Anti cd45r0 antibody

Umbilical cord MSCs (donated to the Acıbadem Labcell Cellular Therapy Center) were suspended as 10,000 cells in 100 µL of MSC NutriStem® XF Supplement (#05-201-1U, Biological Industries) including 1% pen/strep. MSCs in 100 µL of medium was plated in 96-well plates from A to G. The wells were adjusted to have 10 µL, 3 µL, 1 µL, 0.3 µL, 0.1 µL, 0.03 µL, and 0.01 µL of FVIII-LV solution in each 50 µL of the medium, respectively. Subsequently, 50 µL of virus dilution from each concentration was transferred to the MSC-cultured wells, the total volume was adjusted to 150 µL, and cells were incubated for 4 days. Flow cytometry was performed using CytoFLEX (Beckman Coulter) flow cytometry to determine the CD45R0 expression with the anti-CD45R0 antibody (Miltenyi Biotec). Following viral titration analysis, the number of infectious units (IFU) was calculated as the dilution coefficient corresponding to the lowest CD45R0t expression higher than 10% as measured by flow cytometry. Following the titer assay, viruses were stored at -80 °C. Quality control tests of FVIII-Lv were performed, including sterility and purity. Sterility was tested with aerobic-anaerobic (BACTEC), endotoxin (Charles River Laboratories), and mycoplasma analysis (Mycoplasma species 500 PCR kit and GeneAmp PCR System 2700, Applied Biosystems).

The transduction process was evaluated using three different transduction agents. Protamine sulfate (40 µg/mL, Sigma), Vectofusin-1 (10 µg/mL, Miltenyi MACS), and protamine sulfate + Vectofusin-1 were used to determine the efficiency of the FVIII lentiviral transduction of MSCs. MSCs were suspended as 10,000 cells in 100 µL of MSC NutriStem® XF Supplement (Biological Industries) including 1% pen/strep. MSCs in 100 µL of medium were plated in 96-well plates from A to D. These plates were incubated at 37 °C for 24 h for MSC adherence. The wells were adjusted to multiplicity of infection (MOI) levels of 10 MOI, 5 MOI, 3 MOI, and 1 MOI, and the transduction process was initiated with Vectofusin-1, protamine sulfate, and protamine sulfate + Vectofusin-1, with plates subsequently incubated for 4 days. CytoFLEX (Beckman Coulter) flow cytometry was applied to assess CD45R0 expression with the anti-CD45R0 antibody (Miltenyi Biotec).