This work was supported by DFG grant EH 192/5-1 (A.E.), the EU (E-rare-2) project Transposmart (A.E.), the UWH Forschungsf&#246;rderung (E.S. and W.Z), and the PhD programme of the University Witten/Herdecke (P.B.). J.L. was supported by a stipend of the Chinese Scholarship council and T.B. and M.G by the Else Kr&#246;ner-Fresenius foundation (EKFS).

<ol>
	<li>Benihoud, K., Yeh, P., Perricaudet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+vectors+for+gene+delivery.">Adenovirus vectors for gene delivery.</a> Curr Opin Biotechnol. 10 (5), 440-447 (1999).</li><li>Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus:+the+first+effective+in+vivo+gene+delivery+vector.">Adenovirus: the first effective in vivo gene delivery vector.</a> Hum Gene Ther. 25 (1), 3-11 (2014).</li><li>Parks, R. 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=A+helper-dependent+adenovirus+vector+system:+removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> Proc Natl Acad Sci U S A. 93 (24), 13565-13570 (1996).</li><li>Palmer, D., Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+system+for+helper-dependent+adenoviral+vector+production.">Improved system for helper-dependent adenoviral vector production.</a> Mol Ther. 8 (5), 846-852 (2003).</li><li>Morral, N., 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=High+doses+of+a+helper-dependent+adenoviral+vector+yield+supraphysiological+levels+of+alpha1-antitrypsin+with+negligible+toxicity.">High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity.</a> Hum Gene Ther. 9 (18), 2709-2716 (1998).</li><li>Muruve, D. A., 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=Helper-dependent+adenovirus+vectors+elicit+intact+innate+but+attenuated+adaptive+host+immune+responses+in+vivo.">Helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo.</a> J Virol. 78 (11), 5966-5972 (2004).</li><li>Ehrhardt, A., 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=A+gene-deleted+adenoviral+vector+results+in+phenotypic+correction+of+canine+hemophilia+B+without+liver+toxicity+or+thrombocytopenia.">A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia.</a> Blood. 102 (7), 2403-2411 (2003).</li><li>Cots, D., Bosch, A., Chillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper+dependent+adenovirus+vectors:+progress+and+future+prospects.">Helper dependent adenovirus vectors: progress and future prospects.</a> Curr Gene Ther. 13 (5), 370-381 (2013).</li><li>Mizuguchi, H., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+construction+of+a+recombinant+adenovirus+vector+by+an+improved+in+vitro+ligation+method.">Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method.</a> Hum Gene Ther. 9 (17), 2577-2583 (1998).</li><li>Mizuguchi, H., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+constructing+E1-+and+E1/E4-deleted+recombinant+adenoviral+vectors.">A simple method for constructing E1- and E1/E4-deleted recombinant adenoviral vectors.</a> Hum Gene Ther. 10 (12), 2013-2017 (1999).</li><li>Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+adenoviral+helper-dependent+vector+results+in+long-term+therapeutic+levels+of+human+coagulation+factor+IX+at+low+doses+in+vivo.">A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo.</a> Blood. 99 (11), 3923-3930 (2002).</li><li>Jager, L., 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=A+rapid+protocol+for+construction+and+production+of+high-capacity+adenoviral+vectors.">A rapid protocol for construction and production of high-capacity adenoviral vectors.</a> Nat Protoc. 4 (4), 547-564 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. 1-3, (2001).</li></ol>

The authors declare that they have no competing financial interests.

The protocol presented here allows purification of HCAdV vectors based on human adenovirus type 5 based on previously described procedures 4,12. The HCAdV genome within the pAdFTC plasmid is devoid of all adenovirus genes and only carries the 5&#39;- and 3&#39;- ITRs and the packaging signal. In this strategy the HV AdNG163R-2 4 provides all necessary genes for efficient virus production in trans. This offers a packaging capacity of up to 35 kb, which clearly outcompetes first and second ge...

For gene therapeutic applications it is of great importance to avoid cytotoxic and immunogenic side effects caused by expression of viral proteins, the transgene itself, or by incoming viral proteins. Adenovirus vectors (AdV) are widely used to introduce foreign DNA into a wide variety of cells to investigate the impact of transgene expression 1,2. The most advanced version of AdV is represented by high-capacity adenovirus vectors (HCAdV) lacking all viral coding sequences 3,4 and thereby offering a packaging capacity up to 35 kb combined with low immunogenicity and low toxicity 5-8. Due to their high packaging capacity they allow delivery of large or multiple transgenes using a single vector dose. Therefore, they represent a valuable tool for the research community.
In contrast to first- or second-generation AdV lacking the early genes E1 and/or E3 that can be easily produced using commercial kits, vector genome construction and virus production of HCAdV is more complex. The system for the construction of HCAdV genomes is based on the plasmid pAdFTC carrying a HCAdV genome devoid of all viral coding sequences and the shuttle plasmid pHM5 9-12. Any gene of interest of up to 14 kilobases (kb) can be cloned into the shuttle vector pHM5 in which the multiple cloning site is flanked by recognition/cleaving sites of the homing endonucleases PI-SceI and I-CeuI. Therefore, a cloned gene of interest can be released by consecutive PI-SceI and I-CeuI digests for subsequent directed insertion into the same restriction sites present in the HCAdV genome contained in the plasmid pAdFTC. In pAdFTC the transgene insertion site located between the PI-SceI and I-CeuI cleavage sites is flanked by stuffer DNA and the noncoding adenoviral sequences required for genome packaging such as the 5&#39; and 3&#39; inverted terminal repeats (ITRs) at both ends and the packaging signal downstream of the 5&#39;ITR. The additional stuffer DNA provides optimal size of the final HCAdV genome ranging from 27 to 36 kb to ensure efficient packaging during virus production. Since pAdFTC is a large plasmid with up to 45 kb (dependent on the size of the inserted transgene) and the usage of homing endonucleases with comparably long DNA recognition sites exhibits strong DNA binding, several cleanup steps are necessary during transfer of the transgene from pHM5 to pAdFTC. Careful handling avoiding shearing forces is recommended.
The ITRs of the HCAdV genome are flanked by NotI restriction enzyme recognition sites located directly upstream of the 5&#39;ITR and downstream of the 3&#39;ITR 12. Therefore, HCAdV can be released by NotI digest for subsequent transfection of the viral genome into the HCAdV producer cell line. Note that the usage of the restriction enzyme NotI for release of the viral genome from the plasmid pAdFTC implies that the inserted transgene is devoid of NotI DNA recognition sites. The HEK293 cell based producer cells (116 cells) stably express Cre recombinase. For virus amplification 116 cells are co-infected with a helper virus (HV) providing all AdV genes needed for replication and packaging in trans 3,4. The HV is a first-generation AdV with a floxed packing signal which is removed during virus amplification by Cre recombinase expressed in 116 cells 4. This ensures that predominantly HCAdV genomes containing an intact packaging signal are encapsidated.
Pre-amplification of the HCAdV is performed by conducting serial passaging steps in 116 cells grown on surfaces in tissue culture dishes. After each passage viral particles are released from infected cells by conducting three consecutive freeze-thaw steps. With every passage increasing numbers of cells are infected with 1/3 of cell lysate from the preceding passage. Finally lysate from the last pre-amplification step is used to infect producer cells grown in suspension in a spinner flask for large scale amplification. Virions are purified from the suspension cells by performing ultracentrifugation in a cesium chloride density gradient 4,12. With this procedure empty particles and fully assembled particles are separated into two distinct bands. To further concentrate the HCAdV particles a second non-gradual ultracentrifugation step is performed. Subsequently the resulting band containing the HCAdV is collected and dialyzed against a physiological buffer. Final vector preparations are characterized with respect to numbers of absolute viral particles, infectious particles and HV contamination levels. Absolute viral particles can be determined by lysing viral particles and measuring the absorbance at 260 nm or by performing quantitative real-time PCR (qPCR) 12. Infectivity of the purified virus particles can be determined by qPCR measuring HCAdV genomes present within infected cells 3 hr post-infection.

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">I-CeuI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td style="width:123px;">R0699S</td>
			<td style="width:315px;">restriction digest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">PI-SceI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0696S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">T4 Ligase</td>
			<td style="width:147px;">New Engand Biolabs</td>
			<td>M0202S</td>
			<td>ligation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">SwaI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0604S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:312px;">NotI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0189S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Calf Intestinal Alkaline Phosphatase (CIP)</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>M0290S</td>
			<td>dephosphorylation of digested plasmids</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Hygromycin B</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P02-015</td>
			<td>selection of CRE expressing 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM</td>
			<td style="width:147px;">PAN Biotech</td>
			<td>P04-03590&#160;&#160;&#160;</td>
			<td>Hek293T cell culture medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Minimal Essential Medium (MEM) Eagle</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P04-08500</td>
			<td>116 cell culture medium&#160;&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dulbecco&#8217;s phosphate buffer saline (DPBS)</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P04-36500</td>
			<td>washing of cells, resuspension of cells</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:312px;">250 ml storage bottle</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:123px;">CLS430281-24EA</td>
			<td>infection of 116 cells grown in suspension</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">500mL PP CentrifugeTubes</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:123px;">CLS431123-36EA</td>
			<td>sedimentation of cells from suspension culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Spinner flask</td>
			<td style="width:147px;">Bellco</td>
			<td style="width:123px;">1965-61030</td>
			<td>growth of 116 cells in suspension</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Ultra Clear Ultracentrifuge tubes</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">344059</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Ultracentrifuge</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">&#160;--</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">SW-41 rotor</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">&#160;--</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:312px;">Spectrum Laboratories Spectrapor Membrane</td>
			<td style="width:147px;">VWR</td>
			<td style="width:123px;">132129</td>
			<td>dialysis tubing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ready-to-use dialysis cassettes&#160;</td>
			<td style="width:147px;">Thermo</td>
			<td style="width:123px;">66383</td>
			<td>dialysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">one shot DH10B electrocompetent E. coli</td>
			<td style="width:147px;">invitrogen</td>
			<td>C4040-52</td>
			<td>transformation of ligation reactions</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">PureYield&#8482; Plasmid Midiprep System</td>
			<td>Promega</td>
			<td>A2495</td>
			<td>midiprep</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">peqGOLD Tissue DNA Mini Kit&#160;</td>
			<td style="width:147px;">Peqlab</td>
			<td>12-3396-02</td>
			<td>isolation of genomic DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SuperFect Transfection Reagent</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:123px;">301305</td>
			<td>tranfection of 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">opti MEM (10 % FBS)&#160;</td>
			<td style="width:147px;">Gibco</td>
			<td>31985-062</td>
			<td>transfection of 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iQ SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>&#160;170-8882</td>
			<td>q-PCR</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">CFX 96 C1000 touch&#160;</td>
			<td style="width:147px;">Biorad</td>
			<td style="width:123px;"></td>
			<td>qPCR machine</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Phenol/Chloroform/Isoamyl alcohol&#160;</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">A156.1</td>
			<td>purification of DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Cesium chloride</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">8627.1</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium acetate 99%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">6773.2</td>
			<td>DNA precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">LB medium&#160;</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">X968.3</td>
			<td>bakterial growth medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ethanol&#160; 99,8% pure</td>
			<td style="width:147px;">Carl Roth</td>
			<td>9065.5&#160;</td>
			<td>DNA precipitation and washing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SDS 99,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td>2326.2</td>
			<td>lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">EDTA</td>
			<td style="width:147px;">Carl Roth</td>
			<td>8043.2</td>
			<td>lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tris-HCl 99%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">9090.3</td>
			<td>dialysis buffer/ lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">glycerol 99,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3783.1</td>
			<td>dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2 98,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">KK36.2</td>
			<td>dialysis buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3957.1</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">KH2PO4</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3904.2</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">sucrose</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">9286.1</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">Na2HPO4x2H2O</td>
			<td style="width:147px;">Carl Roth</td>
			<td>4984.2</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1,5ml tubes</td>
			<td style="width:147px;">sarstedt</td>
			<td style="width:123px;">72,730,005</td>
			<td>storage of&#160; virus preparations at -80&#176;C</td>
		</tr>
	</tbody>
</table>

cloning and large-scale production of high-capacity adenoviral vectors based on the human adenovirus type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

Here representative examples for cloning, amplification and purification of HCAdV preparations are shown. An overview of the cloning strategy (Figure 1) and representative examples for cloning and release of the HCAdV genome by restriction enzyme digest are provided (Figure 2). A typical restriction pattern after release of the GOI-expression cassette from pHM5 by PI-SceI and I-CeuI digest and subsequent phenol-chloroform extraction and ...

Watch this Scientific Journal Video about Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5 at JoVE.com

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

cloning-large-scale-production-high-capacity-adenoviral-vectors-based

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JoVE Journal

Bioengineering

14.2K Views.  University Witten/Herdecke. A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans. 

Video: Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

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 ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different techniques and varies in price, materials, and purpose. Many of these fabrication methods result in polydisperse populations with non-uniform activation thresholds. Additionally, controlling the droplet sizes typically requires stable perfluorocarbon liquids with high activation thresholds that are not practical in vivo. Producing uniform droplet sizes using low-boiling point gases would be beneficial for in vivo imaging and therapy experiments. This article describes a simple and economical method for the formation of size-filtered lipid-stabilized phase-shift nanodroplets with low-boiling point decafluorobutane (DFB). A common method of generating lipid microbubbles is described, in addition to a novel method of condensing them with high-pressure extrusion in a single step. This method is designed to save time, maximize efficiency, and generate larger volumes of microbubble and nanodroplet solutions for a wide variety of applications using common laboratory equipment found in many biological laboratories.

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...