SuperSpinner D 1000 vs. standard Spinner Flask: efficient cultivation of CHO cells
Application Note
K. Schmale, J. Herrmann and A. Kocourek
Sartorius Stedim Biotech GmbH, Germany
Introduction
Sufficient supply of oxygen during cultivation
processes is one requirement for high cell
densities and product concentrations. The
majority of common laboratory scale cultivation devices, like spinner flasks are surface
aerated. In these flasks the oxygen supply
is limited by the horizontal liquid surface.
This often results in an oxygen limitation and
in low productivities.
The SuperSpinner D 1000 is a single-use &
pre-assembled modified spinner flask for
efficient lab scale cultivation of animal cells.
Main feature is a membrane aeration stirrer
which enables controlled and gentle mixing,
and bubble-free aeration whilst avoiding
foam generation. The gassing membrane
ensures higher oxygen transfer and thus
optimal growth conditions and higher cell
densities compared to the traditional headspace aerated spinner flasks.
A hollow-fiber membrane is wound around
the stirrer bar which contains a magnetic core
driven by a magnetic drive unit. A membrane
gas pump feeds ambient air through a sterile
filter into the flask. Oxygen and carbon dioxide diffuse from the hollow-fiber membrane
into the cell suspension. The multiple
windings of the hollow fiber represent an
enormous increase in the (active) aeration
surface. The cells are not limited by oxygen,
which results in a higher productivity compared to traditional spinner flasks.
In this work we describe the evaluation of
the SuperSpinner D 1000 for the cultivation
of Chinese Hamster Ovary (CHO) cells.
Cell line
CHO DG44 ST1-C6
Media & Additives
– ProCHO5-media (Lonza, BE12-766P)
– L-Glutamin (4mmol, Lonza, 17-605F)
– HT (50-fold, PAN P07-01100)
Equipment
– SuperSpinner D 1000
(Sartorius Stedim Biotech GmbH)
– Membrane pump
(Sartorius Stedim Biotech GmbH)
– Magnetic drive
(Sartorius Stedim Biotech GmbH)
– Spinner Flask
– BIOSTAT® CultiBag RM
(10 l, Sartorius Stedim Biotech GmbH)
– Minisart®
(Sartorius Stedim Biotech GmbH)
– Centrifuge (Sigma)
– CO2- Incubator (Braun)
Seed Culture
Cells are cultivated in ProCHO5 Medium
with 4 mM L-Glutamine and 1-fold HT in a
BIOSTAT® CultiBag RM. Starting cell density is
about 2 + 105 - 5 + 105 cells/ml. Cultivation
is carried out under common conditions
(37°C, 5% CO2, rocking rate 60 rpm, angle 6°,
gassing rate 110 ml/min) over 2 to 3 days.
The achieved biomass is used as seed for
further studies.
Main Culture
Cell Suspension (pre-culture) is centrifuged
at 500 + g for 15 min. Supernatant is discarded and the cells are resuspended in fresh
medium. The starting cell density is about
1 + 106 cells/ml. The SuperSpinner D 1000 is
filled with cells and medium (same compositions as in pre-culture) as described in the
manual. The working volume is 800 ml. CHO
cells are cultivated in a batch culture at 37°C
in a CO2-incubator (5% CO2 saturation,
85% humidity). The aeration is ensured by
connecting the membrane pump with a silicone tube to the sterile vent filter. The stirring rate is 120 rpm. Under same conditions
CHO cells are cultivated in standard spinner
flasks (working volume 800 ml). The stirring
speed is 70 rpm (identical tip speed as in SSD).
A passive head-space aeration via the
horizontal surface takes places.
100
16
90
80
12
70
10
60
50
8
40
6
viability [%]
viable cell number [x 10*6 cells/ml]
14
30
4
20
2
10
0
0
0
24
48
72
96
120
144
cultivation time [h]
viable cell number SSD
viable cell number SF
viability SSD
viability SF
Figure 1
SuperSpinner D 1000 vs. standard spinner flask: cultivation of CHO. Described are the
viable cell numbers [+ 106 cells/ml] as well as the viability [%] during the cultivation.
SSD = SuperSpinner D 1000; SF = standard spinner flask).
Sampling & analysis
Samples were taken in place by connecting a
sterile syringe to the clave adapter.
Cell density and viability were determined
automatically (Nucleocounter™, Chemometec
A/S).
Reference
Lehmann J., Heidemann R., Riese U.,
Lütkemeyer D., Büntemeyer H.,
Der Superspinner: Ein „BrutschrankFermenter“ für die Massenkultur tierischer
Zellen, BioEngineering 5+6 (1992).
Results
The cultivation of CHO cells in the
SuperSpinner D 1000 resulted in a maximum
viable cell number of 6.5 + 106 cells/ml
after 4 days of cultivation. The maximum
cell density attained in a standard spinner
flask cultivation was less than 2 + 106
viable cells/ml.
K. Schmale, SuperSpinner D 1000: a disposable
bioreactor for efficient lab-scale cultivation
of animal cells, Nature Methods, Application
Note, August 2008.
Summary
When cultivating CHO cells in the
SuperSpinner D 1000, a threefold increase
in the viable cell number could be observed
compared to that in a standard spinner
flask. These data clearly show that the
SuperSpinner D 1000 delivers higher cell
densities than a traditional spinner flask and
thus provides a simple solution for efficient
lab-scale cultivation of different cell cultures.
Almost all studies showed a faster growth in
the new SuperSpinner D 1000 compared to
the standard spinner flask. A result which is
probably caused by the efficient and bubble
free aeration of the SuperSpinner D 1000.
K. Schmale, C. Schwiebert, A. Kocourek
and R. Eibl, Innovative Membranbegasung,
transkript, Nr. 8-9, 14. Jahrgang, 2008.
Figure 1
SuperSpinner D 1000 product picture
Sartorius Stedim Biotech GmbH
August-Spindler-Strasse 11
37079 Goettingen, Germany
Phone +49.551.308.0
Fax +49.551.308.3289
www.sartorius-stedim.com
USA Toll-Free +1.800.368.7178
UK +44.1372.737159
France +33.442.845600
Italy +39.055.63.40.41
Spain +34.91.3586102
Japan +81.3.3740.5407
Specifications subject to change
without notice. Printed and copyrighted
by Sartorius Stedim Biotech GmbH
W·G
Publication No.: SL-4049-e09011
Order No.: 85034-536-33
Ver. 01 | 2009