Stem kit reagent

The cancer cell count was assessed by evaluating the EpCAM-associated fluorescence signal, as previously reported [21 (link)]. Staining was carried out on 100 µL aliquots with the antibodies 5 μL anti-EpCAM-PE (CD326, clone HEA-125, Miltenyi Biotec, Bologna, Italy) and 5 μL anti-CD45-FITC-conjugated antibody (Beckman Coulter) for 10 min in the dark at room temperature in order to exclude white blood cells. Before acquisition, each sample was treated with 2 mL of lysing solution (Stem-Kit Reagents, Beckman Coulter, Milan, Italy) to avoid erythrocytes interference. To perform an absolute count of EpCAM-positive (EpCAM⁺) cells, 100 µL of calibration beads (LeukoSure Fluorospherers, Beckman Coulter, Milan, Italy) were added immediately before acquisition (FC 500 flow cytometer, Beckman Coulter, Milan, Italy). Negative control samples were treated as described, without adding monoclonal fluorescent antibodies.

Autologous bone marrow was aspirated through an anterior iliac crest puncture under general anesthesia in the operating theater. The collected volume was 8 ml/kg for patients under 10 kg; [80 ml + (body weight in kg − 10) × 7 ml] for patients above 10 kg, based on safety assessments for that volume derived from our previous studies [19 (link)–21 (link)]. Mononuclear cells and autologous plasma were isolated from the aspirated bone marrow by gradient centrifugation using Ficoll-Paque (GE Healthcare, Sweden) in a cleanroom following the ISO 14644 standard at Vinmec Research Institute of Stem Cell and Gene Technology. The cell suspension was washed with phosphate-buffered saline (PBS) solution and resuspended in 10 ml of autologous plasma for injection. The product sterility was confirmed by microbiological evaluation. Entire blood components before and after Ficoll-Plaque separation were evaluated by a Beckman Coulter LH780 hemocytometer. The hematopoietic stem cell content (CD34⁺ cells) was assessed according to the International Society of Hematotherapy and Graft Engineering (ISHAGE) guideline using StemKit™ Reagent (Beckman Coulter) in a Navios flow cytometer. Before injection, the cell products were examined for endotoxin levels with the Endosafe-PTS Kit (Charles River).

Immunophenotypic analysis of UCB-MSCs was analyzed by flow cytometry using BD Stemflow™ hMSC Analysis Kit (BD Biosciences – USA) with the following markers: CD90, CD73, and CD105 for positive markers; CD45, CD34, CD11b, CD19, and HLA-DR for negative markers. For Muse cell detection, FITC-conjugated SSEA-3 (BD Biosciences – USA) and PE-conjugated CD105 antibodies (BD Biosciences – USA) were used. Surface marker expression was analyzed using the Navios system and FlowJo software. CD34⁺ cell counting was done by using the Stem Kit Reagent (IM3630; Beckman Coulter) containing CD45-FITC and CD34-PE antibodies.

BMMNCs were isolated by gradient centrifugation using Ficoll‐Paque (GE Healthcare, Waukesha, Wisconsin) in our ISO 14644 standard clean room at Vinmec Research Institute of Stem Cell and Gene Technology. The cell suspension was washed with 1× phosphate‐buffered saline solution and resuspended in autologous plasma up to a total of 10 mL for injection. The sterility of the product was confirmed by microbiological evaluation using the BacT/Alert3D microbial detection system (bioMérieux, Durham, North Carolina). The total blood components before and after Ficoll‐Paque separation were evaluated with a Beckman Coulter LH780 hematocytometer. The hematopoietic stem cell CD34⁺ (hHSC CD34⁺) count was assessed using Stem‐Kit Reagent (Beckman Coulter, Brea, California) on a Navios flow cytometer (Beckman Coulter). Before injection, cell products were examined for endotoxin levels using the Endosafe‐PTS kit (Charles River Laboratories, Cambridge, Massachusetts) and Mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

The CD34+ cells were enumerated using Stem-Kit reagent (Beckman coulter) according to the manufacturer’s instructions. After vortexing of EDTA specimen, 100 μL aliquot was incubated for 20 min with 20 μL mixture of CD45-FITC monoclonal antibody and CD34-PE monoclonal antibody in the first tube, and with 20 μL mixture of CD45-FITC and IsoClonic control-PE reagent in the second tube. In each tube, 20 μL of viability dye 7-AAD was added to distinguish between viable and nonviable cells. In order to lyse erythrocytes, 2 mL of NH₄Cl lysing buffer was added and incubated for 10 min. For absolute count of CD34+ cells, 100 μL of stem cell fluorosphere with known concentration was added and incubated for 5 min. After gentle mixing, the product was analyzed on FC500 flow cytometer (Beckman coulter) following manufacturers’ recommendations, based on a gating protocol developed by the International Society of Hematotherapy and Graft Engineering (ISHAGE) [16 (link)]. A total of 75,000 CD45+ events were acquired for each sample. Among viable CD45+ and CD34+ events, cells with dim CD45 expression and low sideward light scatter were selected, and events falling outside the cluster of cells with low-to-intermediate forward light scatter were excluded. Absolute CD34+ cell count was calculated using the ratio of fluorosphere to CD34+ cell numbers.