Extraction and cultivation of the SVF

After obtaining informed consent, we used the IRB-approved protocol to collect human lipoaspirates from 17 healthy donors aged 28–66 years who had undergone liposuction of the abdomen or thighs.

After natural gravity sedimentation, lipoaspirates develop three layers: oil, adipose tissue, and tumescent liquid. The floating adipose tissue layer was extracted, measured, and digested with an equivalent volume of collagenase-based enzyme solution (details below) at 37 °C for 30 min at 120 rpm of reciprocating motion (Yamato Scientific), followed by centrifugation at 800×g for 10 min. The resulting cell pellet was designated the SVF (Fig. 1A). After washing with Hanks’ Balanced Salt Solution (HBSS; Thermo Fisher Scientific; #14175-103), the SVF was sequentially passed through 100-µm and 40-µm sieve cell strainers (Corning). The strained cell suspension was centrifuged again at 800×g for 5 min at 4 °C, and the obtained SVF pellet was washed with HBSS. The nucleated cell number, viability, and total cell-sized particle number were measured using a fluorescent cell counter (LUNA-STEM; Logos Biosystems) after double staining with acridine orange and propidium iodide (Logos Biosystems).

SVF extraction and characterization. (A) The SVF was enzymatically isolated from human lipoaspirates using a collagenase-based enzymatic solution at 37 °C for 30 min at 120 rpm of reciprocating motion, followed by centrifugation at 800×g for 10 min. The open circle indicates the SVF pellet. (B) The freshly isolated SVF was characterized by the expression of CD45, CD34, CD31, CD105, CD146, CD157, and CD200 during the flow cytometry analysis. The experiment was performed independently 3 times (3 donors).

We used flow cytometry to examine the extraction efficiency of AEPCs using three collagenase-based enzyme formulations: (#1) 0.2% (w/v) collagenase (crude type; FUJIFILM Wako Pure Chemical; #032-22364) and 3 mM CaCl₂ (FUJIFILM Wako; #037-24031) in HBSS; (#2) 0.2% (w/v) collagenase, 3 mM CaCl₂, and 1000 U/mL of deoxyribonuclease 1 (DNase1; Worthington Biochemical; #{"type":"entrez-nucleotide","attrs":{"text":"LS002139","term_id":"1321652585","term_text":"LS002139"}}LS002139) in HBSS; (#3) 0.2% (w/v) collagenase, 3 mM CaCl₂, 1000 U/mL DNase1, and 0.1% (v/v) Poloxamer 188 solution (Pol188; Sigma-Aldrich) in HBSS. Pol188 has previously been reported as an additive to elevate the extraction efficiency of ASCs in the SVF²⁴.