thus elucidating the density and viability of the cell population.

For further information, consult the user manual supplied by

the manufacturer.

3. Substrate (glucose, L-alanyl-L-glutamine, L-glutamine, and L-

glutamate) and metabolite (lactate and ammonium) concentra-

tions were measured using the Cedex Bio Analyzer (Roche

Custom Biotech). The analyzer can be fitted with various test

kits, which allows the system to measure the concentration of

multiple media components simultaneously.

4. The expression of specific extracellular hMSC markers serves as

an indicator of cell quality. Thus, marker expression rates were

analyzed at the beginning, during, and upon completion of

cultivation. To determine the prevalence of these CDs,

fluorophore-conjugated primary antibodies corresponding to

the CD or antigen of interest (positive markers: CD73, CD90,

CD105, negative markers: CD36, CD45) were used in con-

junction with the MACSQuant 10 flow cytometer (Miltenyi

Biotec). The evaluation of the results was performed using the

FlowLogic software version 7.2.1 (Inivai Technologies Pty.

Ltd.).

5. An adipose-derived mesenchymal stem cell line, isolated from a

Caucasian female patient, immortalized with human telome-

rase reverse transcriptase (hTERT) (hASC52Telo or ATCC^®

SCRC-4000™) and purchased from ATCC^®.

6. Chemically defined culture media guarantees reproducible cul-

tivations due to the absence of animal-derived serum, proteins,

peptides, or other components. Furthermore, the use of xeno-

free media is essential for the production of cell-based thera-

pies, as it eliminates the risk of zoonotic diseases, such as bovine

spongiform encephalitis, which could potentially contaminate

the final product. To this end, the chemically defined, xeno-

and serum-free medium UrSuppe (Cardiocentro Ticino) was

used for the expansion of the hASCs, as recommended by the

manufacturer. See the publications by Jossen et al. [51] and

Panella et al. [50] for more information.

7. In order to maintain the optimal process conditions for static

cell expansion and subsequent sample work-up, both a CO2

incubator with (Minitron, Infors AG) and without (BBD

6220, Heraeus Group) an integrated orbital shaker platform

(set to 5% CO2, 130 rpm, 25 mm and 37 ^C) were used, while

for the spinner flask cultivations, an agitation platform (Dura-

Mag, Chemglass Life Sciences) was used in addition to the

latter.

8. The Synthemax™II-SC working solution can be prepared by

reconstituting Synthemax™II-SC Substrate (Corning^®) using

sterile cell culture grade water (WFI) to produce a 1 mg mL^1

Mesenchymal Stem Cell Expansion at Benchtop-Scale

105