Ex vivo perfusion bioreactor cultures

Bioreactor cultures under perfusion were performed according to the manufacturer’s instructions (Cellec Biotek AG, Basel, Switzerland). Briefly, fresh, intact GBM tissue explants were placed into ice-cold Neurobasal-A medium (Life Technologies, #21103049) and immediately taken to the laboratory (less than 30 min). Tissues from the center and periphery of the tumor were cut into ~20- to 30-mm³ fragments. 

Tissue fragments were placed between two 10-mm-diameter discs of microfibrillar collagen hemostat sheets (Ultrafoam, Avitene) prewet with phosphate-buffered saline (PBS) for 1 hour at 37°C, 5% CO₂. Silicone adaptors and ethylene-tetrafluoroethylene copolymer mesh grids were arranged on the top and bottom of the collagen scaffold, and the tissues were placed into U-CUP perfusion chambers (Cellec Biotek AG). 

The perfusion media consisted of a 50:50 mix of Neurobasal-A medium (Life Technologies, #21103049) and Dulbecco’s modified Eagle’s medium/F12 medium (Gibco, #21331020) supplemented with nonessential amino acids (1×; Sigma-Aldrich, #M7145), 1 mM sodium pyruvate (Sigma-Aldrich, #S8636), 44 mM sodium bicarbonate (Gibco, #5080094), 25 mM Hepes (Gibco, #156301), 4 mM l-alanyl-l-glutamine (Corning, #25-015-CI), antibiotic-antimycotic (1×; Gibco, #LS15240062), human recombinant epidermal growth factor (20 ng/ml; Gibco, #PHG0314), human recombinant fibroblast growth factor (20 ng/ml; Gibco, #AA10-155), heparin sulfate (10 ng/ml; STEMCELL Technologies, #07980), and 5% human serum (Sigma-Aldrich, #H4522-100ML). 

The perfusion flow rate was set at 0.47 ml/min, resulting in a superficial flow velocity of 100 μm/s. Starting from day 0, bioreactor media were either left untreated or supplemented with the anti–PD-1 antibody nivolumab (Opdivo, Bristol Myers Squibb; 5 μg/ml) or anti-CD47 antibody (Bio X Cell, #BE0019-1; 5 μg/ml), alone or in combination. On day 7, culture media were frozen at −80°C and tissues were fixed in 10% neutral buffered formalin (Sigma-Aldrich, #HT501128) for 24 hours and processed according to standard histopathology procedures for paraffin embedding.

Cellec Biotek for U-Cup bioreactors : http://www.cellecbiotek.com/

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