resuspend the suspension 10 times using a 10-mL sterile

pipette, then filter the suspension through a sterile 70-μm

cell strainer (Corning^®) into a sterile centrifugation tube.

(e)

Use an equal amount of fresh pre-warmed culture media

(see Note 6) to wash the interior of the spinner flask, filter

it through the strainer into the sterile centrifugation tube

containing the initial filtrate, then centrifuge at 300  g

for 5 min.

(f)

Remove the supernatant and resuspend the cells in

pre-warmed DPBS, then centrifuge the cells at 300  g

for 5 min. Repeat this washing step twice.

(g)

Finally, remove the supernatant and resuspend the cells in

the desired amount of either fresh pre-warmed culture

media (see Note 6), for direct use, or pre-cooled cryogenic

medium, for long-term cell storage at 160 ^C (see Note

15).

3.3

hASC Expansion

in Eppendorf’s

Instrumented BioBLU^®

0.3c

1. The following procedure describes the xeno- and serum-free

expansion of hASCs in chemically defined medium and an

instrumented BioBLU^® 0.3c stirred bioreactor (150 mL work-

ing volume). This method makes use of a partial media

exchange approach. Approximately 24 h prior to the inocula-

tion of the BioBLU^® 0.3c, begin with preparations of the

culture medium, MCs, and bioreactor system.

(a)

Calibrate the offset of the dissolved oxygen (DO) probe

using pure N2 gas, as well as the offset and slope of the pH

probe using the appropriate buffers.

(b)

Secure the pH probe on an open Schott flask containing

sterile DPBS in such a way that more than half of the glass

section is submerged. Autoclave the pH probe at 121 ^C

for 20 min to ensure sterility prior to installation. As DO is

measured optically through a membrane, the probe does

not come into contact with the cultivation medium and

does therefore not require autoclaving.

(c)

Pre-warm at least 200 mL of cell culture medium (see

Note 6), per BioBLU^® 0.3c vessel, to 37 ^C for the

expansion procedure.

(d)

Thereafter, transfer at least 1.5 g (¼ 540 cm²) of ProNec-

tin^® F MCs (see Note 10) per BioBLU^® 0.3c to a 100 mL

Schott Flask (Duran) and suspend them in sterile DPBS to

achieve a final MC concentration of 100 g L^1. Autoclave

the MC suspension at 121 ^C for 20 min to ensure

sterility.

(e)

Allow the suspension cool to room temperature and the

MCs to sediment to the bottom of the flask. Remove and

Mesenchymal Stem Cell Expansion at Benchtop-Scale

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