Endosafe pts kit

Divalent scFvF7-Fc Ab was produced through transient transfection of CHO DG44 cells followed by incubation for 6 days in ProCHO5 serum free medium (Lonza). The scFvF7-Fc Ab was purified from the cell medium using a protein A column (Thermo Fisher) with Abs eluted with 100 mM glycine buffer pH 3.0 into a 1 M Tris-HCl eluate at pH 8.0. Abs were dialyzed twice against PBS, concentrated using a JumboSep centrifuge filter with 3 kDa molecular weight cut-off (Pall Laboratories), and sterile filtered with Millex GP 0.22 μm pore size (Millipore). Prior to evaluation of scFvF7-Fc in the in vitro functional recovery assays, Abs were tested with the FDA-licensed Endosafe-PTS kit (Charles River Laboratory) which showed < 5 EU of endotoxins per mg of protein.

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).

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).