Characterizing cell types of fresh mechanically isolated SVF

Abstract

The stromal vascular fraction (SVF) derived from adipose tissue contains a heterogenous mixture of cells, which are highly relevant for regenerative medicine due to their therapeutic potential. Adipose-derived stromal cells (ASCs), owing to their multilineage differentiation potential and their capacity to support tissue repair, are increasingly applied in regenerative and aesthetic medicine. While the standard method for isolating SVF is enzymatic digestion, this approach is time-consuming and involves regulatory challenges. In contrast, mechanical isolation is a fast and gentle method preserving cells in their extracellular matrix (ECM), potentially enhancing therapeutic effects and making this approach more suitable for intraoperative applications. To characterize SVF, flow cytometry is considered the gold standard. However, for mechanical isolation this may not be optimal as this method requires single-cell suspensions, which demand additional enzymatic digestion and filtration steps that disrupt the preserved ECM of mechanically isolated SVF. The aim of this thesis was to investigate the use of immunofluorescence microscopy as an alternative method for characterizing fresh mechanically isolated SVF with minimal manipulation. To achieve this, a stepwise methodological approach was performed, beginning with the establishment of a 2D (cytospin) and a 3D (gel-embedding) SVF preparation method for microscopy analysis. To transfer applicability for surface marker detection from flow cytometry to microscopy, verification via cytospin was carried out, followed by the detection of tissue-specific antigens using both approaches. Second, the flow cytometry protocol was adapted for mechanically isolated SVF in order to enable comparability with microscopy-based results. Using these adapted protocols, both 2D and 3D preparations were successfully established, enabling the visualization of distinct SVF marker expression. In addition, the adapted flow cytometry protocol provided the basis for a first characterization of SVF populations according to their marker expression. In summary, this work demonstrates the feasibility of immunofluorescence microscopy as an alternative approach to flow cytometry for the characterization of fresh mechanically isolated SVF.

Date of Award	2025
Original language	English
Supervisor	Manuel Selg (Supervisor)