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 a variety 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 insect cell cultures, using our Advanced Cell Culture Kit. For this application, we used a standard 3.0L BioFlo 110 Advanced Cell Culture Kit with a magnetic drive and a water-jacketed vessel (NBS Catalog No. M1273- 1212). A Suspension-Cell Spin Filter, with 16-18 micron screen (NBS Catalog # M1273-3202) was used for a high-flow-rate perfusion to grow the insect cells in a continuous mode. Additionally, BioCommand Plus supervisory software (NBS Catalog No. M1291-0000) was used to control the feeding schedule. Insect cells are used to produce a variety of proteins by utilizing the Baculovirus Expression Vector System (BEVS). Insect cell lines such as Sf9, Sf21, Hi-5, etc., are proven to express high levels of end products.
The BioFlo 110 Advanced Cell Culture Kit was equipped with a magnetic drive and water-jacketed 3.0 L vessel with a nominal working volume of 2.25 L. All BioFlo 110 Advanced Cell Culture Kits are configured with a pitched-blade agitation impeller, as well as dissolved oxygen and pH probes (Mettler Toledo), three liquid addition bottles, cables, tubing, clamps and other useful items. Optional direct drive and magnetic-drive agitation systems, as well as water-jacketed and heat-blanketed vessels are also available in the cell culture kits.
In this application we substituted an optional NBS spin filter with a marine blade impeller for the pitched blade impeller, (Fig. 2). 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. (See Appendix.)
With most commonly-available spin filters, screens rapidly become clogged when cells reach high cell density during perfusion. Therefore, NBS designed spin filters with three different screens for various cell sizes. Please contact us for screen size suggestions.
All six control modules included in the Advanced Cell Culture Kit were used for this run:
Materials and Methods
HyQ SFX-Insect (liquid) medium supplied by Hyclone (Logan, UT) (serum-free expression medium).
Hi-5 insect cell inoculum supplied by Hyclone (Logan, UT) Inoculum was cultivated in a NBS Shaker (Innova 2000) which was inside an Incubator (Fisher Scientific, Model No. 10).
Sterilize the vessel with phosphate buffer solution (PBS) for 60 minutes.
Remove the PBS from the vessel.
Add 1.5 L of insect cell media to the vessel.
Inoculate 500 ml of insect cell suspension to provide a starting cell count of 4.1 x 105 cells/ml.
Setpoints were keyed into the controller prior to inoculation. The vessel was allowed to equilibrate prior to inoculation. DO loops current value rem ained higher than setpoint until the culture was introduced.
Temperature . . . . . . . . . . . . . . . . .28C
pH . . . . . . . . . . . . . . . . . . . . . . .. .6.3
DO . . . . . . . . . . . . . . . . . . . . . . . .50%
Agitation . . . . . . . . . . . . . . . . . . .70 - 130 rpm
O2 (Gases) . . . . . . . . . . . . . . . . . .4 Gas
The pH probe was calibrated prior to the autoclave cycle (refer to BioFlo 110 Instruction Manual). CO2 gas and liquid base were used to maintain the pH setpoint. pH control parameters were:
Base . . . . . . . . . . . . . .Sodium bicarbonate, 8% solution
Pump . . . . . . . . . . . . .Pump 3 of the 4-Pump Module
Transfer tubing . . . . .Narrow bore silicone tubing, as supplied (1⁄16" ID & 3⁄16" OD - NBS Part No. P0740-2396)
Vessel inlet . . . . . . . . .Triport adapter in the vessel headplate
Pump 3 plugged into "BASE" Power Controller Outlet
pH Control Selections:
Multiplier = 100% before inoculation
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 set to 4 Gas Mode after the Gas Control (O2 loop) selection. We also changed the P and I value of the DO loop to meet the culture demands, as shown below.
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.
O2 Control Selections: 4 Gas mode
All pumps were calibrated using the standard supplied tubing to keep track of the liquid quantities entering and exiting the vessel. Samples were taken several times a day to measure glucose and cell density.
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 (automatically adds liquid to the vessel with the Bio- Command control program)
Pump B Selection . . .Wet On (removes liquid from the vessel using the level probe as a trigger to turn the pump on)
For this study, we used BioCommand Plus software to control nutrient addition. For the nutrient control program, we calculated the approximate timeframe profile to automatically add fresh media using pump 1. After 2 days of batch process, medium perfusion was started at the rate of 0.5 - 2L working volumes per day.
Results and Discussion
Insect cells generally have a high demand for oxygen when producing protein. Maximum growth rates and high cell densities are achieved by keeping the DO at a given setpoint.
Of course, factors such as substrate concentration and metabolite build-up can also be limiting, but these are often more controllable than the inherent physical limitations of the fermentor.
The BioFlo 110 system allowed for the growth of insect cells to high density: 26.4 x 106 cell/ml (91 g/L DCW).
Inclusion of a BioFlo 110 Gas Mix Controller, standard with our Advanced Cell Culture Kit, has the ability to mix the four gases according to culture needs and further enhances the final cell-density.
With the above in mind, we view the high final cell density of 26.4 x 106 cell/ml as proof of the very good fundamental capabilities of the BioFlo 110 system.
Temperature control was excellent using un-chilled (12.8C) tap water as the coolant.
DO and pH control was excellent and remained constant at setpoint.
Overall, with our protocol, the BioFlo 110 system performed extremely well. The BioFlo 110 Advanced Cell Culture Kit is a highly suitable instrument for growing insect cell cultures.
More About Spin Filters
What is a Spin Filter?
A spin filter is a simple device that allows you to refresh media while retaining cells in the vessel.
Why should you use a Spin Filter?
When you are running a perfusion process, the object is to cultivate the highest yield possible. The spin filter is an efficient means to retain cells in the vessel, encouraging continued growth while media is exchanged.
an you maintain your Spin Filter?
The most important element of the spin filter is the screen, which is sized to keep your cells from escaping into the media. The best maintenance is proper cleaning of the entire filter, especially the screen, to keep it from clogging. If needed, rinse thoroughly in soapy water. From time to time, it will become necessary to replace the screen cage.