Technical Bulletin

EX-CELL™ CD CHO: A Chemically Defined,

Animal-Component Free, Serum-Free Medium
for CHO Cells
Introduction
SAFC Biosciences has developed EX-CELL CD CHO, a
chemically defined, animal-component free, serum-free
medium optimized for the growth of CHO-derived cell lines
producing recombinant proteins, including monoclonal
antibodies (MAb). EX-CELLTM CD CHO is hydrolysate-free
and contains no animal-derived components.
The following study was undertaken to demonstrate the
ability of EX-CELLTM CD CHO to support the growth and MAb
production in a CHO-derived (DXB11) cell line producing
human MAb immunoglobulin G (IgG4). Additionally, the
ability of EX-CELLTM CD CHO to support the growth of a non-
producing CHO-K1 cell line was assessed. Both cell lines were
adapted to EX-CELLTM CD CHO using direct adaptation from
serum-containing suspension cultures prior to the evaluation
of growth and MAb production.
Materials
Cells
• DXB11, American Type Culture Collection, ATCC No.
CRL-11397
• CHO-K1, American Type Culture Collection, ATCC No.
CRL-61
Media and Supplements
• EX-CELLTM CD Cho, with hypoxanthine and thymidine,
SAFC Biosciences, Catalog No. 14361
• Iscove’s Modified Dulbecco’s Medium (IMDM), American
Type Culture Collection, ATCC No. 30-2005
• Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture
F12 (DMEM/F12), SAFC Biosciences, Catalog No. 51448
• Fetal Bovine Serum - Gamma Irradiated (FBS), SAFC
Biosciences, Catalog No. 12107
• 200 mM L-glutamine, SAFC Biosciences, Catalog No. 59202
Antibody Assay Kit
TM • Immuno-Tek Quantitative IgG ELISA Kit, ZeptoMetrix
Corporation, Catalog No. 0801182
Methods
Media/Supplement Preparation and Storage
EX-CELLTM CD CHO was supplemented with 8 mM
L-glutamine at point of use. All media were stored at
2 to 8 C protected from light. Cultures were maintained
using aseptic technique with no antibiotic or fungicide
supplementation.
Culture Techniques
Prior to adaptation, CHO-K1 and DXB11 cell lines were
maintained as suspension cultures in 125 mL shaker flasks in
DMEM/F12 (CHO-K1) and IMDM (DXB11) supplemented
with 10% FBS and 4 mM L-glutamine. After adaptation to
serum-free medium, the cells were transferred to 250 mL
shaker flasks and routinely subcultured every 3 - 4 days at a
density of 3 x 105 cells/mL and 4 x 105 cells/mL respectively
(60 mL volume per 250 mL shaker flask). The flasks were
shaken on an orbital shaker at 120 - 130 rpm and were
maintained at 37 C in a humidified atmosphere with 10%
CO2. Cell densities and viabilities were determined by trypan
blue exclusion.
Adaptation
The CHO-K1 and DXB11 cell lines were adapted to
EX-CELLTM CD CHO by direct adaptation. During the first
2 - 3 passages, suspension cultures previously grown in
basal medium + 10% FBS were seeded directly into
pre-warmed EX-CELLTM CD CHO at a density of
5 x 105 cells/mL. Cells were subcultured every 3 - 4 days and
were considered fully adapted when cell densities achieved
> 2.0 x 106 cells/mL and viabilities of > 95%.

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SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
USA
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Growth Studies and Antibody Production
Growth studies were initiated after adaptation and were
monitored over an additional 6 passages. The cells were
subcultured one additional time and daily cell counts were
determined until culture viabilities dropped below 60%.
Additionally, during the last passage, daily aliquots from the
DXB11 cell suspension were taken for IgG determination.
Each aliquot was micro-centrifuged (1000 rpm) for
2 minutes, followed by collection and storage (-20 C) of the
supernatant. Antibody (human IgG) production was
determined by ELISA (ZeptoMetrix Human IgG ELISA kit).
The appropriate sample dilutions were prepared in dilution
buffer supplied with the kit. The absorbance was read at
405 nm on a VersaMaxTM microplate reader and calculations
were performed using SoftMax® Pro. 4.0 software (both
from Molecular Devices Corporation).
Results
Growth Studies
Both the CHO-K1 and DXB11 cell lines typically reached
densities of at least 3.5 x 106 and 2.5 x 106 respectively when
subcultured every 4 days in EX-CELLTM CD CHO. Figure 1
depicts the typical growth of CHO-K1 and DXB11 cells over
the course of 6 passages in EX-CELLTM CD CHO. Figure 2
illustrates the culture longevity of CHO-K1 and DXB11 cells
over a period of 7 - 8 days in EX-CELLTM CD CHO medium.
CHO-K1 densities increase steadily over the first 4 days, reach
a plateau by day 5 and steadily decline after day 5. Viabilities
start to drop after day 5 and are below 60% on day 7. DXB11
densities increase steadily over the first 5 days, reach a plateau
by day 6 and decline after day 6. Viabilities start to drop
after day 5 and are below 60% on day 7.
Monoclonal Antibody Production
A human IgG ELISA assay kit was used to titer the production
of IgG by the DXB11 cell line. Figure 3 depicts a typical
viability and concurrent IgG production in the DXB11 cell line
cultured in EX-CELLTM CD CHO. Viabilities remain above 90%
until day 5 and proceed to decline thereafter. IgG production
steadily increases with peak productivity on day 8. Additional
evaluations conducted by SAFC Biosciences demonstrate that
IgG production in EX-CELLTM CD CHO is comparable to levels
typically experienced in a basal formulation + 10% FBS (Figure
4). These studies demonstrate that EX-CELL TM CD CHO
supports high density cell growth and MAb production in a
CHO-derived cell line.

Figure 1

CHO-K1
CHO-K1Cell Line
Cell Line DXB11 Cell Line
DXB11 Cell Line

6.00E+06
4.50E+06

4.00E+06
5.00E+06
Density (cells/mL)
(cells/mL)
(cells/mL)

3.50E+06
Density (cells/mL)

4.00E+06 3.00E+06
CellDensity

Cell Density

2.50E+06
3.00E+06
2.00E+06
Cell

Viable Cell

2.00E+06 1.50E+06
Viable
Viable

Viable

1.00E+06
1.00E+06
5.00E+05

0.00E+00 0.00E+00
1 2 3 4 5 6 1 2 3 4 5 6
Passage number
Passage number after
after adaptation
adaptation Passage
PassageNumber
numberAfter
after Adaptation
adaptation

Figure 1: Multiple passage growth of CHO-K1 and DXB11 cells in EX-CELLTM CD CHO.

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 Figure 2

CHO-K1 Cell
CHO-K1 CellLine
Line DXB11
DXB11Cell LineLine
Cell

6.00E+06 120% 5.00E+06 120%

Density 4.50E+06

(cells/mL)
5.00E+06 Viability 100% 100%
4.00E+06
Density (cells/mL)

Density (cells/mL)
(cell/mL)

3.50E+06
4.00E+06 80% Density 80%
3.00E+06 Viability

CellDensity
Cell Density

Viability
Viability
Viability

Viability
3.00E+06 60% 2.50E+06 60%
2.00E+06
Viable Cell

ViableCell
2.00E+06 40% 40%
1.50E+06
Viable

Viable
1.00E+06
1.00E+06 20% 20%
5.00E+05

0.00E+00 0% 0.00E+00 0%
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

Days
Days in
in Culture
culture Days
DaysininCulture
culture

Figure 2: Typical culture longevity of CHO-K1 and DXB11 cells in EX-CELLTM CD CHO.

Figure 3 Figure 4

1.6 120%
DXB11
1.4 Productivity
100%
1.2
Basal + 10% FBS
80%
Basal + 10% FBS
100
100
% Relative to FBS Control

1
IgG (ug/mL)
(µg/mL)

EX-CELL CDCD
CHO
% Relative to FBS Control

EX-CELL CHO
Viability
Viability

75
75
0.8 60%
IgG

0.6
40% 50
50

0.4
25
25
IgG (ug/mL) 20%
0.2 Viability
00
0
4 5 6 7 8
0%
1
Days ininCulture
Days culture

Figure 3: Human IgG production (µ µg/mL) and cell viability in Figure 4: Normalized human IgG production by DXB11 cells
DXB11 cells cultured in EX-CELLTM CD CHO. cultured in EX-CELLTM CD CHO and IMDM + 10% FBS.

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 Warranty, Limitation of Remedies
SAFC Biosciences warrants to the purchaser for a period of one year from date of delivery that this product conforms to
its specifications. Other terms and conditions of this warranty are contained in SAFC Biosciences’ written warranty, a
copy of which is available upon request. ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING THE IMPLIED
WARRANTY OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE, ARE EXCLUDED. In no case will SAFC
Biosciences be liable for any special, incidental, or consequential damages arising out of this product or the use of this
product by the customer or any third party based upon breach of warranty, breach of contract, negligence, strict tort,
or any other legal theory. SAFC Biosciences expressly disclaims any warranty against claims by any third party by way of
infringement or the like. THIS PRODUCT IS INTENDED FOR PURPOSES DESCRIBED ONLY AND IS NOT INTENDED FOR
ANY HUMAN OR THERAPEUTIC USE.
Additional Terms and Conditions are contained in the product Catalog, a copy of which is available upon request.

EX-CELL™ is a trademark of SAFC Biosciences, Inc.

SoftMax® is a registered trademark of Molecular Devices Corporation.
VersaMaxTM is a trademark of Molecular Devices Corporation.

© 2006 SAFC Biosciences, Inc.

Issued March 2006 T067

0404 1105

United States Europe Asia Pacific

SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
USA UNITED KINGDOM AUSTRALIA

www.safcbiosciences.com Phone
Toll free-USA
Fax
+1 913-469-5580
1 800-255-6032
+1 913-469-5584
Phone
Fax
E-mail
+44 (0)1264-333311
+44 (0)1264-332412
info-eu@sial.com
Phone
Toll free-AUS
Fax
+61 (0)3-9362-4500
1 800-200-404
+61 (0)3-9315-1656
E-mail info-na@sial.com E-mail info-ap@sial.com