Non tissue culture coated 24 well plates

Medium containing GFP encoded or human CD19.CAR encoded gamma retrovirus (RV) was concentrated 10-fold by Amicon centrifugation (MWCO 100 kDa, Millipore), 2500 g, 15–20 mins. Dry lyophilized MASTER scaffolds were transferred to non-tissue culture coated 24 well plates (Falcon), and 1 x 10⁶ PBMCs isolated from human buffy coat fractions and concentrated RV at MOI 2 was pipetted onto each scaffold (total volume of virus and PBMC = 300 µl). Control scaffolds were seeded with 1 x 10⁶ PBMCs suspended in cell culture medium. For in vitro studies only, the seeded scaffolds were incubated without any additional medium in the 5% CO₂ incubator at 37°C for 1 h, then 1 ml of complete medium was added. Cells were isolated from scaffolds after 72 h by digesting with 0.25 M EDTA, washed twice with excess PBS and analyzed for GFP expression or CD19.CAR expression by flow cytometry. For in vivo studies, MASTER was seeded with PBMCs and CD19.CAR encoding retrovirus, incubated for ~1 h at 37°C until the media was completely absorbed by the scaffolds and implanted on the same day in the subcutaneous space of NSG mice.