Vent cap

Cultures of the wild-type P. marinus ATCC PRA-240 and P. mediterraneus ATCC PRA-238 [25 (link)] were maintained in DME: Ham's F12 (1:2) supplemented with 5% fetal bovine serum (FBS) in 25 cm² (5–8 ml) polystyrene canted neck cell culture flasks with vent caps (Corning^®, Corning, NY) at 26–28°C in a microbiology incubator as reported elsewhere [26 ].

Prior to freeze-drying, sterilized scaffolds were precultured in alpha minimum essential medium (α-MEM; Cytiva, United States) at 37°C overnight. The scaffolds were washed with PBS and then collected in sterilized 25 cm² cell culture flasks with vent caps (Corning, United States) that were pre-frozen in a freezer at −80°C for 2 h and then placed on the cold shelf of a freeze dryer (Christ ALPHA1-2LDplus). The drying protocols were performed for 24 h at an ice condenser temperature of −51°C and a vacuum of 0.07 mbar. The freeze-dried products were maintained in sealed cell culture flasks and stored at 4°C until further use.

Vials of an IgG expressing industrial GS-CHO cell line (Lonza, UK) were thawed and diluted with 49 mL of warmed CD-CHO (Life-Technologies, UK) containing 25 μM MSX. The cells were then expanded for 7 days in a shake flask with a vent cap (250 mL nominal volume, Corning Life Sciences, USA) mounted on an orbital shaker (Sartorius, UK) at 37 °C, 5% CO₂, and 70% humidity.

Recombinant baculoviruses were produced using the ViraPower BacMam Expression System (Thermo Fisher) according to manufacturer’s protocols. In brief, recombinant bacmid DNA was purified and transfected into adherent Sf9 cells (Thermo Fisher) using Cellfectin reagent to generate P1 recombinant baculovirus stock. Baculoviruses were amplified by inoculation of 50 ml Sf9 suspension cultures (10⁶ cells/ml) in Sf-900 III SFM Medium supplemented with 12.5 U/ml penicillin/streptomycin (Biochrom) in 125 ml polycarbonate Erlenmeyer flasks with vent cap (Corning) with 1 ml virus stock solution and incubation at 27 °C and 130 rpm for 2–3 days. Amplified viruses were purified and concentrated by ultracentrifugation of 27 ml virus-containing supernatant underlayed with 2.7 ml sucrose solution (25% sucrose with 5 mM NaCl and 10 mM EDTA in H₂O) in OptiSeal polypropylene tubes (Beckmann Coulter) at 80,000 g and 4 °C for 80 min. Viral pellets were re-suspended in 0.5 ml PBS and passed through 0.22 μm low protein binding sterile syringe filters (Merck Millipore). Baculovirus preparations were pre-tested on HEK293 cells (seeded at ~3–5 × 10⁴ cells per well in 96-well plates) by adding 1 μl virus per well overnight. Transduction efficiencies of ≥ 90% were regarded appropriate for further use. Otherwise, virus stock was subjected to further amplification cycles.