Impact of processing steps on cellular content of mechanical isolated stromal vascular fraction

Adipose tissue derived stromal vascular fraction (SVF) has emerged as cell therapeutic applicable by point-of-care one-step procedures in autologous settings. Even a mechanical isolation, completed within minutes, is typically followed by multiple steps such as cell washing, filtration, erythrocyte lysis and cryopreservation, which may impact the cell isolate in terms of cell amounts and quality. Using the BioMicroMill, a straightforward device providing mechanically isolated SVF suited for multiple therapeutic doses, we aimed to evaluate the impact of relevant processing steps during isolation and cryopreservation on cell quantity and quality in terms of viability, the presence of regenerative cells, pro-regenerative secretome, vascular network formation. The mechanical isolation yielded in the mean 3.4 × 10⁵ ± 1.42 × 10⁵ SVF cells per ml of lipoaspirate with a mean viability of 38% ± 7.2%. It comprised a heterogeneous mixture of single cells, cell aggregates, extracellular matrix, and microvascular fragments enriched from adipose tissue, providing functionally relevant cell populations (CD31, CD34, CD90, CD105). Adipose-derived stromal cells (ASC) exhibited robust outgrowth, proliferation, and differentiation capacity in vitro. The SVF showed pronounced paracrine activity (IL-10, VEGF-A, HGF, IL-6, IL-8, MCP-1), including proangiogenic factors, and supported 3D vascular network formation, demonstrating strong proangiogenic potential. However, all additional SVF processing steps substantially influenced total cell yield, with cumulative cell losses (erylysis 68% ± 16%, filtration 67% ± 16%, washing 54% ± 9%) but no loss of viability or ASC attachment and proliferation in vitro was observed. In contrast to other processing steps, filtration substantially altered SVF composition towards single cells, which was also reflected in an altered paracrine activity (significant increase in IL-10 and reduction to minimal levels of HGF). Furthermore, despite further cell loss (mean 47% ± 16%), cryopreservation maintained functional cell populations including ASC, paracrine activity and support of 3D network formation in vitro. Taken together, our data demonstrate that processing steps can influence both cell yield and quality. Accordingly, protocol selection should be guided by the intended application and the required functional properties and therefore warrants careful consideration.