(b)

Subsequently, transfer at least 1 g (¼ 360 cm²) of Pro-

Nectin^® F MCs (see Note 10) per spinner flask to a

100 mL Schott Flask (Duran) and suspend them in sterile

Dulbecco’s phosphate-buffered saline (DPBS) to achieve

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

suspension at 121 ^C for 20 min.

(c)

Allow the suspension to cool to room temperature and the

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

discard the supernatant, while ensuring that no MCs are

lost in the process. Replace the discarded supernatant with

pre-warmed culture medium (see Note 13). Repeat this

step twice to obtain a sterile ProNectin^® F MC stock

solution.

(d)

Transfer the packaging containing the spinner flask to the

biosafety cabinet. Open the package and assemble the

spinner flask under the bench using the appropriate tech-

nique to guarantee cultivation vessel sterility.

(e)

Add 90 mL of pre-warmed culture media (see Note 6) and

10 mL of ProNectin^® F MC stock solution to the spinner

flask.

(f)

Transfer the spinner flask back to the incubator containing

the stirrer platform and allow the MCs to equilibrate

under process conditions, i.e., 37 ^C, 5% CO2, 80% rela-

tive humidity and Ns1u (see Notes 7 and 14), for approxi-

mately 24 h prior to inoculation. This step also serves to

ensure vessel sterility prior to cultivation.

2. Inoculation of the spinner flask.

(a)

Calculate the necessary volume of inoculum, following

cell density and quality control (see Note 12), to achieve

an initial cell density of 15,000 cells cm^2 or 5.4  10⁶

cells per spinner flask.

(b)

Transfer the equilibrated spinner flask to the biosafety

cabinet and allow the MCs to sediment. Subsequently

remove the top lid and place it stirrer side up within the

biosafety cabinet.

(c)

Remove the volume of culture medium to be replaced

with the inoculum and transfer it to a sterile 15-mL cen-

trifuge tube (Corning^®). This sample may then be used to

determine the initial substrate and metabolite concentra-

tion within the spinner flask (see Note 3). Add the inocu-

lum to the spinner flask to achieve the target starting cell

density.

(d)

Transfer the spinner flask back to the incubator containing

the stirrer platform and set the agitation rate to Ns1u for

5 min (see Note 14). Subsequently, reduce the speed to

0 rpm and allow the cells to sediment and attach to the

MCs for 12 h.

Mesenchymal Stem Cell Expansion at Benchtop-Scale

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