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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 highdensity
hybridoma cell cultures using NBS Advanced
Cell Culture Kit.
For this application, researchers in NBS cell culture laboratory used a standard 3.0L BioFlo 110 Advanced Cell Culture Kit with a magnetic drive and a water-jacketed vessel (NBS Catalog # M1273-1212). A suspension cell spin filter, with a 12-14 micron screen (NBS Catalog # M1273-3202) was used for perfusion in order to grow the hybridoma cells in a continuous mode. Additionally, BioCommand Plus supervisory software (NBS Catalog # M1291-0000) was used to control the feeding schedule and collect data; and a NucleoCounter (NBS Catalog # M1293-0000) was used to count viable cells.
The Bioreactor
Vessel
This 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 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. Optional direct
drive and magnetic-drive agitation systems, as well
heat-blanketed vessel options, are available.
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 of inoculum suspension to provide a starting cell count of 2.44 x 105 cells/ml.
Use BioCommand software to automatically calculate and control glucose addition throughout the run.
Medium
RPM1640 medium from Invitrogen, supplemented with
5% FBS from Hyclone and 0.1% Pluronic from Gibco.
Inoculum
Hybridoma cell line 3G5 from ATCC
Inoculum was cultivated in an NBS Shaker (Innova 2000), which was inside a CO2 Incubator (Innova CO- 170 NBS Catalog # M1305-0000)
PCU: Select the 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 manuals.
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% 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 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 below.
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 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)
Growth Conditions
Cells were inoculated into the bioreactor from T-flask
cultures with a live cell concentration of 2.44 x 105
cells/ml.
The rate of the perfusion was changed, based on the glucose concentration in the culture, to maintain glucose level at about 1 g/L. See figure 4 for details.
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.
Procedure
During the first 48 hours, the culture was run in a batch
mode. Media perfusion was started on day 2. Samples
were removed at regular intervals during the culture for
determination of live and dead cells using the NBS
NucleoCounter. To verify the accuracy of the
NucleoCounter reading, we also used Trypan blue
exclusion method (Sigma T8154) as well as a 12mm
biomass probe, which was inserted in the vessel and
connected to a biomass monitor. The biomass monitor
was connected to our BioCommand software to provide
an instant on-line cell concentration. See Appendix
A linear correlation of 0.993 was observed between viable cell concentration (X) and the capacitance (pf/cm) range as seen in Figure 3. See Appendix section (2) for details.
In most perfusion processes, cell viability typically decreases over time; but in this experiment viability improved during the process and was maintained at a very high level (95%) in the late perfusion stage due to the superior control of BioFlo 110. A high cell density of 1.27 x 107 cells/ml was achieved after 240 hours of culture.
APPENDIX
(1) The Biomass monitor and sensor used in this
experiment are not available in the marketplace as
they are still undergoing test runs.
When this equipment is available, we will make an announcement in the Culture Club, BioFlo 110 newsletter.
The Biomass sensor uses the principle that under the effect of an electric field between two electrodes, live cells (which have intact plasma membranes) polarize and behave as small condensers. The value of the resulting capacitance is measured by the second pair of electrodes and correlates exactly to the concentration of biomass in the culture environment.
In the first stage of culture (0-30 hrs), the pH and DO were controlled by surface aeration at a rate of 200 ml/min. After 30 hours of culture, the capacitance value began to decrease to 0.42 [pf/cm] at 45 hour, as shown in Figure 3. At this time perfusion was started at 1 liter per day, which rapidly increased the value to 0.73 [pf/cm] after 90 min. This phenomenon occurred three times (and is indicated in Figure 4 as T1, T2 and T3) during the S1 and S2 stages.We believe that the plasma membrane polarization for each cell changed, depending on the cell culture conditions.
(2) The equation below was used to calculate the linear correlation between viable cell densities (determined by NucleoCounter, Trypan Blue) and capacitance (determined by biomass probe).
X [10 6 cells/ml] = 1.386 X C [pf/cm]
Here, X is viable cell density [10 6 cells/ml] and C is capacitance [pf/cm]
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Source:
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