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PROTOCOLS
Growing CHO cells in a benchtop stirred-tank bioreactor using single-use vessels
Sponsored,vendor-submitted protocol    Sponsored by New Brunswick Scientific    Published in BioTechniques 2010 Protocol Guide (p.15) DOI: 10.2144/000113282

Abstract

This protocol describes setup and operation of New Brunswick Scientific’s new CelliGen™ BLU stirred-tank bioreactor, which combines the accurate process control and trusted performance of traditional stirred-tank technology with the convenience of single-use, pre-sterilized vessels to eliminate autoclaving and cleaning. A procedure is described for culturing Chinese hamster ovarian (CHO) cells in a 5.0-L vessel, using CD CHO serum-free medium in a batch culture.

Materials

Bioreactor (CelliGen BLU; New Brunswick Scientific). Pre-assembled, pre-sterilized vessels include pitched-blade impeller, microsparger, tubing, and more. All components in product contact are made of materials that meet USP Class VI standards for cGMP environments. A non-disposable control station provides advanced gas flow control, process management, and monitoring capability, with multiple options for custom configuration.

Rapid set-up, easy operation, and elimination of autoclaving and cleaning between runs are a few of the many advantages of the new CelliGen BLU 5.0- and 14.0-L stirred-tank bioreactor for growth of mammalian cultures.

As configured for this protocol, the control station included one Thermal Mass Flow Controller (TMFC) with 2–100 cu. cm/per min. flow rate for direct sparging of gases, and integrated gas overlay with 0.1–3.0 standard liters per minute (SLPM) flow rate, also regulated by a TMFC. Both are capable of 4-gas mixing for automatic control of pH and dissolved oxygen (DO). Built-in pumps and all process loops were controlled through the bioreactor’s firmware. A non-invasive, reusable polarographic DO probe, and non-invasive optical pH probe and fluorescence sensor, included with the system, were used for monitoring pH and DO.

Chinese hamster ovarian (CHO) cells

CD CHO serum-free medium (10743-029; Invitrogen, Carlsbad, CA, USA)

Orbital Shaker (Innova® 2000; New Brunswick Scientific)

CO2 Incubator (Galaxy® 170 R; New Brunswick Scientific)

Off-line glucose and lactate analyzer (YSI 2700, YSI, Inc., Yellow Springs, OH, USA)

Automated cell counting system (NucleoCounter®; New Brunswick Scientific)

Automated data logging (BioCommand® Batch Control software; New Brunswick Scientific)

Methods

1. The inoculum was grown to a density of 2.0–3.0 × 105 cells/mL, with greater than 90% cell viability. (Full procedure described at www.biotechniques.com/protocols/113282.)

2. One day before cells reached inoculation density, growth medium was warmed to 37°C for 24 h in a CO2 incubator; and the DO probe was connected to the bioreactor controller for =6 h to enable polarization.

3. The bioreactor vessel was removed from its sterile packaging, and the heat blanket, supplied with the unit, was wrapped around the vessel.

4. Culture medium vessel was connected to the bioreactor vessel’s inlet line, and medium was pumped into the bioreactor. Connections to the controller, including sparge, overlay, RTD, pH, and agitation

were made.

5. pH and DO were calibrated through the touchscreen controller, and process setpoints were entered using the values outlined in the full protocol. When setpoints were reached, the inoculum flasks were connected to the bioreactor’s addition line, and contents were pumped.

6. 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 gas were additionally directly sparged using a porous sparger and automatic gas mixing into the bioreactor vessel.

7.Process data were continuously logged. A built-in sampling device enabled sterile sampling. Daily off-line measurements of glucose and lactate concentration were read, and cell density and cell viability were measured.

Results and discussion

Viable cell density steadily grew, peaking on day 5, at 5.55 × 106 cells/mL.

CHO cells reached a maximum viable cell density of 5.55 × 106 cells/mL on day 5. Cell viability ranged between 97.3 and 97.9% through day 5, until the nutrient source, glucose, was depleted from the medium.

The data demonstrates that the CelliGen BLU bioreactor is an efficient system for the culture of CHO cells. No effort was made to optimize either the medium or the process control parameters. This study was only intended to provide a general guide to bioreactor set-up and operation, and present typical results you could expect to achieve with your mammalian cell line. For additional protocols or information on the CelliGen BLU, see www.nbsc.com/BLUa.

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