2.2
hASC Expansion
in Spinner Flasks
1. A sufficient quantity of hASC prepared during the inoculum
production (see Subheading 3.1).
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. 125 mL spinner flasks (Corning®).
4. A CO2 incubator containing a magnetic stirrer platform (see
Note 7).
5. ProNectin® F MCs (see Note 10).
6. The enzymatic dissociation reagent and buffer of choice (see
Note 9).
2.3
hASC Expansion
in the BioBLU®
1. A sufficient quantity of hASC cells prepared during the inocu-
lum production (see Subheading 3.1).
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. BioBLU® 0.3c bioreactor vessels, the corresponding DAS-
GIP® Parallel Bioreactor System (Eppendorf), and conven-
tional compatible pH probes.
4. ProNectin® F MCs (see Note 10).
5. The enzymatic dissociation reagent and buffer of choice (see
Note 9).
2.4
Process
Analytics
1. Sample drawn from the cultivation systems (see Subheadings
3.2 and 3.3).
2. The enzymatic dissociation reagent and buffer of choice (see
Note 9).
3. A CO2 incubator with integrated orbital shaker (see Note 7).
3
Methods
The following protocols, developed for the expansion of the hASC
line hASC52Telo, include the preliminary preparation of the media
and MCs, inoculum production, preparation of the cultivation sys-
tems, inoculation, sampling, analytics, and cell harvest. With minor
modifications, these protocols may be adapted for the expansion of
other patient adipose tissue-derived mesenchymal stem cells.
3.1
T75-Flask-Based
Inoculum Production
1. Prepare 20 mL of cell culture medium (see Note 6) for the
inoculum production in a T75-Flask by pre-warming it to
37 C.
2. Coat the T75-Flasks with 15 mL of Synthemax™II-SC work-
ing solution (see Note 8).
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