Growing CHO Cells in a CelliGen® BLU
Benchtop, Stirred-Tank Bioreactor
Using Single-Use Vessels
The study presents a typical protocol for the setup and
operation of New Brunswick Scientific’s new CelliGen®
BLU single-use, stirred-tank bioreactor, a versatile new
benchtop system for the culture of a wide range of
mammalian cells. This bioreactor has been designed to
provide research and production facilities with a single-
use vessel which combines the benefits of both
traditional stirred-tank design and single-use technology,
capable of seamless process scale-up. The system can
be operated in batch, fed-batch or continuous modes. A
procedure for culturing Chinese Hamster Ovarian (CHO)
cells in a 5.0 Liter (L) vessel, using CD CHO serum-free
medium in a batch culture is described.
Historically, stirred-tank fermentors and bioreactors have
been the trusted design for culturing all types of submerged
cultures including suspension and anchorage-dependent
mammalian cells, insect, yeast, plant and microbial cultures.
The tried and tested tank design offers scalability and proven
reproducibility which is pivotal for cost-saving process
development and productivity. In the last decade, there has
been an increasing acceptance and use of single-use
technologies, due to their convenient operation and low
start-up cost. Single-use systems eliminate the need for
cleaning and sterilization, reduce validation requirements,
provide rapid turn-around between runs, and significantly
reduce the risk of cross contamination and microbial
contamination because the culture vessel is only used once
and then discarded. Although single-use, stirred-tank
systems in the 75 – 2000 L scale have been on the market
for some time, as have small-scale single-use bags that are
gently rocked rather than stirred, until now there has been
no single-use stirred-tank system for small-scale work. The new CelliGen BLU fills that void, offering a proven stirred-
tank design as well as the benefits of single-use technology
in a benchtop system.
Materials and Methods
CelliGen BLU single-use vessels are offered in 5.0 and 14.0 L
total volume capacities. The vessels are delivered pre-
assembled with pitched-blade impeller, porous microsparge,
and all the necessary tubing, filters, and connectors; and
come sterilized, ready for use right out of the package. All
components in product contact are made of materials that meet USP Class VI standards and have been tested for
leachables and extractables, making these vessels
appropriate for cGMP environments. In this protocol, we
describe use of a CelliGen BLU with 5.0 L vessel.
CelliGen BLU’s compact, non-disposable control station is
designed to provide advanced process management and
monitoring capability, ranging from three fixed-speed pumps
for additions and harvesting, to a powerful controller with
15” industrial color touchscreen monitor. Multiple options,
including gas flow control, a weight scale, validation
packages and more, enable customization to your needs.
The control station used in this protocol was configured with
one 2 – 100 cubic centimeters per minute (ccm) Thermal
Mass Flow Controller (TMFC) for direct sparging of gases
and an integrated gas overlay with 0.1 - 3.0 Standard Liters
Per Minute (SLPM) flow rate also regulated by a TMFC.
Both the gas flow and gas overlay are capable of 4-gas
mixing for automatic pH and Dissolved Oxygen (DO) control.
Pumps, temperature control, agitation, as well as all of the
other process loops, were controlled and monitored through
the powerful Reactor Process Controller (RPC) firmware
installed in the controller. DO was monitored using a non-
invasive reusable polarographic DO probe; and pH was
monitored using a non-invasive optical pH probe and
One 2.5 mL vial of CHO cells was thawed and used to
inoculate a 125 mL shake flask which contained 25 mL of
serum-free CD CHO medium (Invitrogen 10743-029) which
was pre-warmed to 37°C. The flask was placed on an orbital
biological shaker (NBS Innova® 2000) placed inside a CO2
incubator (NBS Galaxy® 170 R), and set at 120 rpm.
The CO2 incubator was programmed to provide a gas mix of
5% CO2 and 95% air, at 37.0°C. On day 4, when the viable
cell density reached 1.5x106 cells/mL, the cells were
transferred into a 500 mL shake flask which contained 100
mL of freshly made, pre-warmed medium and allowed to
incubate for 3 additional days at the same conditions as
earlier. The cells were then transferred to two 1 L shake
flasks, each containing 250 mL of the freshly made medium.
The inoculum was grown in the shake flasks until cell
density reached 2.0 - 3.0 x 105 cells/mL, with greater than
90% cell viability, sufficient for the bioreactor inoculation.
Bioreactor Set-Up & Inoculation
One day before the cells reached inoculation density, the
growth medium was warmed to 37°C and the DO probe
was polarized. For this study, 3.0 L of sterile CD CHO
serum-free medium was prepared by pre-warming at 37°C
for 24 hours in a CO2 incubator. During this time, the DO
probe was connected to the controller for at least 6 hours to
enable polarization, as per the manufacturer’s
Once the medium was warmed and the inoculum grown to
sufficient starting density, the CelliGen BLU bioreactor
vessel was removed from its sterile packaging and the heat
blanket supplied with the unit was wrapped around the
outside of the vessel. Next, the vessel containing the cell
culture medium was connected to one of the bioreactor
vessel’s inlet lines using a tube welder. (A tube welder is
offered as an optional accessory to the CelliGen BLU. A
pre-sterilized medium filter with an attached quick connect
or Luer connection can also be used if a tube welder is not
available). Since this was a batch process, all of the medium
was pumped into the bioreactor vessel. All additional
connections to the controller including sparge, overlay, RTD,
pH, and agitation were also made.
pH and DO were calibrated through the touchscreen
controller, and all process setpoints were entered on the
touchscreen using the Control Setpoint values shown on the
next page. Once the parameters were at their setpoints, the
inoculum flasks were connected to the addition line in a
sterile manner using a tube welder and contents were
pumped into the bioreactor vessel.
Cultivation of animal cells in an environment optimal for
manufacture of desired end products require monitoring and
control of a substantial number of physical and chemical
parameters. Physical parameters include temperature, fluid
flow (gas flow and liquid flow) rates and agitation rates.
Chemical parameters include the dissolved oxygen (DO)
concentration and pH.
pH Control Parameters
Agitation .....................80 rpm
pH control was set to Auto mode, which automatically adds
base solution or CO2 gas to the system based on culture
Dissolved Oxygen (DO) Control
PID values......... factory set default values
Base....................Sodium bicarbonate, 7.5% solution
Base Solution Transfer tubing . . .Narrow bore silicone tubing
with Luer-connection (1⁄18" ID & 1/4" OD)
Vessel inlet........1/8” inlet tubing in the vessel headplate
DO control was set to Auto mode, which automatically
regulates gas mixing based on culture demand.
PID values: factory set default values.
The gas control was set to 4-Gas mode, which automatically
maintains DO and pH. The gas flow rate was based on the
Up until day 3, gases were introduced into the vessel
headspace only through the overlay port at a rate of
0.30 L/min using 4-Gas mixing to maintain pH and DO. On
day 3, and for the remainder of the run, 5 - 10 ccm of gas
were directly sparged into the system using a porous
sparger and automatic 4 gas mixing. The overlay gas flow in
the vessel headspace was kept at the previous settings.
A built-in sampling device enabled sterile sampling. Daily off-
line measurements of glucose and lactate concentration
were read using a YSI 2700, and cell density and cell viability
was measured using an Automated Cell Counting System
Results & Discussion
All data was logged via BioCommand® Batch Control PC-
compatible Supervisory Control and Data Acquisition
(SCADA) software (New Brunswick Scientific).
As shown in Figure 1, the CHO cells in this study grew
steadily, reaching a maximum viable cell density of 5.55 x
106 cells/mL on day 5.
Cell viability, shown in Figure 2, ranged between 97.1 and
97.9% through Day 5, until the nutrient source, glucose,
was depleted from the medium, as shown in Figure 3.
As expected, lactate production steadily increased as the
available glucose in the medium was consumed. As glucose
in the medium become exhausted, consumption of lactate
as a secondary carbon source also declined.1
This data presented here, and in Table 1, demonstrates that
the CelliGen BLU bioreactor is an easy-to-use, efficient
system for the culture of CHO cells. No effort was made to
optimize either the medium or the cell culture process
control parameters. This study was only intended to
document a general guide to bioreactor setup and operation,
and present typical results you could expect to achieve with
your mammalian cell line. For protocols on other cell lines, or
for additional information on the CelliGen BLU, see
New Brunswick Scientific, Edison, NJ USA:
Guozheng Wang, Sr. Research Scientist &
Wenying Zhang, Research Scientist Assistant, R&D Lab
Rich Mirro, Product Manager
Vikram Gossain, PhD., Applications & Training Manager
1.732.650.2012 or email@example.com
1. A single nutrient feed supports both chemically defined
NS0 and CHO fed-batch processes: Improved productivity
and lactate metabolism. Ma N, Ellet J, Okediadi C, Hermes P, McCormick E,
Casnocha S.Biotechnol Prog.
2009 Jul 27.