Fig. 2.
a Schematic representation of isolating UA-ADRCs from lipoaspirate with the Transpose RT/Matrase system (InGeneron) used in this pilot study (derived from [28]): (1) recovered lipoaspirate (25 mL) is loaded together with 2.5 mL reconstituted, proprietary enzymatic Matrase Reagent and lactated Ringer solution (preheated to 39 °C) into a processing tube up to the MAX FILL line; (2) the filled processing tubes are subjected in an inverted position inside the Transpose RT system to repetitive acceleration and deceleration for 30 min at 39 °C; (3) the processed lipoaspirate solution is filtered through a 200 μm filter and transferred into a wash tube; (4) after filling the wash tube with saline (room temperature) up to the MAX FILL line, the cells are separated from the rest of the tissue by centrifugation at 600 g for 5 min at room temperature; (5) the ADRCs (approximately 2 mL) are extracted through a swabable luer vial adapter at the bottom of the wash tube, and the remaining substances (fat, debris, and liquid) are discarded; (6) the cells are returned into the empty wash tube and (after adding fresh saline up to the MAX FILL line) centrifugated again for 5 min; (7, 8) the previous washing step is repeated; and (9) finally the concentrated ADRCs (approximately 3 mL) are extracted and slowly pushed through a luer coupler into a new sterile syringe for further application to the subject. This gentle and highly efficient isolation process results in a high cell yield (7.2 × 105 ± 0.90 × 105 ADRCs per mL lipoaspirate in [28]), high cell viability (85.9 ± 1.1% in [28]) and, thus, high number of living cells per mL lipoaspirate (6.25 × 105 ± 0.79 × 105 ADRCs per mL lipoaspirate in [28]). To our knowledge, the latter is the highest value ever reported in studies describing methods for isolating ADRCs [28]. b–e Adipogenic (b), osteogenic (c), hepatogenic (d), and neurogenic (e) differentiation potential of adipose derived stem cells (ASCs) derived from ADRCs isolated from lipoaspirate using the Transpose RT/Matrase system, demonstrated by culturing ASCs on their 3rd (b–d) or 6th passage (e) for 2 weeks (b, c), 10 days (d), or 3 weeks (e) in adipogenic (b), osteogenic (c), hepatogenic (d), or neurogenic (e) differentiation medium (panels taken from [28]). Adipogenic, osteogenic, hepatogenic, and neurogenic differentiation was demonstrated by respectively presence of intracytoplasmic lipids (triglycerides) using Oil red-O staining (b; the yellow arrows in b indicate single Oil red-O positive cells), presence of calcific deposits using Alizarin red staining (c), presence of structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, and proteoglycans) using Periodic Acid Schiff staining (d), or immunofluorescence for the detection of beta III Tubulin (β3TUB) (e) (details are provided in [28]). The scale bar in E represents 100 μm in (b, c) and 50 μm in (d, e)