Collagenase 4

BM mononuclear cells (BMMNCs) were obtained by conducting equal-volume Ficoll centrifugation (2000 rpm, 40 minutes) (GE Healthcare, UK). The BMMNCs were then seeded in a flask at a cell density of 2×10⁶/cm². The basic growth medium consisted of α-MEM (GIBCO, CA, USA)+20% FBS (Corning, NY, USA)+4 ng/mL of bFGF (Millipore, MA, USA)+1xPS (GIBCO, CA, USA). The paired subcutaneous adipose tissues were dissected into small pieces and digested with collagenase IV (SERVA Electrophoresis GmbH, Heidelberg, Germany) and hyaluronidase (Sigma-Aldrich, MO, USA), and were cultured in the basic growth medium (α-MEM+20% FBS+4 ng/mL bFGF+1xPS) at 37°C for 50 minutes with intermittent shaking. The resulting suspensions were filtered using a 70-µm strainer (BD Bioscience, NJ, USA) to remove debris and then centrifuged at 2000 rpm for 10 minutes. The supernatants were discarded, and the cell pellets were resuspended in the basic growth medium and cultured at density of 1.3×10⁵/cm² in a tissue flask (37°C, 5% CO₂). The basic growth medium was changed every 3 days and the cells were maintained at subconfluent levels (Passage 0, P0). The attached cells (BMMSCs and ADMSCs) were then harvested using trypsin-EDTA (GIBCO, CA, USA), and were subcultured at a density of 2–3×10³ cells/cm² under the same conditions used in the primary culture (Passage 1, P1).

Half of the renal allograft (cortex and medulla) was processed towards a single cell suspension, the other half was used for routine diagnostic assessment. The renal allograft was thoroughly rinsed with PBS to remove peripheral cells and afterwards dissected into small pieces (<0.5 cm³) and incubated for 60 minutes at 37 °C with 1.1 mg/ml collagenase IV (Serva, Heidelberg, Germany). The collagenase treatment was stopped by the addition of heat-inactivated fetal bovine serum with an end concentration of 10%. The tissue suspension was filtered through different sieves up to a 100 μm sieve (Greiner Bio-One, Kremsmünster, Austria) followed by a Ficoll-paque Plus procedure (GE healthcare, Uppsala, Sweden). Isolated cells were stored at −190 °C until further use. Afterwards, cells were thawed and analysed by flow cytometry and reverse transcription-qPCR (RT-qPCR) to determine their phenotype and gene expression profile.
Human splenocytes were disrupted and filtered through a 70 μm cell strainer (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) to obtain a single-cell suspension. Ficoll-paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient was used to collect mononuclear cells. Human PBMCs were isolated with the Ficoll-paque density gradient method.

MFAT samples were treated with collagenase IV (Serva GmbH) to retrieve SVF and analyze its various cell components, in particular the ASCs, expressing CD105 + , CD73 + , CD90 + and CD44 + and negative for HLA-DR, CD34, CD31 and CD45; CD146 + pericytes, CD31 + endothelial cells, CD34 + hematopoietic cells. The standard labelling protocol was performed with the following fluorochrome-conjugated antibodies and isotypic controls: ASCs were marked with MSC phenotyping kit, human CD44 PE, CD45 PerCP, IgG1 PE, IgG1 FITC, IgG1 APC, and IgG2a PerCP (Miltenyi Biotec, Bergisch Gladbach, Germany), CD31PE, CD34FITC, CD146APC and CD90 PerCP (Biolegend, San Diego, CA, USA). About 10⁵ events/sample were used for capture with MACsQUANT10 and data were analyzed with MACs quantify software (Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of ASCs in the MFAT volume was determined, and then the number of ASCs for ml of MFAT was normalized on the absolute number of viable cells.