discard the supernatant, while ensuring that no MCS are

lost in the process. Replace the discarded supernatant with

pre-warmed culture medium (see Note 13). Repeat this

step twice to obtain a sterile ProNectin^® F MC stock

solution.

(f)

Transfer the packaging containing the bioreactor to the

biosafety cabinet. Open the package and assemble the

BioBLU^® 0.3c under the bench using the appropriate

technique to guarantee bioreactor sterility.

(g)

Remove one of the two port lids and add 135 mL of

pre-warmed culture media (see Note 6) and 15 mL of

ProNectin^® F MC stock solution to the bioreactor. Install

the sterilized pH probe, by securing it in place of the

port lid.

(h)

Transfer the bioreactor back to the control unit and pro-

ceed to install the exhaust gas cooler, DO probe, temper-

ature probe, pH probe cable, inlet gas piping, and mount

the stirrer. Then begin operation of the bioreactor by

activating the various control loops, i.e., 37 ^C, pH 7.2,

DO >30%, 0.1 vvm overlay and Ns1u (see Notes 14, 16,

and 17 for more information).

(i)

Allow the MCs to equilibrate under process conditions for

approximately 24 h prior to inoculation. This step also

serves to ensure system sterility prior to cultivation.

2. Inoculation of the BioBLU^® 0.3c.

(a)

Set the slope of the DO probe by using the saturated

culture medium as a reference to calibrate 100% DO.

(b)

Calculate the necessary volume of inoculum, following

cell density and quality control (see Note 12), to achieve

an initial cell density of 15,000 cells cm^2 or 8.1  10⁶

cells per bioreactor.

(c)

Deactivate the control loops. Transfer the equilibrated

bioreactor to the biosafety cabinet and allow the MCs to

sediment. Subsequently remove the remaining port lid

and place it right side up on a sterile surface within the

biosafety cabinet.

(d)

Remove the volume of culture medium to be replaced

with the inoculum and transfer it to a sterile 15-mL cen-

trifuge tube (Corning^®). This sample may then be used to

determine the initial substrate and metabolite concentra-

tion within the bioreactor (see Note 3). Add the inoculum

to the bioreactor to achieve the target starting cell density.

(e)

Refasten the sterile port lid, transfer the vessel back to the

control unit, and reactivate all the necessary control loops.

Subsequently, deactivate agitation after 5 min to allow the

cells to sediment and attach to the MCs for 12 h.

98

Misha Teale et al.