during the process step. For example, if the weight of the

suspension remains unchanged after removing and replacing

the supernatant with fresh cell culture media, DPBS or TrypLE

Select 1, and no MCs were lost during aspiration, one may

assume that the microcarrier suspension has maintained its

initial concentration.

14. The Ns1u criterion describes the impeller speed required to

locate the particles at the bottom of the bioreactor, while

ensuring that none are at rest. This criterion not only guaran-

tees sufficient mass transfer, but also reduces the shear stress

which

cells

may

be

subjected

to

during

cultivation

[38, 52]. Studies have shown the criterion is met for ProNec-

tin^® F MCs at agitation speeds of 49 rpm in 125 mL spinner

flasks (Corning^®) and between 70 and 80 rpm in the BioBLU^®

0.3c (Eppendorf) with marine impeller and down-pumping

set-up [47].

15. The use of an appropriate cryogenic medium for the long-term

storage of the cells at 160 ^C is beneficial to maintaining cell

quality during and following the thawing process. To this end,

cells were resuspended in Bambanker™(Nippon Genetics

Europe GmbH) to achieve a concentration of 1  10⁶ cells

mL^1 prior to freezing.

16. To reduce and maintain a pH of 7.2 within the instrumented

BioBLU^® 0.3c bioreactor, CO2 was added to the overlay of the

inlet gas.

17. The process parameters used for the cultivation in the instru-

mented stirred bioreactor system are summarized in Table 3.

18. The BioBLU^® 0.3c is sold with a sampling valve which allows

for the sterile sampling of the bioreactor, while it is coupled to

the control unit. However, this port was developed for sam-

pling single-cell suspension cultures and is therefore only par-

tially compatible with MC processes. The strong aggregation

of the MCs towards the end of the cultivation could potentially

result in clogging of the sample valve, adversely impacting

sample homogeneity. Therefore, sampling of the bioreactor

over the spare probe port, while within a biosafety cabinet is

highly recommended.

Table 3

Parameter settings for temperature, pH, DO, and CO2 control loops

Parameters

Setpoint

Deadband

P value

I value

Temperature

37 ^C

0.00

10.0

240.0 s

pH

7.20

0.02

50.00

1800 s

DO

30.0%

0.00

0.10

300 s

P proportional, I integral

Mesenchymal Stem Cell Expansion at Benchtop-Scale

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