This work was supported by the European Union&#39;s Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134. Zsuzsanna&#160;Izsv&#225;k was funded by the European Research Council, ERC Advanced (ERC-2011-ADG 294742). The authors would like to thank Anna Dobias and Antje Schiefer (Department of Ophthalmology, University Hospital RWTH Aachen) for excellent technical support, and the Aachen Cornea Bank (Department of Ophthalmology, University Hospital RWTH Aachen) for providing the human donor eyes.

Human donor eyes were obtained from the Aachen Cornea Bank of the Department of Ophthalmology (University Hospital RWTH Aachen) after obtaining informed consent in accordance with the Declaration of Helsinki protocols. Procedures for the collection and use of human samples have been approved by the institutional ethics committee.
1. Isolation of primary human RPE cells
<ol>
	<li>Lay out sterile protective clothing and gloves. Place a sterile drape under a laminar flow.</li>
	<li>Place sterile preparation instruments and other necessary sterile equipment under the laminar flow.</li>
	<li>Record the receipt of the eye globes, the beginning of the preparation as well as possible abnormalities. Register donor age, gender, cause of death, and the period between time of death and eye removal.</li>
	<li>Place the eye globe in a sterile gauze compress and hold it in one hand.</li>
	<li>Separate the anterior segment from the posterior segment by a circumferential cut approximately 3.5 mm posterior to the limbus using a scalpel and an extra fine pointed eye scissor.</li>
	<li>After tilting the posterior segment downwards, carefully remove the vitreous and the retina using colibri forceps.</li>
	<li>Discreetly rearrange the collapsed RPE/choroidea complex using straight iris forceps and subsequently hold it in position with curved iris forceps.</li>
	<li>Fill the posterior eye cup with 1 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium/Ham&#39;s F-12 Nutrient Mixture (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 80 U/mL penicillin and 80 &#181;g/mL streptomycin (Pen/Strep), and 2.5 &#181;g/mL amphotericin B (AmphoB). 
	&#8203;NOTE: Contrary to the isolation of primary RPE cells from pig or bovine eyes36,37, the posterior eye cup doesn&#39;t have to be filled and incubated with trypsin.</li>
	<li>Harvest the RPE cells by gently brushing the retinal pigment epithelium from the optic nerve toward the limbus with a fire-polished curved glass Pasteur pipette, while fixating the RPE/choroidea complex at the limbus using colibri forceps. Transfer the cell suspension into a Petri dish.</li>
	<li>Repeat the steps 1.8 and 1.9 and collect all cells in the Petri dish. Resuspend the cell suspension carefully by pipetting up and down using a single channel pipette (100-1,000 &#181;L).</li>
	<li>Seed the RPE cell suspension of each eye globe into three wells of a 24-well cell culture plate and fill them up to 1 mL with DMEM/F12 supplemented with FBS, Pen/Strep, and AmphoB.</li>
	<li>Maintain the RPE cell cultures at 37 &#176;C in a humidified atmosphere of 95% air and 5% CO2 until confluence is reached. Change the cell culture medium twice a week.</li>
</ol>
2. Electroporation of primary human RPE cells
<ol>
	<li>Preparation of the SB100X transposase/PEDF transposon plasmid mixture
	<ol>
		<li>Purify the SB100X transposase and PEDF transposon plasmid DNA using customary plasmid purification kits, which are identified to be suitable for the use in applications such as transfection, according to the protocol of the manufacturer.</li>
		<li>Quantify the plasmid DNA contents using a microvolume spectrophotometer and adjust them to a concentration of 250 ng/&#181;L with 10 mM Tris-HCl (pH 8.5).</li>
		<li>Mix one portion of 250 ng/&#181;L SB100X transposase plasmid DNA (e.g., 2.5 &#181;L) with 16 portions of 250 ng/&#181;L PEDF transposon plasmid DNA (e.g., 40 &#181;L). Residual plasmid mixture can be stored at -20 &#176;C. 
		&#8203;NOTE: Multiple repeated freezing and thawing cycles of the plasmid mixture should be avoided.</li>
		<li>Place 2 &#181;L of the SB100X transposase/PEDF transposon plasmid mixture, containing 29.4 ng of SB100X transposase plasmid and 470.6 ng of PEDF transposon plasmid, in a sterile 1.5 mL safe-lock microcentrifuge tube. Keep on ice until mixed with cells.</li>
	</ol>
	</li>
	<li>Set-up of the capillary transfection system 
	NOTE: Electroporation of primary human RPE cells has been performed via a capillary transfection system using the 10 &#181;L kit according to the manufacturer&#39;s protocol. The system comprises the transfection device, the transfection pipette, and the pipette station. The kit contains 10 &#181;L transfection tips, buffer tubes, electrolytic buffer E (buffer E), and resuspension buffer R (buffer R).
	<ol>
		<li>Connect the pipette station to the transfection device.</li>
		<li>Fill a buffer tube with 3 mL of buffer E and insert it into the pipette station until a click sound is heard. 
		NOTE: Ensure that the side electrode of the buffer tube is connected to the side ball plunger of the pipette station.</li>
		<li>For the electroporation of primary human RPE cells, set the following pulse conditions on the transfection device: 1,100 V (pulse voltage), 20 ms (pulse width), two pulses (pulse number).</li>
	</ol>
	</li>
	<li>Preparation of the cultivated primary human RPE cells
	<ol>
		<li>Document shape and morphology of the primary RPE cell cultures via phase contrast microscopy. Take low magnification micrographs to demonstrate the growth of the RPE cells to a confluent and integrated monolayer as well as higher magnification micrographs to point out the typical cobblestone morphology of the RPE cells.</li>
		<li>Aspirate the cell culture medium and wash the cells twice with 1 mL buffered phosphate saline (PBS, pH 7.4).</li>
		<li>Trypsinize primary human RPE cells with 0.5 mL 0.05% trypsin-EDTA at 37 &#176;C in a humidified atmosphere of 95% air and 5% CO2 for 7-15 min (maximum). Check the cell detachment microscopically.</li>
		<li>Stop trypsinization with 1 mL of DMEM/F12 supplemented with FBS. Take a 20 &#181;L aliquot for cell counting using a hemocytometer. Centrifuge the RPE cell suspension at 106 x g for 10 min.</li>
		<li>Mix the 20 &#181;L aliquot with 20 &#181;L trypan blue solution. Pipette 10 &#181;L in each half of the hemocytometer and count the cells under a phase contrast microscope.</li>
		<li>After centrifugation, resuspend the RPE cell pellet in 1 mL of PBS.</li>
		<li>Take 10,000-100,000 RPE cells per transfection reaction and centrifuge them at 106 x g for 10 min. 
		&#8203;NOTE: Beside the transfection reactions with electric field application and addition of plasmid DNA, each approach also includes two different control cultures: (1) without electric field application and without plasmid DNA; (2) with electric field application, but without plasmid DNA.</li>
		<li>Resuspend the cell pellet in 11 &#181;L of buffer R and combine it with 2 &#181;L of the SB100X transposase/PEDF transposon plasmid mixture (see step 2.1.4). 
		&#8203;NOTE: After resuspension in buffer R, the cells must be processed within 15 min to avoid a reduction in cell viability and transfection efficiency.</li>
		<li>Insert the head of the transfection pipette into the 10 &#181;L transfection tip until the clamp fully picks up the mounting stem of the piston. Draw up the cell/plasmid solution into the transfection tip. 
		NOTE: Avoid generation of air bubbles and their aspiration into the transfection tip.</li>
		<li>Provide 1 mL of DMEM/F12 supplemented with FBS, but without Pen/Strep and without AmphoB, into the required number of wells of a 24-well cell culture plate.</li>
	</ol>
	</li>
	<li>Electroporation process
	<ol>
		<li>Insert the transfection pipette into the buffer tube placed in the pipette station (see step 2.2.2) until a click sound is heard. 
		&#8203;NOTE: Ensure the metal head of the transfection pipette is connected to the ball plunger inside of the pipette station and to the buffer tube.</li>
		<li>Press Start on the touchscreen of the transfection device. Before application of the electric pulse, the device automatically checks whether the buffer tube and the transfection pipette are properly inserted. After delivery of the electric pulses, Complete is displayed on the touchscreen.</li>
		<li>Carefully remove the transfection pipette from the pipette station and immediately release the electroporated cells from the 10 &#181;L transfection tip by pipetting the cell/plasmid solution into the prepared wells of the cell culture plate (see step 2.3.10).</li>
		<li>Maintain transfected RPE cell cultures at 37 &#176;C in a humidified atmosphere of 95% air and 5% CO2. Add Pen/Strep and AmphoB with the first medium exchange 3 days after electroporation.</li>
	</ol>
	</li>
</ol>
3. Analyses of transfected primary human RPE cells
<ol>
	<li>Sample preparation
	<ol>
		<li>After a cultivation time of 3 weeks, ultimately incubate the RPE cell cultures in a defined volume of 1.0 mL DMEM/F12 supplemented with FBS, Pen/Strep, and AmphoB for a defined time of 24 h.</li>
		<li>Take the cell culture supernatants and store them at -20 &#176;C until further near-time processing or at -80 &#176;C for thermally unstable samples and long-term storage.</li>
		<li>Trypsinize transfected RPE cell cultures with 0.5 mL of 0.05% trypsin-EDTA at 37 &#176;C in a humidified atmosphere of 95% air and 5% CO2 for 10 min.</li>
		<li>Stop trypsinization with 1 mL of DMEM/F12 supplemented with FBS. Take small aliquots for cell counting using a hemocytometer (see step 2.3.5). Centrifuge the RPE cell suspensions at 106 x g for 10 min.</li>
		<li>Store the RPE cell pellets at -80 &#176;C until further processing.</li>
	</ol>
	</li>
	<li>Evaluation of PEDF secretion by western blotting 
	NOTE: For a qualitative assessment, cell culture supernatants (see step 3.1.2) are analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent western blotting. Depending on the PEDF transposon plasmid DNA used, the supernatants have to be processed differently.
	<ol>
		<li>Purification of His-tagged PEDF fusion proteins from cell culture supernatants
		<ol>
			<li>Take 30 &#181;L nickel-nitrilotriacetic acid (Ni-NTA) slurry per sample using a bevel-cut tip and pellet the Ni-NTA resin by centrifugation (2,660 x g for 30 s).</li>
			<li>Carefully resuspend the Ni-NTA resin with 200 &#181;L of 1x incubation buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and pellet it by centrifugation (2,660 x g for 30 s).</li>
			<li>Repeat the previous step.</li>
			<li>Carefully resuspend the Ni-NTA resin with 40 &#181;L 4x incubation buffer (200 mM NaH2PO4, 1.2 M NaCl, 40 mM imidazole, pH 8.0) per sample.</li>
			<li>Mix 900 &#181;L of cell culture supernatant with 260 &#181;L of 4x incubation buffer and 55 &#181;L of pretreated Ni-NTA slurry (see step 3.2.1.4).</li>
			<li>Incubate the mixture on a rocking shaker at room temperature for 60 min.</li>
			<li>Pellet the Ni-NTA resin by centrifugation (2660 x g for 60 s).</li>
			<li>Carefully resuspend the Ni-NTA resin with 175 &#181;L of 1x incubation buffer and pellet resin by centrifugation (2,660 x g for 60 s).</li>
			<li>Repeat the previous step.</li>
			<li>Carefully resuspend the Ni-NTA resin with 30 &#181;L of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) and incubate the mixture on a rocking shaker at room temperature for 20 min.</li>
			<li>Pellet the Ni-NTA resin by centrifugation (2,660 x g for 30 s).</li>
			<li>Carefully take the supernatant and mix it with 2x SDS sample buffer47.</li>
			<li>Heat the mixture at 95 &#176;C for 5 min and separate the Ni-NTA purified proteins on a 10% SDS-polyacrylamide gel.</li>
			<li>Perform western blotting using anti-Penta-His antibodies (mouse monoclonal, 1:500) and horseradish peroxidase (HRP)-conjugated anti-mouse antibodies (rabbit polyclonal, 1:1,000) as previously described36,37.</li>
		</ol>
		</li>
		<li>Direct analysis of non-tagged PEDF proteins
		<ol>
			<li>Take 15 &#181;L of cell culture supernatant and mix it with 2x SDS sample buffer47.</li>
			<li>Heat the mixture at 95 &#176;C for&#160;5 min and separate the proteins on a 10% SDS-polyacrylamide gel.</li>
			<li>Perform western blotting using anti-human PEDF antibodies (rabbit polyclonal, 1:4,000) and HRP-conjugated anti-rabbit antibodies (goat polyclonal, 1:2,000) as previously described38.</li>
		</ol>
		</li>
		<li>Quantification of PEDF secretion by ELISA
		<ol>
			<li>Analyze cell culture supernatants (see step 3.1.2) using a human PEDF ELISA kit according to the manufacturer&#39;s protocol.</li>
			<li>Relate the amount of secreted PEDF to the time and the cell number determined for each transfection reaction (see step 3.1.4).</li>
		</ol>
		</li>
		<li>Analysis of PEDF gene expression in transfected RPE cells
		<ol>
			<li>Isolate total RNA from the RPE cell pellets (see step 3.1.5) using a commercially available kit according to the manufacturer&#39;s protocol.</li>
			<li>Quantify RNA contents using a microvolume spectrophotometer.</li>
			<li>Carry out reverse transcription on 0.1 &#181;g RNA using a reverse transcription system according to the manufacturer&#39;s protocol.</li>
			<li>Perform real-time qPCR as previously described38.</li>
		</ol>
		</li>
		<li>Analysis of transgene insertion sites in transfected RPE cells
		<ol>
			<li>Isolate genomic DNA from the RPE&#160;cell pellets (see step 3.1.5) using a commercially available kit according to the manufacturer&#39;s protocol.</li>
			<li>Quantify DNA contents using a microvolume spectrophotometer.</li>
			<li>Generate insertion site libraries using a computation-assisted hemi-specific PCR scheme as previously described38.</li>
			<li>Perform computational analysis as previously described38.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Zolt&#225;n Ivics and Zsuzsanna Izsv&#225;k are inventors on several patents on SB transposon technology

In our project, we aim for the non-viral production of genetically modified primary human RPE cells that continuously overexpress and secrete an effective factor in order to use the transfected cells as long-term therapeutic for the establishment and maintenance of a protective environment. We have established the introduction of the gene encoding PEDF, an ubiquitously expressed multi-functional protein with anti-angiogenic and neuroprotective functions. The protocol described here can be used to stably and reproducibly ...

Advanced age is described to be the main risk for neurodegenerative diseases. Age-related macular degeneration (AMD), a polygenic disease leading to severe vision loss in patients older than 60 years of age, belongs to the four most common causes of blindness and vision impairment1 and is expected to increase to 288 million people in 20402. Dysfunctions of the retinal pigment epithelium (RPE), a single layer of tightly packed cells located between the choriocapillaris and the retinal photoreceptors, contribute to the pathogenesis of AMD. The RPE fulfills multiple tasks that are essential for a normal retinal function3 and secretes a variety of growth factors and factors essential to maintain the structural integrity of the retina and the choriocapillaris, thereby supporting photoreceptor survival and providing a basis for the circulation and the supply of nutrients.
In healthy eyes, pigment epithelium-derived factor (PEDF) is responsible for balancing the effects of vascular endothelial growth factor (VEGF) and protects neurons against apoptosis, prevents endothelial cell proliferation, and stabilizes the capillary endothelium. A shifted VEGF-to-PEDF ratio is related to ocular neovascularization, which was observed in animal models4,5 as well as in samples of patients with choroidal neovascularization (CNV) due to AMD and proliferative diabetic retinopathy6,7,8,9,10. The enhanced VEGF concentration is the target for the current standard treatment. The anti-VEGF pharmaceuticals bevacizumab, ranibizumab, aflibercept and, most recently, brolucizumab improve visual acuity in about one third of CNV patients or rather stabilize vision in 90% of cases11,12,13. However, the frequent, often monthly, intravitreal injections bear the risk of adverse events14, impair patient compliance, and represent a significant economic burden to healthcare systems15. Moreover, a certain percentage of patients (2%-20%) do not respond or only poorly react to the anti-VEGF therapy16,17,18,19. These negative concomitants necessitate the development of alternative treatments, e.g., intraocular implants, cell and/or gene therapeutic approaches.
Gene therapy has evolved as promising treatment for hereditary and non-hereditary diseases and intends to restore non-functional gene sequences or suppress malfunctioned ones. For polygenic diseases, where identification and replacement of the causative factors is hardly possible, strategies aim for the continuous delivery of a protective factor. In the case of AMD, various additive therapies have been developed, such as the stable expression of endostatin and angiostatin20, the VEGF antagonist soluble fms-like tyrosine kinase-1 (sFLT-1)21,22, the complement regulatory protein cluster of differentiation 59 (CD59)23 or PEDF24,25. The eye, and especially the retina, is an excellent target for a gene-based medication due to the enclosed structure, good accessibility, small size, and immune privilege, thus allowing for a localized delivery of low therapeutic doses and making transplants less susceptible to rejection. Moreover, the eye enables non-invasive monitoring, and the retina can be examined by different imaging techniques.
Viral vectors are, because of their high transduction efficiency, the main vehicle to deliver therapeutic genes into target cells. However, depending on the viral vector used, different adverse reactions have been described, such as immune and inflammatory responses26, mutagenic and oncogenic effects27,28, or dissemination in other tissues29. Practical limitations include a restricted packaging size30 as well as difficulties and costs associated with the production of clinical grade lots31,32. These drawbacks have promoted the further development of non-viral, plasmid-based vectors that are transferred via lipo-/polyplexes, ultrasound or electroporation. However, genomic integration of the transgene into the host genome is usually not promoted with plasmid vectors, thus resulting in a transient expression.
Transposons are naturally occurring DNA fragments that change their position within the genome, a characteristic that has been adopted for gene therapy. Due to an active integration mechanism, transposon-based vector systems allow for a continuous and constant expression of the inserted transgene. The Sleeping Beauty (SB) transposon, reconstituted from an ancient Tc1/mariner-type transposon found in fish33 and further improved by molecular evolution resulting in the hyperactive variant SB100X34, enabled efficient transposition in various primary cells and was used for the phenotypic correction in different disease models35. At present, 13 clinical trials have been initiated using the SB transposon system. The SB100X transposon system consists of two components: the transposon, which comprises the gene of interest flanked by terminal inverted repeats (TIRs), and the transposase, which mobilizes the transposon. Following plasmid DNA delivery to the cells, the transposase binds the TIRs and catalyzes the excision and integration of the transposon into the cell&#39;s genome.
We have developed a non-viral cell-based additive therapy for the treatment of neovascular AMD. The approach comprises the electroporation-based insertion of the PEDF gene into primary pigment epithelial (PE) cells by means of the SB100X transposon system36,37,38. The genetic information of the transposase and PEDF are provided on separate plasmids, thereby enabling the adjustment of the ideal SB100X-to-PEDF transposon ratio. Electroporation is performed using a pipette-based capillary transfection system that is characterized by a maximized gap size between the electrodes while minimizing their surface area. The device was shown to achieve excellent transfection rates in a wide range of mammalian cells39,40,41. The small electrode surface area provides a uniform electric field and reduces the various side effects of electrolysis42.
The anti-angiogenic functionality of PEDF secreted by transfected pigment epithelial cells was shown in various in vitro experiments analyzing the sprouting, migration, and apoptosis of human umbilical vein endothelial cells43. In addition, transplantation of PEDF-transfected cells in a rabbit model of corneal neovascularization44 as well as a rat model of CNV43,45,46 showed decline of neovascularization.
Here, we describe a detailed protocol for the stable insertion of the PEDF gene into primary human RPE cells via the SB100X transposon system using a capillary transfection system. The transfected cells were kept in culture for 21 days and subsequently analyzed in terms of PEDF gene expression by quantitative polymerase chain reaction (qPCR) and in terms of PEDF protein secretion by immunoblotting and enzyme-linked immunosorbent assay (ELISA, Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Isolation of primary human RPE cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Cell Culture Plate</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030722019</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B [250 &#181;g/mL] (AmphoB)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri Forceps</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-18950</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Iris Forceps&#160;</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-18856</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel (No. 11)</td>
			<td>Feather, Osaka, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Ham&#8217;s F-12 Nutrient Mixture (DMEM/F12)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P04-41150</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Pointed Eye Scissor&#160;</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-19405</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum [0.2 &#181;m Sterile Filtered] (FBS)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipettes</td>
			<td>Brand, Wertheim, Germany</td>
			<td>747715</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1000 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (100-1000 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Drape</td>
			<td>Lohmann &#38; Rauscher, Rengsdorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze Compress&#160;</td>
			<td>Fink-Walter, Merchweiler, Germany</td>
			<td>321063</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gloves</td>
			<td>Sempermed, Wien, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri Dish (Falcon 60 mm x 15 mm)</td>
			<td>Corning, Corning, NY</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Surgical Gown</td>
			<td>Halyard Health, Alpharetta, GA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Iris Forceps&#160;</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-18855</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation of primary human RPE cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl (pH 8.5)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-Well Cell Culture Plate</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>150628</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Cell Culture Plate</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030722019</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B [250 &#181;g/mL] (AmphoB)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Ham&#8217;s F-12 Nutrient Mixture (DMEM/F12)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P04-41150</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes (1.5 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum [0.2 &#181;m Sterile Filtered] (FBS)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Leica Mikrosysteme, Wetzlar, Germany</td>
			<td>Leica DMi8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometer (NanoDrop Spectrophotometer)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Transfection System (Neon Transfection System)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>MPK5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer (Neubauer Chamber)</td>
			<td>Paul Marienfeld, Lauda-K&#246;nigshofen, Germany</td>
			<td>0640110</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco w/o Ca2+ w/o Mg2+</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L182-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (10 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1000 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (200 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Maxi Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>12163</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (0.1-10 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (100-1000 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (10-200 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,05&#160;%)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Analyses of transfected primary human RPE cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% SDS-Polyacrylamide Gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x Incubation Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x SDS Sample Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4x Incubation Buffer (200 mM NaH2PO4, 1.2 M NaCl, 40 mM imidazole, pH 8.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Protran Supported 0.2 &#181;m&#160;Nitrocellulose Blotting Membrane</td>
			<td>Cytiva, Marlborough, MA</td>
			<td>10600015</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B [250 &#181;g/mL] (AmphoB)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PEDF Antibodies (Rabbit Polyclonal)</td>
			<td>BioProducts, Middletown, MD</td>
			<td>AB-PEDF1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Penta-His Antibodies (Mouse Monoclonal)</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>34660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Ham&#8217;s F-12 Nutrient Mixture (DMEM/F12)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P04-41150</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum [0.2 &#181;m Sterile Filtered] (FBS)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer (Neubauer Chamber)</td>
			<td>Paul Marienfeld, Lauda-K&#246;nigshofen, Germany</td>
			<td>0640110</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish Peroxidase-Conjugated Anti-Mouse Antibodies (Rabbit Polyclonal)</td>
			<td>Agilent Dako, Santa Clara, CA</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish Peroxidase-Conjugated Anti-Rabbit Antibodies (Goat Polyclonal)</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>Human PEDF ELISA Kit&#160;</td>
			<td>BioProducts, Middletown, MD</td>
			<td>PED613</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-3000 Imaging System</td>
			<td>Fujifilm, Minato, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 1.2 Instrument</td>
			<td>Roche Life Science, Penzberg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler FastStart DNA Master SYBR Green I</td>
			<td>Roche Life Science, Penzberg, Germany</td>
			<td>12239264001</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler&#160;Capillaries (20 &#956;l)</td>
			<td>Roche Life Science, Penzberg, Germany</td>
			<td>4929292001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometer (NanoDrop Spectrophotometer)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN&#160;Tetra Cell Casting Module</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1658015</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN&#160;Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>30410</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (10 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1000 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (200 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>51304</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcription System&#160;</td>
			<td>Promega, Madison, WI</td>
			<td>A3500</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit&#160;</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker</td>
			<td>Cole-Parmer, Staffordshire, United Kingdom</td>
			<td>SSM3</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes (1.5 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (0.1-10 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (100-1000 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (10-200 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1704150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,05&#160;%)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>25300054</td>
			<td></td>
		</tr>
	</tbody>
</table>

electroporation-based genetic modification of primary human pigment epithelial cells using the sleeping beauty transposon system

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

Cultivation and electroporation of primary human RPE cells 
We have shown that seeding of a sufficient number of primary RPE cells of animal origin allows for the cultivation and growth to an integrated monolayer of pigmented, hexagonally shaped cells36,37,48. Their capability to form tight junctions, to exhibit phagocytic activity, and to express specific marker genes in vi...

Watch this Scientific Journal Video about Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System at JoVE.com

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

electroporation-based-genetic-modification-primary-human-pigment

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Medicine

1.9K Views.  University Hospital RWTH Aachen. We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Video: Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Measurement of Carotenoids in Perifovea using the Macular Pigment Reflectometer

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical density (L-OD) and zeaxanthin optical density (Z-OD) in the central 1 degree of the fovea. A modification of the technique was developed to evaluate in vivo carotenoid density eccentric to the fovea. An adjustable track system with red LED lights was placed 6.1 m away from the participant to facilitate ocular fixation. Lights were spaced appropriately to create increments of 1 degree retinal disparity during the reflectometry measurements. All reflectometry measurements were obtained with pupillary dilation. The mean MPR-MPOD value for the central measurement was 0.593 (SD 0.161) with an L-OD to Z-OD ratio of 1:2.61. The MPR-MPOD value at 1 degree was 0.248 and the mean MPR-MPOD value at 2 degrees in the parafoveal region was 0.143. The L-OD to Z-OD ratio at 1 degree and 2 degrees off center was 1.38:1.0 and 2.08:1.0, respectively. The results demonstrate that MPOD measurements obtained using the MPR decrease as a function of retinal eccentricity and that there is a higher concentration of zeaxanthin centrally compared to lutein. The L-OD to Z-OD ratio changes with foveal eccentricity, with twice more lutein than zeaxanthin at 2 degrees off center. Our technique successfully provides a quick in vivo method for the measurement of macular pigment optical density at various foveal eccentricities. The results agree with prior published in vivo and in vitro xanthophyll carotenoid density distribution measurements.

We present a protocol to determine the levels of overall macular pigment, lutein, and zeaxanthin optical density in the central and parafoveal regions of the retina. The protocol includes a novel adjustable track system used to measure macular pigment optical density in the foveal eccentricity.

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...