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 border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 3&#160;</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single Use Assembly</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Human Lung Carcinoma Cells (A549)</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:119px;">&#160;CCL-185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose Medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA, 0.25%</td>
			<td style="width:124px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytodex 1 Microcarriers</td>
			<td style="width:124px;">GE</td>
			<td align="right">3781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antifoam C</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:119px;">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/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/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/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 ...

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Bioengineering

24.2K 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

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...