Navigation Links
Hybridoma Cell Culture Application Using a BioFlo 110 Benchtop Bioreactor

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


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


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.

RPM1640 medium from Invitrogen, supplemented with 5% FBS from Hyclone and 0.1% Pluronic from Gibco.

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 (116" ID & 316" 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.

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.


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]



Page: All 1 2 3 4 5 6

Related biology technology :

1. Production of Hybridomas by Electrofusion
2. Production of Hybridomas by Electroporation
3. A Mix-and-Read Cell-Based Assay for Hybridoma Screening Using the FMAT 8100 HTS System
4. Big Blue Rodent Cell Cultures for Mutagenesis Studies
5. Simple Isolation of RNA from Tissue and Cultured Cells
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. The QIAGEN Guide to Animal Cell Culture
10. The QIAGEN Guide to Animal Cell Culture
11. The QIAGEN Guide to Animal Cell Culture
Post Your Comments:
(Date:7/10/2014)... 1960s, theatergoers have shelled out for crude 3-D ... their viewing experience. These basic devices, used to ... reality, may soon be rendered obsolete with the ... Aviv University researchers. , TAU doctoral students Yuval ... highly efficient holography based on nanoantennas that could ...
(Date:1/15/2014)... device," Nobel laureate Herbert Kroemer famously observed, referring ... the junctures where layers of different materials meet. ... between layers of metal oxides are becoming increasingly ... spintronics, high-temperature superconductors, ferroelectrics and multiferroics. Realizing the ...
(Date:1/15/2014)... Look inside the new Preferred Solutions® ... lab, from fluid handling to instruments to supplies. Many ... you order. , Preferred Solutions features a full ... L/S® model for precise flow control and dispensing to ...
(Date:1/15/2014)... This webinar will focus on EMA ... assessment in biosimilars. , Regulatory frameworks are evolving many ... however the complex nature of biopharmaceuticals makes the demonstration ... extremely challenging. Based on the specific aspects of biosimilar ...
Breaking Biology Technology:Projecting a 3-dimensional future 2Projecting a 3-dimensional future 3A deeper look at interfaces 2Cole-Parmer Begins 2014 with the Release of Preferred Solutions 2Xtalks Life Sciences Webinar Examines Safety Assessment of Biosimilars 2
... BioLife Solutions, Inc.,(OTC Bulletin Board: BLFS), a ... storage and cryopreservation media products for cells,tissues, ... joined the Biomedical,Excellence for Safer Transfusion (BEST) ... and commercial partners whose,mission is to explore ...
... AlloSource, one of the nation,s largest non-profit providers of ... surgical procedures, will this year sponsor one of the ... in the January 1 Tournament of Roses Parade(R). The ... to the millions of people touched by organ, tissue ...
... California (USC) have, for the first time in history, derived ... will enable scientists to create far more effective animal models ... , The research will be published in the Dec. 26 ... is a major development in stem cell research because we ...
Cached Biology Technology:BioLife Solutions Joins Prestigious Biomedical Excellence for Safer Transfusion Collaborative 2BioLife Solutions Joins Prestigious Biomedical Excellence for Safer Transfusion Collaborative 3AlloSource, Tissue Recipient Join 'Stars of Life' Affected by Organ and Tissue Donation Aboard Donate Life Float in 2009 Rose Parade(R) 2AlloSource, Tissue Recipient Join 'Stars of Life' Affected by Organ and Tissue Donation Aboard Donate Life Float in 2009 Rose Parade(R) 3USC researchers derive first embryonic stem cells from rats 2USC researchers derive first embryonic stem cells from rats 3
(Date:7/10/2014)... , June 27, 2014  The American Academy ... Institute of Standards and Technology (NIST) and the ... academic research and forensic science expert members to ... is an element of the NIST,s Organization of ... among the first appointments made to the new ...
(Date:7/10/2014)... N.Y. , July 2, 2014 It ... (ABD) to announce the appointment of the world-renowned Thoracic ... Board of Directors. Dr. Ginsburg, with over ... facilities: NY Presbyterian Hospital and Good Samaritan Regional Medical ... Pacemaker since the late 1980,s and is considered an ...
(Date:7/10/2014)... 7, 2014 According to a ... Access Control Systems Market Global Forecast, Market Share, Size, ... Electronic Access Control systems market was valued at USD ... at a CAGR of 12.6% from 2014 to 2019, ... in 2019. Browse the full Electronic Access ...
Breaking Biology News(10 mins):Ten Members of the American Academy of Forensic Sciences Appointed to Forensic Science Standards Board (FSSB) 2Ten Members of the American Academy of Forensic Sciences Appointed to Forensic Science Standards Board (FSSB) 3Avery Biomedical Devices, Inc. announces the appointment of Mark E Ginsburg, M.D. to its Board of Directors 2Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 2Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 3Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 4Electronic Access Control Systems Market is Expected to Reach USD 31.2 Billion Globally in 2019: Transparency Market Research 5
... 1980s, the idea has become predominant that the best ... value for it in the framework of markets. The ... offered a glimpse of many possibilities for money-earning uses ... the Convention on Biological Diversity, a measure adopted in ...
... Helios Education Foundation today awarded $6.5 million to the ... new partnership that extends the Helios Scholars Program at ... cultivate new scientific and technical talent across the state ... a long term partnership with TGen for student training ...
... to count monkeys? To Tasmania to track devils Down Under? ... No worries, theres a field guide for you, in ... but mostly practical field guides to all matter of, ... English churchyard lichens. Diane Schmidt, the biology librarian at ...
Cached Biology News:Markets of biodiversity and equity in trade: An illusion? 2Markets of biodiversity and equity in trade: An illusion? 3Helios Education Foundation invests $6.5 million 2Helios Education Foundation invests $6.5 million 3U. of I. librarian launches comprehensive Web database of field guides 2U. of I. librarian launches comprehensive Web database of field guides 3
One-step, microplate or cuvet, colorimetric, linear detection range 0.6 mM to 100 mM. Procedure: 2 min....
assays for phosphatase, lipase, nucleoside triphosphatases (ATPase, GTPase etc). HTS and follow up studies for phosphatase inhibitors. Quantitation of phosphate content in protein, lipids, and DNA....
... Design with Purpose -- ... MS provide the highest level ... performance in an affordable, easy-to-use ... autosampler and workstation guarantees success ...
Homo sapiens 4-aminobutyrate aminotransferase Antigen: Recombinant Protein...
Biology Products: