6.0  10⁵ cells mL^1 (1.7  10⁵ cells cm^2) after 6 d, while the

spinner flasks required 7 d to achieve a density of 5.3  0.5  10⁵

cells mL^1 (1.5  10⁵ cells cm^2). Due to the difference in maxi-

mum cell densities achieved and the working volumes of the culti-

vation vessels, 70% more viable cells could be produced in the

BioBLU^® 0.3c, compared to a spinner flask. The slight deviation

in the CD105 marker expression rates between cell populations

sampled from the two cultivation vessels following the 7 d cultiva-

tion period, highlight the importance of harvesting prior to reach-

ing the stationary phase (between 5 and 6 days) to assure cell

quality. Harvesting the cells 24 h prior to reaching the stationary

phase would result in viable cell densities of 3.9  10⁵ cells mL^1

for the BioBLU^® 0.3c and 3.4  10⁵ cells mL^1 for the spinner

flask, superseding or matching values reported for hASCs cultivated

in similar stirred bioreactor systems, namely the 1.3 L Bioflo^®

(0.6  10⁵ cells mL^1) and 2 L UniVessel^® SUB (4.1  10⁵ cells

mL^1), using chemically defined xeno- and serum-free media

[48, 49]. It is worth mentioning that subsequent studies should

also include investigations to determine the differentiation potency

of the harvested hASCs, as described by Panella et al. [50].

2

Materials

During preparation of the cultivation systems, inoculum, media,

and microcarriers, be sure to use at least research grade material

from a reputable supplier. Diligently follow all biosafety regula-

tions, especially when discarding waste which has come into contact

with biological material. The materials listed below were necessary

for the expansion of the hASCs in the single-use spinner flasks and

BioBLU^® 0.3c. In addition, the following standard cell culture

equipment and materials were routinely used: biosafety cabinet

class II, centrifuge, water bath, magnetic stirrer platform, CO2

incubators, pH meter, sterile pipettes, and pipette tips. Further-

more, the following devices were required for routine analytics:

microscope (see Note 1), cell counter (see Note 2), media compo-

nent analyzer (see Note 3), and flow cytometer (see Note 4).

2.1

hASC Inoculum

Production in T75-

Flasks

1. The hASC cell line of interest (see Note 5).

2. The corresponding chemically defined, xeno-free cultivation

medium prepared according to the manufacturer’s instructions

and stored at 4 ^C in the dark until use (see Note 6).

3. T75-Flasks with filtered venting caps.

4. A CO2 incubator (see Note 7).

5. Synthemax™II-SC working solution (see Note 8).

6. The enzymatic dissociation reagent and buffer of choice (see

Note 9).

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Misha Teale et al.