( Introdu...)CHO Cell Culture Application Using a BioFlo 110 Benchtop Bioreactor

Introduction
This Application Report is one in a series documenting simple protocols for culturing cells in a BioFlo 110 benchtop fermentor/bioreactor system. The modular BioFlo 110 is offered with an extensive choice of vessels, impellers and control modules to enable growth of a wide variety of cultures, including mammalian, insect, plant, yeast and bacterial cells. Complete kits are also available. This report concentrates on achieving high-density CHO cell cultures using NBS Advanced Cell Culture Kit.

For this application, researchers in NBS cell culture laboratory used a:
BioFlo 110 Advanced Cell Culture Kit with a magnetic drive and a 3.0L total volume water-jacketed vessel, (nominal working volume 2.2L.) (NBS Catalog # M1273-1212)
Suspension Cell Spin Filter with a 12-14 micron screen (NBS Catalog # M1273-3202) for continuous perfusion culture
BioCommand Plus Supervisory Software (NBS Catalog # M1291-0000) to control the feeding schedule and collect data
NucleoCounter Automated Cell Counting System (NBS Catalog # M1293-0000) to count viable cells.

The Bioreactor
All BioFlo 110 Advanced Cell Culture Kits are configured with six control modules, a complete vessel assembly including a pitched blade agitation impeller, foam/level sensors, as well as dissolved oxygen and pH probes (Mettler Toledo), three liquid addition bottles, cables, tubing, clamps and other related items. Comes with choice of direct drive or magnetic-drive agitation systems, as well choice of water-jacketed or heat-blanketed vessel options.

For this application we substituted an optional NBS spin filter impeller for the standard pitched-blade impeller. The spin filter impeller is very useful for a continuous perfusion process because it is a simple device that lends itself to media exchange and can be easily scaled up. With most commonly available spin filters, screens rapidly become clogged when cells reach high cell density during perfusion. NBS, however, has designed spin filters with three different screen sizes that are suitable for most cell types. Feel free to contact NBS for screen size suggestions.

Control System
Listed below are six control modules included in an Advanced Cell Culture Kit, which were also used for this application:

Materials and Methods

Overview
Sterilize the vessel with phosphate buffer solution (PBS) for 60 minutes.
Remove the PBS from the vessel.
Add 1.5 L of media into the vessel.
Key in control setpoints.
Inoculate 500 mL
Use BioCommand software to automatically calculate and control glucose addition throughout the run.

Medium
EX-CELL 325 PF CHO from JRH.

Cell Line: CHO/B4C7

PCU: Select Cell Culture Application
On the initial set-up screen, the USE selector button will allow you to change the default Fermentation application to Cell Culture application. For further details refer to the Operation Section in the BioFlo 110 manual.

Control Setpoints
Setpoints were keyed into the controller prior to inoculation. The vessel was allowed to equilibrate prior to inoculation. The DO loops Current Value remained higher than setpoint until the culture was introduced.
Temperature . . . . . . . . . . . . . . . .37C
pH . . . . . . . . . . . . . . . . . . . . . . . .7.2
DO . . . . . . . . . . . . . . . . . . . . . . . .50%
Agitation . . . . . . . . . . . . . . . . . . .70-130 rpm
O₂ (Gases) . . . . . . . . . . . . . . . . . .4 Gas

PH Control
The pH probe was calibrated prior to the autoclave cycle (refer to BioFlo 110 Instruction Manual). CO₂ gas and a liquid base were used to maintain the pH setpoint. pH control parameters were:
Base . . . . . . . . .. . .Sodium bicarbonate, 8% solution
Pump . . . . . . .. . . .Pump 4 of the 4-Pump Module
Transfer tubing . . .Narrow bore silicone tubing, as supplied (¹⁄₁₆" ID & ³⁄₁₆" OD - NBS Catalog # P0740-2396)
Vessel inlet . . . . .. .Triport adapter in the vessel headplate

PCU setup: (pH loop)
Pump 4 plugged into "BASE" Power Controller Outlet
pH Control Selections:
Auto mode
Multiplier = 100% until base feed is started, after inoculation. Then, 25%
Multiplier = 25% after inoculation
Dead-band = 0.1
PID values: factory defaults

Dissolved Oxygen (DO) Control
The DO probe was calibrated after the autoclave cycle (refer to BioFlo 110 Instruction Manual). DO control was set to Auto mode, which automatically defaults to 4-Gas Mode after the Gas Control (O₂ loop) selection is made. We also changed the P and I value of the DO loop to meet the culture demands, as shown on the next page.

PCU Setup: (DO loop)
DO Control Selections: Auto (4 Gas mode)
PID values: P = 2.00 and I = 0.67

Gas Control (O₂ loop)
The control was set to 4-Gas mode, to automatically maintain DO and pH setpoints, using 3 gases (Air, O₂, N₂) to control the DO setpoint and CO₂ to control pH setpoint.

PCU Setup: (O₂ loop)
O2 Control Selections: 4 Gas mode

Continuous Feed
All pumps were calibrated using the standard tubing supplied to keep track of the liquid quantities entering and exiting the vessel.

Samples were taken several times a day for off-line measurement of glucose and cell density.

PCU setup: (Pump 1 & Pump 2 loop)
Pump 1 . . . . . . . . . . .. .plugged into "Pump A" Power Controller outlet
Pump 2 . . . . . . . . . .. . .plugged into "Pump B" Power Controller outlet
Pump 1 (Nutrient) . .. ..Vessel inlet - Triport adapter in the vessel headplate
Pump 2 (Harvest) . . . .Vessel outlet - 12mm port using the dip tube
Pump A Selection . .. . .Manual mode (automatically adds liquid to the vessel with the BioCommand control program)
Pump B Selection . .. . .Wet On (removes liquid from the vessel using the level probe as a trigger to turn the pump on)

Control Program
For this study, we used NBS BioCommand Plus software to control nutrient addition. For the nutrient control program, we calculated the approximate time frame profile to automatically add fresh media using pump 1.

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