3. Initial and routine in process control of the BioBLU^® 0.3c

during cultivation.

(a)

Following the attachment phase, transfer the bioreactor

to the biosafety cabinet, remove the remaining port lid as

previously described, resuspend the MCs using a 10 mL

sterile pipette, and take a homogenous 7 mL sample (see

Note 18). Subsequent sample treatment prior to the

analyses is more closely described in Subheading 3.4.

(b)

Transfer the bioreactor back to the control unit and reac-

tivate all the control loops to maintain the predefined

process conditions, such as the Ns1u criteria 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 media exchange (see Fig. 3).

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

the accumulation of inhibitory cell metabolites and the glucose

concentration

from

dropping

below

5.55

mmol

L^1

(¼ 1 g L^1).

(a)

Transfer the bioreactor to the biosafety cabinet and allow

the MCs to sediment.

(b)

Open the remaining port lid as previously described and

remove 50% of the supernatant (approximately 61.5 mL)

while ensuring that no MCs are aspirated, and replace with

fresh pre-warmed culture media (see Note 6 and 13).

(c)

Following the media exchange, refasten the sterile port lid

and return the bioreactor to the control unit and reacti-

vate all the control loops.

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

stationary phase of cell growth (within 5–6 days post-

inoculation), at which point a final harvest may be performed.

(a)

Deactivate all the control loops and allow the MCs to

sediment, then transfer the bioreactor to the biosafety

cabinet and remove the remaining port lid of the bio-

reactor as previously described.

(b)

Proceed to remove the supernatant without aspirating

MCs and replace with pre-warmed DPBS. Allow the

MCs

to

sediment,

then

replace

the

DPBS

with

pre-warmed TrypLE Select 1 (see Note 13).

(c)

Refasten the sterile port lid and return the bioreactor to

the control unit. Reattach the necessary instruments and

periphery, reactivate all the control loops and set the

agitation rate to Ns1u for 15 min (see Note 14).

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