| HOME >> BIOLOGY >> TECHNOLOGY |
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.
'"/>
Source:
Related biology technology :
1. Big Blue Rodent Cell Cultures for Mutagenesis Studies
2. Simple Isolation of RNA from Tissue and Cultured Cells
3. The QIAGEN Guide to Animal Cell Culture
4. The QIAGEN Guide to Animal Cell Culture
5. The QIAGEN Guide to Animal Cell Culture
6. The QIAGEN Guide to Animal Cell Culture
7. The QIAGEN Guide to Animal Cell Culture
8. The QIAGEN Guide to Animal Cell Culture
9. RNAi in Non-Mammalian Cultured Cells
10. Total RNA Isolation: 100 l Viral Sample From Cultured-Cell Supernatant Containing Virus
11. Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment
| Copyright © 2003-2012 Bio-Medicine. All rights reserved. ABOUT | CONTACT US | DISCLAIMER | PRIVACY POLICY | TERMS AND CONDITIONS |  |