Inoculation of the Bioreactor

Assemble the MiniPERM bioreactor according to the manufacturer's instructions (see Note 9), disinfect the universal roller, and install in a CO2 incubator. Media for the production module (production medium) and the nutrient module (nutrient medium) should be prepared before the inoculation (see Notes 1-4). The production medium is complete medium as described previously but supplemented with a further 5% FBS and 0.1% v/v CellPROTECT (see Note 10). The nutrient medium contains the same components as complete medium, with the exceptions of 5% FBS instead of 10% FBS and with the addition of 0.2% v/v AntiFOAMa (see Note 11). Both the nutrient media and the production media are prewarmed to 37 °C to prevent expansion of the MiniPERM membrane when introduced into the incubator.

1. Detach the hybridoma cells by tapping and gentle pipetting from a 225-cm2 tissue culture flask, wash by centrifugation at 300g for 10 min, and resuspend the resulting cell pellet in 5 mL of production medium.

2. Calculate the cell density by diluting 10 ^L of suspension in 90 ^L of nigrosin then count the cells using a haemocytometer. Adjust the density to 5 x 105 cells/mL in production medium and inoculate the reactor with 35 mL of this suspension using the following procedure (see Note 12).

3. Draw the suspension slowly up into a 50-mL hypodermic syringe (see Note 13). Unscrew the two Luer-Locks on the assembled MiniPERM and inject the cell suspension carefully into the production module while the module is slowly rotated (see Note 14). This ensures that displaced air is expelled through the open Luer-Lock.

4. Replace both Luer-Locks then add 400 mL of nutrient medium to the nutrient module. Close the cap and place the MiniPERM module inside an incubator on the Universal turning device set at 5 rpm (see Note 15).

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