Name: culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research
Uploaded: 2016-06-10T13:00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

The authors would like to acknowledge the staff at Biospherix for their help in learning to use the Xvivo enclosed cell culture system, especially Matt Freeman; the staff of Miles &#38; Kelley Construction Company, Inc. for their work in setting up the laboratory infrastructure, especially Russ Hughes; the staff of Children&#39;s Hospital of Orange County department of Facilities and Support Services for their work in coordinating the laboratory remodel, especially Adam Lukhard and Devin Hugie; the staff of Children&#39;s Hospital of Orange County department of Information Systems for their help in setting up the data management infrastructure and remote access, especially Viet Tran; the Children&#39;s Hospital of Orange County Executive Management Team for their longstanding support of the project, especially Dr. Maria Minon and Brent Dethlefs. This work was funded by Children&#39;s Hospital of Orange County and the California Institute for Regenerative Medicine through grant TR3-05476 to PHS. All authors contributed equally to this work.

<ol>
	<li>Brick, D. J., 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=The+Autism+Spectrum+Disorders+Stem+Cell+Resource+at+Children's+Hospital+of+Orange+County:+Implications+for+Disease+Modeling+and+Drug+Discovery.">The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.</a> Stem Cells Transl.Med. 3 (11), 1275-1286 (2014).</li><li>Nethercott, H. E., Brick, D. J., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+induced+pluripotent+stem+cells+by+lentiviral+transduction.">Derivation of induced pluripotent stem cells by lentiviral transduction.</a> Methods Mol.Biol. 767, 67-85 (2011).</li><li>Pistollato, 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=Hypoxia+and+HIF1alpha+repress+the+differentiative+effects+of+BMPs+in+high-grade+glioma.">Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.</a> Stem Cells. 27 (1), 7-17 (2009).</li><li>Pistollato, F., Chen, H. L., Schwartz, P. H., Basso, G., Panchision, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+controls+the+expansion+of+human+CNS+precursors+and+the+generation+of+astrocytes+and+oligodendrocytes.">Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes.</a> Mol.Cell Neurosci. 35, 424-435 (2007).</li><li>Raval, J. S., Koch, E., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+non-viable+airborne+particles+correlates+with+airborne+colonies+and+represents+an+acceptable+surrogate+for+daily+assessment+of+cell-processing+cleanroom+performance.">Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.</a> Cytotherapy. 14 (9), 1144-1150 (2012).</li><li>Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+stem+cell+therapies+for+neurological+diseases.">The potential of stem cell therapies for neurological diseases.</a> Expert.Rev.Neurother. 6, 153-161 (2006).</li><li>Schwartz, P. H., Brick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+the+lysosomal+storage+diseases+-+the+quintessential+neurodegenerative+diseases.">Stem cell therapies for the lysosomal storage diseases - the quintessential neurodegenerative diseases.</a> Curr.Stem Cell Res.Ther. 3 (2), 88-98 (2008).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol.Biol. 767, 107-123 (2011).</li><li>Stover, A. E., 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=Process-based+expansion+and+neural+differentiation+of+human+pluripotent+stem+cells+for+transplantation+and+disease+modeling.">Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.</a> J Neurosci.Res. 91 (10), 1247-1262 (2013).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods Mol.Biol. 767, 137-146 (2011).</li><li>Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5 (3), 237-241 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Cells grown within the CPF see no changes in oxygen or carbon dioxide concentrations as they move from incubator to processing chamber to microscope chamber and back. It is critical that conditions in each chamber are matched to the particular incubator in which the cells are kept before the cells are removed from the incubator. The atmosphere within the apparatus is continuously HEPA-filtered and is customizable with regard to oxygen and carbon dioxide concentrations. Cells can be grown at standard concentrations for PSCs or NSCs, 5% and 9%, respectively; or alternative concentrations can be chosen for different cell types or for specific experiments. Thus, the apparatus is supplied with constant sources of medical grade oxygen, carbon dioxide, and nitrogen (Figure 4). All three of these gases are supplied by gas-specific manifold systems that ensure constant supplies. The apparatus is also supplied with a calibration gas mixture consisting of 10% (&#177; 0.01%) carbon dioxide in oxygen. The manifold systems are housed outside the cell production facility and the gases are piped into the facility through the ceiling. The calibration gas is housed within the facility. The apparatus is additionally supplied with house vacuum, also through the ceiling. Using an electronic monitoring system and wireless sending units, the output pressures of all manifolds are constantly monitored. In the event that any pressure falls out of range, the cell production facility operators are automatically telephoned and notified such that appropriate action may be taken.
The power requirements of the apparatus are met by six dedicated 120 V circuits descending from the ceiling and connected to the hospital&#39;s back-up generators to ensure a constant supply. Operation of the apparatus is controlled via software on a PC-based computer powered through an uninterruptible power supply. These power and computer arrangements ensure that the system functions continuously even in the event of a public power system failure. The software controlling the apparatus has a user-friendly graphical interface (Figure 1) which allows for the control of oxygen and carbon dioxide concentrations as well as temperature, humidity, and chamber pressures. The values of all these parameters are continuously recorded to provide a running record of all apparatus parameters. This data is backed up onto a remote server every night to protect their integrity. The computer and software can be accessed remotely by administrative users to assess and/or change any parameter. Additionally, the computer and software can be accessed remotely, allowing interactive assessment of apparatus parameters and troubleshooting with local users. An additional alarm sending unit is connected to the apparatus such that cell production facility operators are notified of any out-of-range condition of the apparatus. The remote access capabilities allow log in and assessment of the specifics of the out-of-range condition.
The apparatus is designed as a modular system both in a macro and a micro sense. Individual cell culture modules, such as incubators and processing chambers, can be customized in regard to their dimensions and requirements as well as in their layout with respect to each other. Additionally, most of the control functions of the individual modules are themselves modular such that individual atmospheric gas controllers, for example, may be easily replaced without significant disruption to the system.
Specialized processing chambers, such as one for microscopic visualization and manipulation of cell cultures, are easily adapted to the system. Both phase-contrast and fluorescence microscope are inside the system (Figure 6) so that cells can be live stained, and colonies can be dissected in the same atmospheric conditions as inside the incubators. Routing of cables through sealed grommets in the side walls of the processing chamber allows equipment such as power supplies and computers to be kept outside of the apparatus, usually on a cart (Figure 6).
The processing chambers in the cell production facility have a different airflow pattern than conventional BSCs. In conventional BSCs, airflow flows down from a central exhaust vent and splits into two separate streams, which are then taken up by two different intake vents in the forward and aft portion of the cabinet&#39;s floor. By contrast, the CPF has a single vent in the forward portion of the ceiling. Air flows downwards and towards the back of the chamber, where it is then drawn upwards into an intake vent. Although the CPF is inherently very clean, this unique airflow pattern means that technicians have to slightly adjust their technique to reduce the risk of contamination. As with a conventional BSC, a lab worker should avoid placing their hands upstream of open cell culture plates and media bottles. However, the direction which is upstream has been altered in the CPF
The cell production facility laboratory itself is fairly standard and comes equipped with a -20 &#176;C freezer, a -80 &#176;C freezer, a 4 &#176;C refrigerator, a centrifuge, and a water bath. The laboratory also has a sink with foot controls for convenient hands-free operation. In order for this laboratory to become a functional clinical cell production facility, however, several additional modifications must still be made. Firstly, the apparatus itself must be upgraded to have the capacity to monitor volatile organic compounds, particulates, and concentrations of chlorine dioxide which is used for decontamination. Secondly, a processing chamber containing a FACS machine may be housed and connected to the rest of the apparatus via a buffer module. This will allow for cell sorting and purification of transplantable cell populations under the appropriate environmental conditions. Lastly, the entire apparatus must be housed within a soft wall clean room. This provides an International Organization for Standardization (ISO) class 8 environment for the apparatus5.
The high sterility and computer-controlled nature of the CPF makes it an ideal system for future applications with cell-based therapy and good manufacturing processes. The risk of contamination is greatly mitigated, but more importantly, the conditions of cell expansion are automatically recorded and archived by the computer system. Deviations in gas concentrations, temperature, humidity, and all events of access into the system are rigorously documented. This can greatly help when investigating product quality problems. However, there are still limitations. The usage of any and all reagents and supplies (e.g., media components, pipettes, plates) must be documented separately. Additionally, there are a multitude of potential problems (including many forms of human error) that can arise which are completely unrelated to the variables documented by the CPF&#39;s monitoring system. Thus, the need for highly trained personnel and detailed manual documentation of tasks remains in place.

Standard stem cell culture techniques suffer from several environmental constraints that place undue stresses on the cells and expose the cells to unacceptable risks of contamination. Among the stresses that cells may endure under standard cell culture conditions are precipitous changes in the levels of carbon dioxide and oxygen concentrations3,4. This occurs when the cells are moved from the incubator to the biosafety cabinet and/or microscope which may not be optimal for the cells. Previous studies have confirmed the advantages of culturing both pluripotent and neural stem cells in hypoxic conditions4,11, and for best results, these conditions need to be continuous. Moreover, risks of cellular contamination are higher as the laboratory environment and personnel impinge upon the cells at almost every step of their culture and manipulation. Traditional clean rooms comprise one effective method to greatly decrease contamination risks but they are expensive, have a large footprint and fail to address stressors related to carbon dioxide and oxygen concentrations. 
A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria9 provides high quality cells for basic science research as well as clinical applications1,6,7. Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules. Using this basic infrastructure, a wide variety of tasks can be performed, such as standard feeding and passaging of pluripotent stem cells and multipotent neural stem cells, as well as more specialized methods like Sendai virus-based reprogramming, in vitro migration studies, and differentiation of neural stem cells for electrophysiological characterization.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xvivo System</td>
			<td>Biospherix</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Xvivo Software</td>
			<td>Biospherix</td>
			<td>version i.o.2.1.2.1</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 Manifold</td>
			<td>Amico</td>
			<td>P-M2H-C3-S-U-OXY</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Manifold</td>
			<td>Amico</td>
			<td>M2H-C3-D-U-CO2</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Manifold</td>
			<td>Western Innovator</td>
			<td>CTM75-7-2-2-BM</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope with DP21 camera and fluorescence</td>
			<td>Olympus Corporation</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 Glutamax</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro hESC Supplement</td>
			<td>Life Technologies</td>
			<td>A100006-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Millipore</td>
			<td>SCR005</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Sodium</td>
			<td>Hyclone</td>
			<td>9236</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor 2</td>
			<td>R&#38;D Systems</td>
			<td>AFL233</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Protide</td>
			<td>PP1130</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s-based Cell dissociation Buffer</td>
			<td>Life Technologies</td>
			<td>13150-016</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>R&#38;D Systems</td>
			<td>AFL236</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct-3/4 Antibody</td>
			<td>Millipore</td>
			<td>AB3209</td>
			<td></td>
		</tr>
		<tr>
			<td>TRA-1-60 Antibody</td>
			<td>Millipore</td>
			<td>MAB4260</td>
			<td></td>
		</tr>
		<tr>
			<td>SSEA4 Antibody</td>
			<td>Millipore</td>
			<td>MAB4304</td>
			<td></td>
		</tr>
		<tr>
			<td>BIT-9500 Serum Supplement</td>
			<td>Stemcell Technologies</td>
			<td>9500</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Consumable Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2mL Serological pipet</td>
			<td>VWR</td>
			<td>89130-894</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-102</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-104</td>
			<td></td>
		</tr>
		<tr>
			<td>25mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-106</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-107</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>TPP</td>
			<td>90026</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>TPP</td>
			<td>90076</td>
			<td></td>
		</tr>
		<tr>
			<td>20uL pipet tips</td>
			<td>Eppendorf</td>
			<td>22491130</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL pipet tips</td>
			<td>Eppendorf</td>
			<td>22491148</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 pipet tips</td>
			<td>Eppendorf</td>
			<td>22491156</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Scientific</td>
			<td>5000.102</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>BDH</td>
			<td>BDH1164-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sanimaster 4</td>
			<td>Ecolab</td>
			<td>65332960</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>A714239</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

This manuscript describes in some detail a cell production facility, and the unique aspects of culturing cells inside a closed system. The bulk of the cell production facility comprises a custom-made, enclosed, cell culture apparatus which has a small footprint and can be easily housed within a 6 x 7.5 m2 room (Figure 2). At no time are the cells within the apparatus exposed to laboratory personnel or environment. The apparatus consists of several modules: a process chamber, a laminar flow hood, 2 buffering airlock chambers in between the hood and the process chamber, two cell culture incubators, and a microscope chamber adjacent to the process chamber (Figure 3). The system is controlled by software allowing environmental variables to be controlled and monitored (Figure 1). The computer running this software is on an uninterruptible power supply. Gases are fed into the system by a set of manifolds for oxygen, nitrogen and carbon dioxide (Figure 4). Gases for the device are supplied by manifold systems that have two sets of tanks each - an active set and a reserve set. When the active set is drawn down, the manifold automatically switches to the reserve set and staff order replacement tanks. The power is on a backup generator system.
Pluripotent and neural stem cells can be grown in hypoxic conditions in this facility using previously developed protocols9, with the added complications inherent to the novel equipment. Pluripotent stem cells can be derived using Sendai virus, using pluripotency-specific antibodies to identify fully transfected colonies, which are then isolated and expanded. (Figure 5A and B), From these iPSCs, neural stem cells and neurons can be derived using dual SMAD inhibition, and their phenotype verified using NSC-specific antibodies (Figure 5C and D).
<img alt="Figure 1" src="/files/ftp_upload/53685/53685fig1.jpg" /> 
Figure 1.&#160;Graphical Interface for the Cell Production Facility. (A) The graphical representation of the CPF is the default screen and matches the CAD drawing shown in Figure 3. A click of the mouse over the Buffer Module 1 (2F in Figure 1) opens its control screen (B) where O2 values are set to match the processing chamber (3 in Figure 3). Similarly, clicking on the Process Chamber or Incubator 1 brings up their respective control screens (C and D, respectively), where values may be changed or monitored as appropriate. <a href="https://www.jove.com/files/ftp_upload/53685/53685fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53685/53685fig2.jpg" /> 
Figure 2.&#160;The Cell Production Facility. (A) The system as seen from a front right view. Note the power and gas connections in the ceiling and the cart with the microscope accessories and computer to the right. (B) The laminar flow hood with the access doors to the buffer modules seen at the right. (C) The process chamber with the two incubators (black doors) seen at the rear. (D) A view through the right side of the system showing the microscope and monitor with the doors to the buffer chambers seen in the distance. <a href="https://www.jove.com/files/ftp_upload/53685/53685fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53685/53685fig3.jpg" /> 
Figure 3.&#160;A CAD Drawing of the Cell Production Facility. All items entering the device are first wiped down with alcohol and air dried in the laminar flow hood (1). Items are then transitioned to the appropriate atmosphere in the front buffer module (2F) before being passed into the UCPC module, or Process Chamber (3). Cells are cultured in the two incubators (4, 5). Live cell staining, colony picking, assessment of general cellular morphology and migration analysis takes place in the UPC module, or Microscope Chamber (6). Finally, waste exits the system through the rear buffer module (2R). <a href="https://www.jove.com/files/ftp_upload/53685/53685fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53685/53685fig4.jpg" /> 
Figure 4.&#160;Sources of Gases and Power. (A) The CO2 manifold is a 4 x 4 setup as it supplies all the incubators of the laboratory. (B) The O2 manifold is a 2 x 2 setup and supplies only the cell production facility. Nitrogen for the system is generated in a vaporizer (C, just below the Exit sign) attached to a liquid nitrogen manifold (to the right) that also automatically fills cryofreezers (bottom left). The manifold is a 2 x 2 setup being supplied by two pairs of 160 L liquid nitrogen tanks. CO2, O2, and N2 are supplied from the ceiling through shutoffs (D). House vacuum is also supplied at this location. Seen just behind the supply gas shutoffs are a pair of power cables that supply power to the system. <a href="https://www.jove.com/files/ftp_upload/53685/53685fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53685/53685fig5.jpg" /> 
Figure 5.&#160;Pluripotent Stem Cells Derived in Hypoxia with Sendai virus. (A) SC187-SF4-2I0-E3 iPSCs iPSCs (nomenclature found in Stover et al.9), phase contrast 10X. (B) SC88.1-UH1-2I0 iPSCs live-stained with AF-594-labeled Tra-1-60, 1:100 dilution in media. All iPSCs were transduced in the cell production facility in 5% O2 with Sendai virus. (C) Phase contrast of SC68.1-UH0-2I0-M0S13-N2G6 iPSC-derived NSCs, grown at 5% O2 in chamber slides. Cells were then fixed in 4% paraformaldehyde and stained with anti-human Nestin primary antibody, and Alex-488 secondary antibody. All scale bars are at 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53685/53685fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research (Video) | JoVE

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Research

JoVE Journal

Developmental Biology

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells (Video) | JoVE

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol (Video) | JoVE

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System (Video) | JoVE

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells (Video) | JoVE

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells (Video) | JoVE

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells (Video) | JoVE

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition (Video) | JoVE

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells (Video) | JoVE

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...