3. Initial and routine in-process control of the spinner flask during

the cultivation process.

(a)

Following the attachment phase, transfer the spinner flask

to the biosafety cabinet, open the spinner flask as previ-

ously described, resuspend the MCs using a 10-mL sterile

pipette and take a homogenous sample (7 mL). Sample

treatment prior to analyses is more closely described in

Subheading 3.4.

(b)

Refasten the lid and transfer the spinner flask back to the

incubator containing the stirrer to ensure process condi-

tions can be maintained for the remainder of the

cultivation.

(c)

Subsequent routine sampling (5 mL) should be per-

formed every 24 h post-inoculation, especially prior to

and following the medium exchange (see Fig. 3).

4. Perform a 50% media exchange 4 days post-inoculation to

prevent the accumulation of inhibitory cell metabolites or the

glucose concentration from falling below 5.55 mmol L^1

(¼ 1 g L^1).

(a)

Transfer the spinner flask to the biosafety cabinet and

allow the MCs to sediment.

(b)

Open the spinner flask as previously described, then

remove 50% of the supernatant (approximately 36.5 mL)

and replace with fresh pre-warmed culture media (see

Note 6).

(c)

Following the media exchange, refasten the lid of the

spinner flask and return it to the incubator containing

the stirrer platform to maintain process conditions (see

Note 7).

5. The harvest criterion is met at least 24 h prior to reaching the

stationary phase of cell growth (between 5 and 6 days post

inoculation). At this point perform a final harvest (see Fig. 3).

(a)

Transfer the spinner flask to the biosafety cabinet and

allow the MCs to sediment.

(b)

Open the spinner flask as previously described, then

remove the supernatant without aspirating MCs and

replace it with pre-warmed DPBS. Allow the MCs to

sediment, then replace the DPBS with pre-warmed

TrypLE Select 1 (see Note 13).

(c)

Return the spinner flask to the incubator containing the

stirrer platform and increase the agitation rate to Ns1u for

15 min (see Notes 7 and 14).

(d)

Following enzymatic dissociation, transfer the spinner

flask back to the biosafety cabinet, open and gently

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Misha Teale et al.