3. Following the coating procedure, remove the remaining

Synthemax™II-SC working solution from the T75-Flask and

add the 20 mL of pre-warmed cell culture medium.

4. Expand the hASC cells (see Note 5) in T75-Flasks to produce

enough cells to inoculate the desired cultivation systems.

(a)

Thaw the hASC cells at least 7 days prior to their expan-

sion in the stirred systems. Remove one cryovial contain-

ing approximately 1 mL cell suspension with a density of

at least 1  10⁶ cells mL^1 from the cryogenic storage

tank and immediately thaw the vial in a pre-warmed water

bath (37 ^C). Prevent the lid from touching the surface of

the water bath to prevent contamination. As soon as only

small ice crystals are left, wipe the vial down with a tissue

soaked in ethanol. The entire contents of the cryovial

should be transferred to the T75-Flask, containing the

pre-warmed media, under sterile conditions to achieve a

final cell density of 50,000 cells mL^1 (or approximately

13,333 cells cm^2).

(b)

Incubate the cells under static conditions at 37 ^C, 5%

CO2, and 80% relative humidity for 6 h (see Note 7).

(c)

Following a 6 h incubation period, inspect the T75-Flask

under the microscope (see Note 1) and determine cell

attachment to the substrate. Once confirmed, replace the

supernatant with fresh pre-warmed cell culture media.

(d)

Monitor the T75-Flask by microscope every 24 h until the

cells achieve 80–90% confluency (see Note 1). At this

point, passage the cells (see Notes 9, 11, and 12) into

2–5 freshly coated T75-Flasks (see Note 8), depending on

the desired inoculum quantity. Inoculate each flask con-

taining 20 mL of pre-warmed culture medium (see Note

6), at a density of 10,000 cells cm^2.

(e)

Again, monitor the T75-Flasks every 24 h by microscope

until the cells have achieved 80–90% confluency (see Note

1). Subsequently, passage the cells (see Notes 9 and 11),

determine their cell density and quality (see Note 12),

then transfer the required cell quantity to the cultivation

systems of choice.

3.2

hASC Expansion

in Corning’s Spinner

Flasks

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

expansion of hASCs in chemically defined medium and 125 mL

spinner flasks (100 mL working volume) using a partial media

exchange approach. Approximately 24 h prior to the inocula-

tion of the spinner flasks, begin with preparations of the culture

medium, MCs, and spinner system.

(a)

Pre-warm at least 150 mL of cell culture medium (see

Note 6) to 37 ^C for the expansion procedure per spinner

flask.

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Misha Teale et al.