Name: expansion of two-dimension electrospun nanofiber mats into three-dimension scaffolds
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds at JoVE.com

This work was supported by grants from the National Institute of General Medical Science (NIGMS) at the NIH (2P20 GM103480-06 and 1R01GM123081 to J.X.), the Otis Glebe Medical Research Foundation, NE LB606, and startup funds from the University of Nebraska Medical Center.

All in vivo procedures outlined below were approved by the IACUC Committee at the University of Nebraska Medical Center.
1. Prepare the Solutions for Standard Electrospinning
<ol>
	<li>In a 20 mL glass tube, dissolve 2 g of poly(&#949;-caprolactone) (PCL, Mw= 80 kDa) in a solvent mixture of dichloromethane (DCM) and N,N-dimethylformamide (DMF) with a 4:1 ration (v/v) at a concentration of 10% (w/v). 
	CAUTION: Handle DCM and DMF in a well-ventilated hood to avoid exposure to fu...

<ol>
	<li>Chen, 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=Recent+advances+in+electrospun+nanofibers+for+wound+healing.">Recent advances in electrospun nanofibers for wound healing.</a> Nanomedicine. 12 (11), 1335-1352 (2017).</li><li>Khandalavala, K., Jiang, J., Shuler, F. D., Xie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+Nanofiber+Scaffolds+with+Gradations+in+Fiber+Organization.">Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization.</a> Journal of Visualized Experiments. (98), e52626 (2015).</li><li>Xie, J., Li, X., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Put+electrospun+nanofibers+to+work+for+biomedical+research.">Put electrospun nanofibers to work for biomedical research.</a> Macromolecular Rapid Communication. 29 (22), 1775-1792 (2008).</li><li>Xie, 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=Nanofiber+membranes+with+controllable+microwells+and+structural+cues+and+their+use+in+forming+cell+microarrays+and+neuronal+networks.">Nanofiber membranes with controllable microwells and structural cues and their use in forming cell microarrays and neuronal networks.</a> Small. 7 (3), 293-297 (2011).</li><li>Xie, 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=Radially+aligned,+electrospun+nanofibers+as+dural+substitutes+for+wound+closure+and+tissue+regeneration+applications.">Radially aligned, electrospun nanofibers as dural substitutes for wound closure and tissue regeneration applications.</a> ACS. 4 (9), 5027-5036 (2010).</li><li>Xie, 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="Aligned-to-random"+nanofiber+scaffolds+for+mimicking+the+structure+of+the+tendon-to-bone+insertion+site.">"Aligned-to-random" nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site.</a> Nanoscale. 2 (6), 923-926 (2010).</li><li>Jiang, 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=Expanded+3D+Nanofiber+Scaffolds:+Cell+Penetration.">Expanded 3D Nanofiber Scaffolds: Cell Penetration.</a> Neovascularization, and Host Response. Advanced Healthcare Materials. 5 (23), 2993-3003 (2016).</li><li>Jiang, 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=Expanding+Two-Dimensional+Electrospun+Nanofiber+Membranes+in+the+Third+Dimension+by+a+Modified+Gas-Foaming+Technique.">Expanding Two-Dimensional Electrospun Nanofiber Membranes in the Third Dimension by a Modified Gas-Foaming Technique.</a> ACS Biomaterials Science &amp; Engineering. 10 (1), 991-1001 (2015).</li><li>Jiang, 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=CO2-expanded+nanofiber+scaffolds+maintain+activity+of+encapsulated+bioactive+materials+and+promote+cellular+infiltration+and+positive+host+response.">CO2-expanded nanofiber scaffolds maintain activity of encapsulated bioactive materials and promote cellular infiltration and positive host response.</a> Acta Biomaterialia. 68, 237-248 (2018).</li><li>Chen, 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=Nanofiber-based+sutures+induce+endogenous+antimicrobial+peptide.">Nanofiber-based sutures induce endogenous antimicrobial peptide.</a> Nanomedicine. 12 (10), 2597-2609 (2017).</li><li>Dhand, C., 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=Bio-inspired+crosslinking+and+matrix-drug+interactions+for+advanced+wound+dressings+with+long-term+antimicrobial+activity.">Bio-inspired crosslinking and matrix-drug interactions for advanced wound dressings with long-term antimicrobial activity.</a> Biomaterials. 138, 153-168 (2017).</li><li>Jiang, 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=Local+sustained+delivery+of+25-hydroxyvitamin+D3+for+production+of+antimicrobial+peptides.">Local sustained delivery of 25-hydroxyvitamin D3 for production of antimicrobial peptides.</a> Pharmaceutical Research. 32 (9), 2851-2862 (2015).</li><li>Jiang, 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=1&#945;,+25-dihydroxyvitamin+D3-eluting+nanofibrous+dressings+induce+endogenous+antimicrobial+peptide+expression.">1&#945;, 25-dihydroxyvitamin D3-eluting nanofibrous dressings induce endogenous antimicrobial peptide expression.</a> Nanomedicine (Lond). 13 (12), 1417-1432 (2018).</li><li>Ma, B., Xie, J., Jiang, J., Shuler, F. D., Bartlett, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+nanofiber+scaffolds+for+orthopedic+tissue+repair+and+regeneration.">Rational design of nanofiber scaffolds for orthopedic tissue repair and regeneration.</a> Nanomedicine. 8 (9), 1459-1481 (2013).</li></ol>

Transforming traditional 2D electrospun nanofiber mats into expanded 3D scaffolds via CO2 depressurization was investigated. Traditional 2D nanofiber mats are successfully expanded via subcritical CO2 fluid. The critical steps are to fabricate 2D nanofiber mats under an optimized condition and cut the mats without deforming the edges (e.g., using sharp surgical scissors). This CO2-expanded nanofiber scaffolds have many benefits over traditional 2D mats including...

The concept of developing a synthetic scaffold that can be implanted into patients to aid in tissue repair and regeneration is one that has permeated the regenerative medicine field for decades. The ideal synthetic scaffold serves to induce cell migration from surrounding healthy tissue, provides an architecture for cell seeding, adhesion, signaling, proliferation, and differentiation, supports vascularization, allows for adequate oxygenation and nutrition delivery, and promotes host immune activity to ensure success after implantation1. Additionally, it can be used as a carrier for embedding antimicrobial molecules to assist in wound healing1,3,6,7,8,9. The ability to control the temporal release of these biologic molecules from the synthetic scaffold is another desirable attribute that is considered when engineering scaffolds1.
Electrospinning has been a well-utilized technique for producing nanofiber scaffolds1,2,3,4,5,6. Previous attempts to create a nanofiber scaffold such as the one discussed here have been done to varying degrees of success. However, traditional nanofiber scaffolds have limited abilities to achieve these goals. Traditional nanofiber scaffolds have been mostly two-dimensional mats1,3. These nonexpanded scaffolds are densely packed with small pore sizes; this limits cell infiltration, migration, and differentiation as it does not promote an environment similar enough to those found in vivo1,7,8,9. For this reason, newer techniques of 3D electrospun nanofiber scaffold preparation have been established to amend the inherent flaws that come with 2D nanofiber mats. These techniques result in 3D scaffolds; however, they have limited applicability due to the production methods requiring aqueous solutions and freeze-drying procedures. This processing results in the random distribution of the nanofibers without restricted organization, proper thickness, and/or desired porosity to provide the adequate nanotopographic cues that are necessary for cell migration and proliferation. These factors result in the previous 3D electrospun nanofiber scaffolds that lack adequate mimicry of living tissues1,7,8,9.
More recent attempts at developing an expanded, 3D scaffold with better biomimicry of extracellular matrix (ECM) have been performed using an aqueous sodium borohydride (NaBH4) solution treatment and predesigned molds to aid in better control of the shape of the resulting scaffold7,8. However, this method is not ideal as it requires the use of aqueous solutions, chemical reactions, and freeze-drying that may interfere with polymers and any encapsulated biomolecules that are water-soluble. The additives used may also cause side effects during tissue regeneration8,9. The CO2 expansion method outlined in this article greatly reduces processing time, eliminates the need for aqueous solutions, and preserves the amount and functionality of biologically active molecules to a greater extent than the previously established methods9.
In previous studies, antibiotics, silver, 1&#945;,25 dihydroxyvitamin D3, and antimicrobial peptide LL-37 were loaded into the nanofiber scaffolds individually and in combination to investigate the potential of these scaffolds to release agents to further aid in wound healing9,10,12,13. For the purpose of demonstrating this method of nanofiber scaffold expansion, Coumarine 6, a fluorescent dye, will be loaded into the scaffold to demonstrate the potential of embedding the scaffold with various desired compounds. This method of expanded nanofiber scaffold fabrication in conjunction with encapsulated bioactive molecules holds great potential in tissue regeneration, wound healing, the creation of 3D tissue models, and the topical delivery of drugs.

<table>
	<tbody>
		<tr>
			<td>Polycaprolactone</td>
			<td>Sigma-Aldrich</td>
			<td>440744</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethlyformamide</td>
			<td>Fisher Chemical</td>
			<td>D-199-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Chemical</td>
			<td>AC61093-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coumarin 6</td>
			<td>Sigma-Aldrich</td>
			<td>546283</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotating Steel Drum</td>
			<td>customized</td>
			<td></td>
			<td>This serves as a collector during electrospinning.</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Fisher Scientific</td>
			<td>14-831-200</td>
			<td>Coaxial spinning requires two single syringe pumps.</td>
		</tr>
		<tr>
			<td>Revolver</td>
			<td>Lab Net International</td>
			<td>H5600</td>
			<td>Adjustable lab rotator for mixing solutions</td>
		</tr>
		<tr>
			<td>Hypodermic Needle (27G x 1 1/2&#34;)</td>
			<td>EXCELINT International Co</td>
			<td>26426</td>
			<td>This is part of the example customized coaxial nozzel shown.</td>
		</tr>
		<tr>
			<td>Hypodermic Needle (21G x 1 1/2&#34;)</td>
			<td>EXCELINT International Co</td>
			<td>26416</td>
			<td>This is part of the example customized coaxial nozzel shown.</td>
		</tr>
		<tr>
			<td>High Voltage DC Power Supply</td>
			<td>Gamma High Voltage Research</td>
			<td>ES30</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>FEI</td>
			<td>Nova 2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager 2</td>
			<td></td>
		</tr>
		<tr>
			<td>LL 37 ELISA Kit</td>
			<td>Hycult Biotech</td>
			<td>HK321-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

expansion of two-dimension electrospun nanofiber mats into three-dimension scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

The efficacy of expanding traditional 2D electrospun nanofiber mats into 3D scaffolds via depressurization of subcritical CO2 fluid was demonstrated in different capacities: the thickness of the scaffolds increased from 1 mm when untreated to 2.5 mm and 19.2 mm with one and two CO2 treatments, respectively (Figure 3A-C). The porosity-a characteristic of the architecture critical for cell seeding-also increased in a manne...

Watch this Scientific Journal Video about Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds at JoVE.com

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

This protocol reported the transformation of traditional electrospun nanofibremembranes from 2D to 3D, via depressurization of subcritical CO2 fluid which was not realized previously. This method eliminates many issues associated with previous approaches, including the use of aqueous solutions and chemical reactions, multiple step processes, loss of activity of encapsulated biological molecules, and limitations of hydrophobic polymers. Demonstrating this procedure is Shixuan Chen, a postdoc from my lab.In a 20 mililitre glass tube, dissolve two grams of PCL in a solvent mixture of dichloromethane and DMF, with a four to one ratio at a concentration of 10 percent. Place the glass tube into a lab rotator until the solution becomes clear. The solution may mix over night.To set up the Electrospinning Apparatus, first, add the PCL solution to a 20 milliliter syringe with a 21 gauge blunt needle attached. Ensure there is no air in the syringe, and dissociated tubing. Place a rotating steel drum with the ground collector 12 centimeters from the needle tip.Using alligator clips, connect the direct-current high-voltage power supply to the needle, and ensure that the collector is grounded. For the 20 milliliters of PCL solution, set the parameter of the syringe pump using a diameter of 20.27 millimeters, and a flow rate of 0.5 milliliters per hour. Check if the droplets are forming at the tip of the needle.Apply an electric potential of 20 kilovolts between the spinneret and a ground collector located 20 centimeters away from the spinneret. Collect the aligned nanofiber mats in a drum, rotating at 2, 000 RPM. Collect the PCL nanofiber mats once they reach a thickness of approximately one millimeter.Immerse the PCL nanofiber mats in liquid nitrogen for five minutes. Keep the PCL nanofiber mats in liquid nitrogen, and punch PCL nanofiber mats with a 0.5 millimeter diameter punch. Place the PCL nanofiber mats into liquid nitrogen for five minutes.Cut the mats into one centimeter by one centimeter squares using sharp surgical scissors while submerged in liquid nitrogen to avoid deformation of the edges. Place the cut mat in a 30 milliliter centrifuge tube with approximately one gram of dry ice. Tightly cap the lid, and allow for the dry ice to change into liquid carbon dioxide.Once the liquid has formed in the tube, quickly release the pressure by opening the cap. Remove and observe the puffed scaffold from the tube. Place the scaffold in a new centrifuge tube with dry ice, and repeat util the desire thickness is achieved.Sterilize the expanded nanofiber scaffolds in ethylene oxide prior to incubation with cells. The effectiveness of expanding traditional 2D electrospun nanofiber mats into 3D scaffolds via depressurization of subcritical CO2 fluid is shown on the left after the second treatment. The thickness of the scaffold increased from one millimeter when untreated to 2.5 millimeters with one CO2 treatment, to 19.2 millimeters with two CO2 treatments.The porosity of the scaffolds increased from 79.5 percent for the untreated mats, to 92.1 percent after the first treatment, to 99.0 percent after the second treatment. This is significant because the degree of cell penetration into a scaffold, and thus its efficacy to induce regeneration, is largely dependent on the porosity. SEM images reveal that the densely-packed fibular structure of untreated 2D mats were transformed into ordered, layered structures with aligned nano fibers after expansion with CO2.In Vevo studies were carried out by subcutaneous implantation of CO2-expanded nano fiber scaffolds with square-arrayed holes to rats. This allows for cellular migration and proliferation within the holes, as well as further infiltration within the nano fiber layers that were created during expansion. From week one to four post-implantation, the expanded scaffolds showed a significant increase in the number of blood vessels formed, and multinucleated giant cells when compared to a traditional nano fiber mat.Following this procedure, different molecules including growth factors, amino-modulating compounds, hemostatic agents, and anti-microtubular agents can be incorporated into the nano fiber mats and expanded in subcritical CO2 fluid. Such functionalized expanded nano fiber scaffolds could be used to explore new questions in other scientific fields, such as hemostasis, prevention and treatment of infection, immunology, and tissue regeneration and repair. Organic solvents are toxic, and should be handled in a chemical hood.Furthermore, a container that can endure the high pressure of subcritical CO2 fluid should be used for the expansion.

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Genetics

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.