Autologous Bone Marrow Aspiration and Isolation

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).