Maximizing pediatric stem cell harvest: A novel two-step digestion approach for adipose tissue

Figure 1 This figure illustrates a two-step enzymatic digestion of adipose tissue using collagenase type IV to isolate stromal vascular fractions. The first digestion yields stromal vascular fraction 1, and the residual tissue undergoes a second digestion to obtain stromal vascular fraction 2. Both fractions were collected after centrifugation and used for downstream assays such as viability testing, flow cytometry, and gene expression analysis. SVF: Stromal vascular fraction; CFU: Colony forming unit; qRT-PCR: Quantitative reverse transcription polymerase chain reaction.

Figure 2 Comparison of viability and cell yield. A-D: The viability of cells (A), total number of cells (B), total number of mononucleated cells (C) and percentage of mononucleated cells (D) in stromal vascular fraction 1 and stromal vascular fraction 2. Results have been presented as mean ± SD and statistical significance was measured between stromal vascular fraction 1 and stromal vascular fraction 2 by unpaired two-tailed Student’s t-test. ^aP < 0.05; ^bP < 0.001. SVF: Stromal vascular fraction.

Figure 3 Comparison of colony forming units and clonogenic potential of stromal vascular fraction 1 and stromal vascular fraction 2. A-C: Figure shows total number of colony forming units (CFU) (A), plating efficiency (B), and number of CFUs per 1000 mononucleated cells (C) in stromal vascular fraction 1 and stromal vascular fraction 2. Graphs were presented as mean ± SD and statistical significance was measured between stromal vascular fraction 1 and stromal vascular fraction 2 by unpaired two-tailed Student’s t-test. ^aP < 0.05. SVF: Stromal vascular fraction; CFU: Colony forming unit.

Figure 4 Flow cytometric analysis of CD44, CD73 and CD146 in stromal vascular fraction fractions. A-C: Representative flow cytometry plots showing the expression of regenerative surface markers in stromal vascular fractions (SVF). Dot plots and histograms represent gating and marker expression profiles for CD44, CD73, and CD146 in SVF1 and SVF2; D and E: Bar graph comparing the percentage of CD44+, CD73+, CD44+CD73+ (double-positive), and CD146+ cells in SVF1 and SVF2. SVF: Stromal vascular fraction.

Figure 5 Mean gene expression of target genes. Mean gene expression of target genes normalized to GAPDH in adipose tissue (AT), AT1, AT2, stromal vascular fraction 1, stromal vascular fraction 2. A-H: Mean gene expression of vascular endothelial growth factor (A), stromal cell-derived factor (B), nitric oxide synthase-3 (C), CD14 (D), matrix metalloproteinase 3 (E), interleukin-4 receptor (F), P16 (G), and P53 (H). Differences between all fractions that are shown in the graphs of all genes are not significant statistically as P > 0.05. VEGF: Vascular endothelial growth factor; SDF: Stromal cell-derived factor; NOS3: Nitric oxide synthase-3; MMP3: Matrix metalloproteinase 3; IL4R: Interleukin-4 receptor; CDKN2A: Cyclin-dependent kinase inhibitor 2A; AT: Adipose tissue; SVF: Stromal vascular fraction.

Figure 6 Histological evaluation of adipose tissue structure at different stages of collagenase digestion. Representative photomicrographs of adipose tissue sections captured at 4 × (left) and 20 × (right) magnifications. A: Undigested adipose tissue showing a well-preserved and compact structure of densely packed, adipocytes with intact morphology and minimal extracellular matrix (ECM) disruption; B: Adipose tissue after the first round of collagenase digestion reveals partial ECM degradation and moderate adipocyte disruption, with some cells retaining morphology and others beginning to dissociate; C: Tissue after the second round of digestion demonstrates substantial structural breakdown, with complete loss of adipocyte integrity and prominent fibrous ECM remnants. This stage reflects advanced digestion and effective decellularization.

Figure 7 Graphical abstract. SVF: Stromal vascular fraction.