Name: cultivation of mammalian cells using a single-use pneumatic bioreactor system
Uploaded: 2014-10-10T14:00:00.000+00:00
Duration: 11 min 42 s
Description: Watch this Scientific Journal Video about Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System at JoVE.com

This project was support in part by Johns Hopkins University, Office of the Provost through the Gateway Science Initiative.

1. Login
<ol>
	<li>Power up the Bioreactor. Click anywhere to open the login page. Select user name and enter password and click &#8220;Login&#8221;.</li>
</ol>
2. Calibration
<ol>
	<li>Calibrate pH sensor (2 point prior to autoclaving).</li>
	<li>Inspect pH sensor and confirm sensor tip is filled with electrolyte solution. Prepare two beakers with pH calibration solutions (electrolyte) pH 4 and pH 7 and have available a wash bottle with distilled water.</li>
	<li>Connect the pH cable to the pH sensor. Navigate to the &#8220;Actions&#8221; tab on the Hello interface and click &#8220;calibrate&#8221;. Enter buffer temperature in the Calibration Solution Temp field.</li>
	<li>Place pH sensor in buffer 1 (pH 4) and enter value in the &#8220;zero&#8221; field. Wait for the graph to stabilize and the click the &#8220;calibrate 1&#8221; button. Rinse the pH sensor with distilled water.</li>
	<li>Place sensor in buffer 2 (pH 7). Enter buffer 2 value in the &#8220;span&#8221; field. Wait for graph to stabilize and click calibrate 2 button.</li>
	<li>Click &#8220;Save&#8221; then click &#8220;Close&#8221;.</li>
	<li>Calibrate the Dissolved Oxygen (DO) sensor.</li>
	<li>Ensure the DO sensor has been polarized by being connected to the system for several hours. Navigate to the &#8220;Actions&#8221; tab on the Hello interface and click calibrate. Click the &#8220;DO A&#8221; button.</li>
	<li>Disconnect the DO sensor and enter 0 in the &#8220;zero&#8221; field. Wait for the graph to stabilize and the click the &#8220;Calibrate 1&#8221; button.</li>
	<li>Reconnect the DO sensor and enter 100 in the &#8220;Span&#8221; field. Wait for the graph to stabilize and the click the &#8220;Calibrate 2&#8221; button.</li>
	<li>Click &#8220;Save&#8221; then click &#8220;close&#8221;.</li>
</ol>
3. Autoclave and Install Sensors and Reagent Vessels
<ol>
	<li>After calibration, place sensors and thermal well in autoclave pouches and autoclave for 30 min at 121 &#176;C, 15 psi.</li>
	<li>Sanitize autoclave pouches with 70% Isopropyl Alcohol (IPA) and transfer pouches to biological safety cabinet (BSC). Remove outer packaging of vessel.</li>
	<li>Sanitize inner packaging with 70% IPA and transfer vessel to biological safety cabinet (BSC). Remove inner packaging and inspect the vessel and tubing for damage inflicted during shipping.</li>
	<li>Install the pH and DO sensors in the two front ports. Install the thermal well to the left back port. Open the sensor cap.</li>
	<li>Guide the sensor through the sensor port. Thread the sensor tightly into the port.</li>
	<li>Transfer vessel out of BSC.</li>
	<li>Hang the DO and pH sensor cables outside the vessel sleeve and check that nothing is in the sleeve. Slide the vessel into the sleeve, feet first.</li>
	<li>Carefully fit the temperature sensor into the vessel thermal well. Ensure that the bottom of the vessel rests against the heaters.</li>
	<li>Remove the tubing sets from their bags. Match color coding on the tubing to the corresponding connectors and pumps on the bioreactor control unit.</li>
	<li>Install the main gas line by pressing the connector into its gas outlet. Install the micro gas line by twisting the connector clockwise into the gas outlet. Install the exhaust filter tubing:</li>
	<li>Open the filter oven. Secure the exhaust filter on the U-channel so its tubing goes through the two hooks to the filter and out of the oven.</li>
	<li>Install the tubing by condenser bag in the tubing holder. Close the door.</li>
	<li>Route addition lines A and B, both media lines, and the harvest line behind the DO sensor and onto the bench next to the bioreactor control unit.</li>
	<li>Connect the cables to the DO and pH sensors.</li>
</ol>
4. Adding Medium and Micro-carriers
<ol>
	<li>Navigate to the &#8220;actions&#8221; tab and click &#8220;Control Pumps&#8221; on the computer interface.</li>
	<li>Form a sterile connection between an unused medium addition line (1 orange band) and the medium bottle/bag source by welding the tubing or using Luer fittings.</li>
	<li>Click the slider to turn on the media pump on. Click the slider to turn the media pump off after addition desired amount of medium.</li>
	<li>Place microcarrier beads in Ca2+, Mg2+ free PBS for 3 hr at RT.</li>
	<li>Wash beads several times with Ca2+, Mg2+ free PBS.</li>
	<li>Autoclave for 15 min at 115 &#176;C, 15 psi. 
	NOTE: Add 3 g/L&#160;(dry weight) in this experiment.</li>
	<li>Pump in micro-carriers that have been hydrated, washed and autoclaved into the reactor in the same manner the medium was added in step 4.1.</li>
</ol>
5. Equilibration and One-point DO Calibration
<ol>
	<li>Set the controllers to Auto and enter the desired setpoints. Here, use Agitation Set Point (SP) = 15 rpm, Temperature SP = 37.0 &#176;C, pH = 7.2, DO = 100%. Wait for the parameters to equilibrate.</li>
	<li>Confirm sensor is fully polarized. Confirm DO present value has stabilized.</li>
	<li>Navigate to the &#8220;Actions&#8221; tab and click &#8220;calibrate&#8221;. Click &#8220;DO A&#8221;, click &#8220;One-point&#8221;.</li>
	<li>Enter &#8216;100&#8217; in the &#8220;Span&#8221; field. Click the &#8220;Calibrate 1&#8221; button, click &#8220;Save&#8221; and click &#8220;close&#8221;:</li>
</ol>
6. Starting a Run
<ol>
	<li>Navigate to the Actions tab. Click &#8220;Batch.&#8221; Use the on-screen keyboard or an external keyboard to enter a batch name 16 characters or less.</li>
	<li>Click the on-screen keyboard&#8217;s &#8220;Hide&#8221; button. Click the &#8220;Start batch&#8221; confirm by clicking &#8220;Start&#8221; in the overlay.</li>
</ol>
7. Inoculate with Cells
<ol>
	<li>Form a sterile connection between an unused medium addition line (1 orange band) and the cell bottle/bag source by welding the tubing or using the Luer fittings.</li>
	<li>Install the silicone section of the tubing in the media pump so the arrow points toward the tubing between the pump and vessel.</li>
	<li>Check tubing clamp is open and its branched tubing clamp is closed. Click the slider to turn the media pump on and click to &#8220;Off&#8221; after adding cells.</li>
	<li>Navigate to the &#8220;Actions&#8221; tab and click &#8220;Control Pumps&#8221;.</li>
</ol>
8. Sampling
<ol>
	<li>After inoculating and as frequently as desired to monitor the culture, draw a sample from the culture in the following manner:</li>
	<li>Navigate to the &#8220;actions&#8221; tab and click &#8220;take sample&#8221;. Place the sampling tubing in the sampling pump and manipulate the sampling stopcock according to the on-screen instructions.</li>
	<li>Perform at least a daily microscopic observation and cell count on these samples.</li>
	<li>When the cells have reached the desired density, in this case 1.2 x 106 cells/ml, infect the cells with the addition of a virus inoculum.</li>
	<li>Aseptically add the inoculum to a 20 ml syringe, and connect the syringe to one of the spare addition ports on the reactor. Introduce the inoculum to the reactor by pressing on the syringe plunger.</li>
	<li>Continue sampling and analyzing the culture for the adenovirus intracellular particle concentration.</li>
</ol>

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells A Manual of Basic Techniques and Specialized Applications. , (2010).</li><li>Healthcare, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+Principles+and+Methods.">Microcarrier Cell Culture Principles and Methods.</a> GE Healthcare Data File 18-1140-62. , (2005).</li><li>Simaria, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+cell+therapy+bioprocess+economics+and+optimization+Single-Use+cell+expansion+Technologies.">Allogeneic cell therapy bioprocess economics and optimization Single-Use cell expansion Technologies.</a> Biotechnol and Bioeng. 111 (1), 69-83 (2014).</li><li>Lee, B., Fang, D., Croughan, M., Carrondo, M., Paik, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+novel+pneumatic+mixing+for+single-use+bioreactor+application.">Characterization of novel pneumatic mixing for single-use bioreactor application.</a> BMC Proc. 5 (S8), O12 (2011).</li><li>Eibl, R., Eibl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+bioreactors+in+cell+culture-based+upstream+processing.">Disposable bioreactors in cell culture-based upstream processing.</a> BioProcess Int. 7 (S1), 18-23 (2009).</li><li>Chaubard, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+bioreactors+for+viral+vaccine+production+challenges+and+opportunities.">Disposable bioreactors for viral vaccine production challenges and opportunities.</a> Biopharm Int Supp. , (2010).</li><li>Croughan, M. S., Hamel, J. F., Wang, D. I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Effects+on+Animal+Cells+Grown+in+Microcarrier+Cultures.">Hydrodynamic Effects on Animal Cells Grown in Microcarrier Cultures.</a> Biotechnol and Bioeng. 95 (2), 295-305 (2006).</li><li>DePalma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-use+Equipment+on+Cusp+of+Industrialization.">Single-use Equipment on Cusp of Industrialization.</a> Genet Eng Biotechnol News. 32 (1), (2012).</li><li>Baltz, R. H., Demain, A. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Industrial Microbiology and Biotechnology. , (2010).</li><li>Sousa, M. F., Giroux, D., Clemente, J., Lee, B., Carrondo, M. J., Alves, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Impact of Bioreactor Design on the Performance of Microcarrier Cultures. , (2012).</li></ol>

Authors D. Giroux and Y. Hashimura are employees of PBS Biotech that produces reagents and instruments used in this article.

This single-use bioreactor system is relatively simple to use and provides real-time analytics for reactor monitoring and analysis. It is extremely well suited for mammalian and insect cell culture with cell densities reaching over 30 million cells/ml. Besides A549 cells described in this report11, we have grown SF-9 insect cells in the bioreactor as well. The gentle mixing provided by the pneumatic air wheel reduces cell damage. Several steps are critical when setting up this reactor. First, proper calibration of the pH and DO sensors is important for optimal monitoring of the culture and for addition of reagents to adjust the pH or the oxygen in the system. Second, the reagent and seed bottles must be filled and the luer attachments made in a sterile environment such as a BSC. Once the reagent bottles are moved out of the sterile environment, the connections to the bioreactor feed lines must be made with care to avoid microbial contamination.
While this bioreactor system works well for mammalian and insect cell lines it is not designed for bacterial cultures. The system cannot provide the rapid mixing and oxygenation that is required for bacterial cells. Bacterial growth is best accomplished in a stirred tank bioreactor. As compared to other single use bioreactors for mammalian or insect cell culture, this system is easy to use, provides sufficient data for analysis of runs, and has similar or better cell growth than the other single use system we have evaluated.
The single use pneumatic bioreactor system has the potential to meet many of the research and clinical applications in the fields of biotherapeutics, vaccines, stem cells, and personalized medicine4. In addition, the flexibility of this system allows for Batch, Fed-batch, Perfusion, and Transfection based bioreactor applications5. Finally, single-use disposable bioreactor systems have the potential to meet the needs of large scale industrial production and to adhere to guidelines and recommendations of national and international regulatory agencies6-10.

Mammalian cells lines can be classified into one of three categories based on their growth characteristics: cells that grow in suspension, cells that grow as aggregates, and cells that grow anchored to a substrate. Although the air-wheel bioreactor demonstrated in this video is able to grow all three types of cells, this video will demonstrate use of the bioreactor to grow anchorage dependent cells on micro-carriers. Anchorage dependent mammalian cells can be grown for the purpose of producing more cells &#8212; where the cells themselves are the product. For example, human bone marrow derived mesenchymal stem cells are currently being cultivated with the purpose of harvesting the cells and injecting them into diseased tissue. The pneumatic bioreactor demonstrated in this video has proven suitable for the production of such mesenchymal stem cells for this application (Serra et al., personal communication, 2013).
Anchorage dependent mammalian cells are typically grown small scale in 2D culture vessels such as cell culture plates, cell culture flasks, or roller bottles, where they adhere to a specially treated growth surface1. When more cells are desired, the plates or flasks can be expanded by using more or larger vessels. However, for more cost-effective cultivation of large quantities of anchorage dependent cells, increasing the surface area for cell attachment can be accomplished by using small solid beads called micro-carriers. Depending on the attachment characteristics of the cell, several different types of micro-carriers are commercially available, such as dextran, peptide, or collagen coated. Micro-carriers have a large surface area to volume ratio providing a larger surface area for cell growth; and the micro-carriers can be maintained in suspension with agitation, which allows the cells to be cultivated to high densities in bioreactor systems2. Currently, the types of bioreactors where adherent cells are grown on micro-carriers include spinner flasks and stirred tank systems, which use axial impellers to maintain suspension of the cell coated micro-carriers.
Several factors are important to the successful cultivation of cells including oxygen tension, shear stress, surface matrix, and nutrient and metabolite concentrations. The use of bioreactors allows for real-time monitoring of the growth conditions and the potential to significantly lower production costs1. There are several common bioreactor designs for in vitro cell cultivation including, stirred suspension, rotating wall vessel, hollow-fiber, bag bioreactor on a rocker platform, and fluidized bed systems3. Many of these systems present unique problems for cell cultivation and scale &#8211;up, such as high cost, nutrient concentration gradients, hydrodynamic shear, cell aggregation, and difficulty in sampling, monitoring, and controlling cell scale-up.
Various adherent cell lines are used in the production of viruses, either in the production of viral vaccines or for the production of viral vectors for gene therapy applications. In this video, using the single use pneumatic (Air-Wheel) bioreactor system, we demonstrate the culture of human lung carcinoma cells (A549) cells on micro-carriers for production of an oncolytic adenovirus. The pneumatic bioreactor design uses a vertical agitation wheel that is powered by the buoyancy of gas sparged into the bottom of the bioreactor. This gentle stirring method limits hydrodynamic shear forces, but still ensures optimal medium and cell mixing4. As compared to the stirred tank reactor, the pneumatic reactor has low wall shear stress even with high volume Air-Wheel bioreactor systems (Figure 1). In contrast to stirred tank bioreactors, the vertical impeller of this single use reactor is turned by a stream of gas bubbles within the vessel, which allows for gentle and uniform medium mixing (Figure 2).

<table><tbody><tr><td>PBS 3&amp;nbsp;</td><td>PBS</td><td></td><td></td></tr><tr><td>Single Use Assembly</td><td>PBS</td><td></td><td></td></tr><tr><td>Human Lung Carcinoma Cells (A549)</td><td>ATCC</td><td>CCL-185</td><td></td></tr><tr><td>DMEM High Glucose Medium</td><td></td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td></td><td></td><td></td></tr><tr><td>Trypsin EDTA, 0.25%</td><td></td><td></td><td></td></tr><tr><td>Cytodex 1 Microcarriers</td><td>GE</td><td>3781</td><td></td></tr><tr><td>Antifoam C</td><td>Sigma</td><td>A8011</td><td></td></tr></tbody></table>

cultivation of mammalian cells using a single-use pneumatic bioreactor system

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

In Figure 3, the parameters for initiation of the bioreactor run are shown. This figure shows the screen prior to setting the parameters of temperature, dissolved oxygen, pH and agitation. Once the parameters are set, the run parameters are continuously monitored and adjustments can be made to maintain the required conditions. The software produces a continuous readout that allows for easy identification of problems. In this experiment using 2.5 L of DMEM containing 10% FBS, 250 ppm of SAFC Anti-foam C, 2 mm L-glutamine and 3 g/L of micro-carriers, the system is stabilized so the percent oxygen is 50%, the temperature is 37 &#176;C, and the pH is 7.2. The reactor is inoculated with A549 cells at a concentration of 7 x 104 cells/ml (or 10 cells/micro-carrier). The parameters chosen for this run resulted in the majority of the cells adhering to the micro-carriers within 2 hr (Figure 4). After 12 hr, the cells are demonstrating signs of flattening and spreading on the micro-carrier surface (Figure 5). By 24 hr, micro-carriers have a relatively even distribution of cells, with no micro-carriers without cells and no large clumps of cells on the micro-carriers (Figure 6). The percentage of micro-carrier colonization by A549 cells is 75% by 24 hr and 90% thereafter (Figure 7). The cells continued to grow exponentially to ~1 million cells/ml after 48 hr (Figure 8). After infection by a dose of oncolytic adenovirus (2 x 108 virions/ml) at 50 hr, the density increased to 1.2 million cells/ml and then began to decrease as the lytic infection progressed. There was ~10,000 fold amplification of the viral inoculum.
In previous experiments, we have found that monitoring and adjusting gas flow is critical to maximizing cell growth (unpublished data). Rapidly growing cells can deplete the system of oxygen with the DO level dropping to zero. While the cells continued to grow, it was at a much slower rate. It is critical to monitor and adjust the oxygen flow to meet the cell demands during the logarithmic growth phase. Each run can be analyzed for optimal growth by comparing the pH, DO, and temperature with daily cell counts.
<img alt="Figure 1" src="/files/ftp_upload/52008/52008fig1highres.jpg" /> 
Figure 1. Fluid dynamics of the Pneumatic Bioreactor System (PBS) compared with a Stirrer tank bioreactor measuring wall shear forces at various reactor volumes.
<img alt="Figure 2" src="/files/ftp_upload/52008/52008fig2highres.jpg" /> 
Figure 2. Pneumatic Air-Wheel reactor impeller.
<img alt="Figure 3" src="/files/ftp_upload/52008/52008fig3highres.jpg" /> 
Figure 3. Pneumatic Air Wheel software screen shot of parameters to initiate the bioreactor run.
<img alt="Figure 4" src="/files/ftp_upload/52008/52008fig4highres.jpg" /> 
Figure 4. Micro-carrier colonization with A549 cells 2 hr post inoculation. (Average micro-carrier diameter ~180 &#181;m.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52008/52008fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52008/52008fig5highres.jpg" /> 
Figure 5. 12 hr after the initiation of the culture, the A549 cells are attaching, flattening, and spreading onto the micro-carriers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52008/52008fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52008/52008fig6highres.jpg" /> 
Figure 6. Micro-carriers coated with cells after 24 hr in culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52008/52008fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52008/52008fig7highres.jpg" /> 
Figure 7. Percentage of micro-carrier colonization by A549 cells post inoculation.
<img alt="Figure 8" src="/files/ftp_upload/52008/52008fig8highres.jpg" /> 
Figure 8. The number of viable A549 cells in culture pre and post infection with adenovirus. Note that the viral infection step occurred at 50 hr.

Watch this Scientific Journal Video about Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System at JoVE.com

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

cultivation-mammalian-cells-using-single-use-pneumatic-bioreactor

Nanyang Technological University

Research

JoVE Journal

Bioengineering

24.3K Views.  Johns Hopkins University. Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells. 

Video: Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biology

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

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In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

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A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

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Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

Closed System Production of Adherent Cells Based on Dissolvable Microcarriers

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

Author Spotlight: Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers 

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using The Single-cell Microliter-droplet Culture Omics System

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.

This protocol describes how to use the Single-cell Microliter-droplet Culture Omics System (MISS cell) to perform microbial monoclonal isolation, cultivation, and picking. The MISS cell achieves an integrated workflow based on droplet microfluidic technology, which offers excellent droplet monodispersity, high parallel cultivation, and high-throughput biomass detection.

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

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