Erlenmeyer shake flasks

OvC-PDE cultures were maintained at 5 explants/mL, in 20 mL culture medium (DMEM supplemented with 10% FBS and 1% P/S), in 125 mL regular Erlenmeyer shake flasks (Corning). Cultures were kept under orbital shaking (IKA KS 260 basic) at 100 rpm, to prevent adhesion and increase oxygen and nutrients diffusion, in an incubator (Nuaire US Autoflow) at 37 °C, 5% CO₂ in air. Cultures were maintained up to 30 days. Culture medium was renewed once a week (50% of the total volume). OvC-PDE were collected at day 0 (surgery day, after sample processing), 10, 20 and 30 of culture. Due to restriction of primary material, and the destructive nature of several endpoints, not all OvC-PDE could be used for all read-outs, at all timepoints: all OvC-PDE cultures were evaluated by H&E in all timepoints; as for the remaining read-outs, they were selected taking into account the work phase and availability of material, as identified in each method.

CHO-S cells (Thermo Fisher Scientific) were cultured in CD CHO medium supplemented with 8 mM L-Glutamine (Thermo Fisher Scientific) and 2 μL/mL Anti-Clumping reagent (AC, Life Technologies). Cells were maintained in Erlenmeyer shake flasks (Corning Inc., Acton, MA), incubated in a humidified incubator at 37°C, 5% CO2 at 120 rpm and passaged every 2-3 days. Viable cell density (VCD) and viability were monitored using the NucleoCounter NC-200 Cell Counter (ChemoMetec, Denmark). One day before transfection, cells were seeded into the culture medium without AC. On the day of transfection, cells were transfected using 3.75 μg of DNA packaged with 3.75 μL of FreeStyle Max transfection reagent (Thermo Fisher Scientific) per well of a 6-well plate (BD Biosciences) in 3 mL culture medium without AC and with 1E6 cells/mL. Specifically, for VF gRNA library transfection, 150 μg Bxb1 recombinase plasmid and 450 μg gRNA library donor were transfected into 480 mL CHO-attp-mCherry cells at the density of 1E6 cells/mL. The cells were divided into several 6-well plates and one day after transfection, the cells were combined into two 500 mL flasks. Transfection efficiencies were monitored using the transfection reagent included pmax-GFP plasmid and was generally observed to be between 60-90%.

HEK-293T cells (ATCC; ATCCCRL-11268) and
SKOV3 cells (ATCC; ATCC HTB-77) were cultured in Iscove’s modified
Dulbecco’s medium (IMDM) supplemented with 10% FBS (Labtech)
and 2 mM GlutaMAX (Invitrogen). SupT1 cells (ECACC; 95013123) were
cultured in complete RPMI (RPMI-1640, Lonza) supplemented with 10%
FBS and 2 mM GlutaMAX. SupT1 cells were genetically modified by transduction
with an SFG vector to express human EGFR. ExpiCHO cells were cultured
in ExpiCHO medium (Gibco) using Erlenmeyer shake flasks (Corning)
and maintained in a Kuhner shaker at 37 °C and 8% CO₂ at 225 rpm.

A CHO cell line (CHO-HCG) overexpressing recombinant hCG was provided by Organon/Merck (Oss, The Netherlands). CHO-HCG cells were cultured between at 0.2×10⁶ and 1×10⁶ cells/ml in 250 ml sterile Erlenmeyer shake flasks (Corning Life Sciences) on an orbital shaker at 200 rpm in a humidified atmosphere at 37°C with 5% CO₂. Cell culture supernatant from CHO-HCG cells was used as a source of recombinant HCG for glycan analysis and was prepared by harvesting cells at 130 g for 10 min at 4°C.

The adherent cell lines HEK293 (ATCC-CRL-1573), HEK293T (ATCC-CRL-3216) and 293E (ATCC-CRL-10852) were obtained from ATCC and propagated in DMEM (D6429) supplemented with 10% FBS at 37 °C in a humidified incubator with 5% CO₂ in air. Suspension cell lines 293-F, 293-H and Freestyle 293-F (Gibco) were obtained from Thermo Fisher Scientific and cultivated in 293 SFM II medium (Gibco) supplemented with Glutamax at a final concentration of 4 mM (Gibco). Suspension cells were cultivated in 125-ml Erlenmeyer shake flasks (Corning) at 37 °C and 120 rpm in a humidified incubator with 8% CO₂ in air. All cells were propagated from frozen stocks for no longer than 20 passages.