the expansion of primary hMSCs, direct inoculation of the bio-

reactor with the cells thawed from the WCB is strongly recom-

mended to ensure that the population doubling level (PDL) is kept

below 15.

The stem cell expansions, which were accompanied by sam-

pling, to determine cell density, viability and distribution on the

MCs, substrate and metabolite concentrations (see Subheading

3.4), as well as flow cytometry analysis, and a partial media

exchange (50%) on the fourth day of cultivation, were concluded

with a final cell harvest. The harvest was followed by product

processing and freezing to enable the long-term storage of the

expanded cells, prior to their application. Cell application is, how-

ever, not the focus of the procedures described in this chapter.

The stem cell expansions were preceded by, as already described

by Kaiser et al. (2013) [47] and Jossen et al. (2014) [38] and shown

in Fig. 2b, (a) screening to determine the optimal medium-MC

combination, (b) elucidation of the fluid dynamic characteristics

(tip speed, Reynolds number, specific power input, local normal

and shear stresses, as well as Kolmogorov length), and (c) the

identification of the cultivation vessel dependent suspension criteria

(Ns1, Ns1u) under the defined process conditions (working volume,

agitation rate, inoculation density, and MC concentration). Here

an Ns1 of approximately 60 rpm could be determined for the

spinner flask and 130 rpm for the BioBLU^® 0.3c. According to

Jossen (2020) [6], the NS1u criterion (or the lower limit of Ns1,

where several microcarriers are located on the floor of the cultiva-

tion vessel, but none are at rest) is the most suitable for predicting

the agitation rate which guarantees the maximum achievable live

cell density and retention of stem cell properties during hMSC

expansion. For more information regarding experimental realiza-

tion, the interested reader is referred to the five part instructional

video “Expansion of human adipose tissue-derived mesenchymal

stem cells in stirred single-use bioreactors” (see https://youtu.be/

PZv7sx4gY9Y).

Regarding the screening for optimal medium-MC combina-

tions, results showed that ProNectin^® F MCs (Pall^® SoloHill^®)

performed best in combination with the UrSuppe medium formu-

lation (Cardiocentro Ticino). This combination was subsequently

used to optimize cell attachment to the MCs at spinner scale,

conduct fluid dynamic studies of both the spinner flasks and Bio-

BLU^® 0.3c, including sedimentation studies to determine optimal

agitation rates, and experiments to achieve at least a 90% harvest

efficiency. The experimentally determined Ns1u criteria were met at

agitation rates of 49 rpm and between 70–80 rpm for the spinner

flask and BioBLU^® 0.3c, respectively.

The ideal point for cell harvest was determined to lie between

days 5 and 6. To further increase the number of harvested cells for

clinical application, the operation at the maximum working volume

of the BioBLU^® 0.3c vessel and a subsequent scale-up to the

90

Misha Teale et al.