(d)
Following enzymatic dissociation, deactivate all the con-
trol loops and return the bioreactor to the biosafety
cabinet.
(e)
Remove the remaining port lid as previously described
and gently resuspend the cell suspension 10 times using
a 10 mL sterile pipette, then filter the suspension through
a
70
μm
cell
strainer
(Corning®)
into
a
sterile
centrifugation tube.
(f)
Use an equal amount of fresh pre-warmed culture media
(see Note 6) to wash the interior of the bioreactor, filter it
through the strainer into the sterile centrifugation tube
containing the initial filtrate, then centrifuge at 300 g
for 5 min.
(g)
Remove the supernatant and resuspend the cells in
pre-warmed DPBS, then centrifuge the cells again at
300 g for 5 min. Repeat this washing step twice.
(h)
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 the long-term cell storage at 160 C (see
Note 15).
3.4
Sampling and
Quality Control
This section describes the sampling procedure for the two cultiva-
tion systems and their corresponding workup, as well as basic
quality control measures, which may be performed in an upstream
setting. A simplified overview of the routine sample work-up is
given in the Fig. 4.
1. Directly following the attachment phase, a 7 mL sample is
taken to determine cellular attachment efficiency, cell distribu-
tion on the MCs, as well as the prevailing substrate and metab-
olite concentrations.
(a)
Transfer the homogenous sample to a 15-mL centrifuge
tube (Corning®) for intermediate storage.
(b)
Remove 1 mL of the homogenous sample and transfer to
a 1.5-mL microtube (Eppendorf®) and allow the MCs to
sediment in both containers.
(c)
Transfer the supernatant to another 1.5-mL microtube in
such a way as to not aspirate any MCs. This sample may
then be analyzed to determine substrate and metabolite
concentrations (see Note 3).
(d)
To the pellet remaining in the 1.5-mL microtube, add
1 mL of formalin and incubate at room temperature for
30 min. Subsequently, the sample may be either used
directly
to
determine
cell
distribution
and
MC
100
Misha Teale et al.