Edge of Clinical Application

“…Yet, antigen-specific Tregs were also produced and as a consequence, self-reactive T effector responses were suppressed (González et al, 2009b). The stromal vascular fraction (SVF) of an adipose tissue contains not only ASCs but also different cell types, such as endothelial cells, pericytes, lymphocytes, monocytes macrophages, fibroblasts, and smooth muscle cells (Dominici et al, 2006;Bourin et al, 2013;Nürnberger et al, 2019). In vitro, freshly isolated SVF containing ASCs secrete trophic and pro-regenerative factors, such as cytokines, growth factors, anti-inflammatory factors and extracellular vesicles harboring proteins or even microRNAs, collectively known as secretome (Fu et al, 2017).…”

“…In fact, several studies have shown that the paracrine effects of ASCs and not the cells themselves, are pivotal players for tissue repair along with angiogenic and immunomodulatory properties occurring at the site of the damaged tissue (Tögel et al, 2005;Krampera, 2011;Eleuteri and Fierabracci, 2019). For example, high levels of two immunomodulatory mediators, indoleamine-pyrrole 2,3-dioxygenase (IDO) and Prostaglandin E2 (PGE2), were detected at 24 h in the supernatants from freshly isolated SVF (Nürnberger et al, 2019). It is known that IDO is an immunomodulatory enzyme produced by macrophages with immunosuppressive functions for T-cells and natural killers (NK).…”