We wish to thank Alison Dorsey for helping to develop the subretinal injection technique. We also acknowledge the National Eye Institute (NEI grants EY11254 and EY021416), California Institute for Regenerative Medicine (CIRM grant TR1-01219), and the Lowy Medical Research Institute (LMRI) for very generous funding for this project.

NOTE: All animals were treated in accordance with the ethical guidelines established by the Scripps Research Institute.
1. Preparation of Materials for the Injection (~20 min)
<ol style="margin-left: 40px;">
	<li>Pre-warm cell dissociation solution (preferably one that is inactivated through dilution, not with serum), sterile PBS, and culture media (Table 1).</li>
	<li>Sterilize the syringe with a blunt needle by disassembling it and boiling the parts in water for 15 min.</li>
</ol>
2. Preparation of the RPE Cells for Injection (~30 min to 1 hr)
<ol style="margin-left: 40px;">
	<li>Detach the RPE cells using pre-warmed cell dissociation solution for 5-8 min at 37 &#176;C.</li>
	<li>Scrape the cells gently to release any that are still attached.</li>
	<li>Dilute the cells with a large volume of culture media (fill up a 15 ml tube) to inactivate the dissociation solution and count them.</li>
	<li>Centrifuge at 800 x g for 5 min to pellet the cells.</li>
	<li>Resuspend the cells at 200,000 cells/&#181;l (to deliver 100,000 cells in a 0.5 &#181;l volume) in sterile pre-warmed PBS and transfer them into a 1.5 ml microcentrifuge tube.</li>
	<li>Optionally, add a live cell transient fluorescent marker and incubate at 37 &#176;C for 30-45 min.</li>
	<li>Load the syringe with a blunt needle with 0.5 &#181;l of cells. Inject the cells as soon as possible.</li>
</ol>
3. Sub-retinal Injection (~5 min per Injection)
NOTE: If possible, learn the technique with adult albino rats since the limbus vessels are much easier to visualize. Inject Fast Green solution when learning (before trying to inject cells) to more easily facilitate visualization of the injection site.
<ol style="margin-left: 40px;">
	<li>Anesthetize the rodent. Use intraperitoneal injections of 100&#160;mg/ml ketamine and 10&#160;mg/ml xylazine (20 &#181;l/10 g body weight) over isofluorane inhalation since it is difficult to maneuver the rodent and inject into the eye with the snout in the inhaler.
	<ol>
		<li>Ensure that the animal is deeply anesthetized by pinching one of its paws. If it flinches, wait several more minutes and try again before beginning the subretinal injection.</li>
	</ol>
	</li>
	<li>Position the rodent onto its side so that the eye that will be injected is facing the ceiling.</li>
	<li>Under a dissecting microscope gently stretch the skin so the eye pops slightly up out of the socket (temporary proptosis) and becomes more accessible by holding its head with two fingers just above the ear and by its jaw and gently stretch the skin parallel to the eyelids so that the eye pops slightly up out of the socket (See Figure 1C). Do not grasp the rodent too close to the throat.</li>
	<li>With a 30 G sharp disposable pre-sterilized needle, make a hole immediately below the limbus (if the vessels are hit, bleeding will be significant and it be difficult to find the hole later) and at an angle to avoid touching the lens with the needle (Figure 1D). Avoid touching the lens with the sharp (or blunt) needle or immediate cataract formation will occur. 
	NOTE: Injections work better with two people. In this way one person can pass the syringe with the blunt needle to the person performing the injection after they have created the first hole with the sharp disposable needle to maintain focus on the where the hole is.</li>
	<li>Retract the disposable sharp needle from the eye while maintaining the grip on the head. Remember exactly where the hole is.</li>
	<li>After either mounting the pre-loaded syringe with a blunt needle on a micromanipulator or holding it by hand, insert the tip of the syringe with the blunt needle through the hole, taking care again not to touch the lens and gently push it through the eye very gently until feeling resistance (Figure 1D).</li>
	<li>Keeping all movements to a minimum, carefully inject the RPE cells slowly into the subretinal space. 
	NOTE: RPE/retina detachment will be induced; this is unavoidable. However, a cleaner injection minimizes the detachment and greatly improves the chances of reattachment (Figure 1E). Any exaggerated movements may move the needle back into the retina, and sideways movements can damage the retina. The use of an injection pump is optional but allows for a very precise delivery.</li>
	<li>Retract the syringe slowly. Apply eye moisturizing drops to keep the eye hydrated.</li>
	<li>Continue to monitor the animal until it regains sternal recumbency. Do not leave animal unattended or return to a cage with other alert animals until it regains sternal recumbency.</li>
</ol>

None of the authors have any commercial disclosures to declare.

In this article we describe a relatively simple method for performing subretinal injections of RPE cells in suspension in rats and mice. The protocol is easy to learn and more experience with the technique will translate in fewer traumas (Figure 3; this represents one of the better injections), especially if a micromanipulator is used (Figure 1A). Any trauma can be monitored in vivo with a cSLO and OCT system (Figure 2) if available. If higher resolution and les...

The sensory retina is organized in functional tiers of neurons, glia, and endothelial cells. Photoreceptors at the back of the retina are activated by light; through phototransduction they convert photons into electrical signals that are refined by interneurons and transmitted to the visual cortex in the brain. Phototransduction cannot occur without the coordinated efforts of Mueller glia and retinal pigment epithelium (RPE) cells. RPE are organized in a monolayer directly behind the photoreceptors and perform multiple and diverse functions integral to photoreceptor function and homeostasis. In fact, RPE and photoreceptors are so co-dependent that they are considered to be one functional unit. Death or dysfunction of RPE results in devastating secondary effects on photoreceptors and is associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly1,2.
While no cure has been discovered for AMD, several clinical studies have shown that RPE cell replacement may be a promising therapeutic option3-13. With the advent of stem cell technology, it is now possible to generate large numbers of RPE cells in vitro from embryonic and induced pluripotent stem cells (hES and hiPS) that strongly resemble their somatic counterparts functionally and anatomically14-26. Stem cell-derived RPE have also been shown to function in vivo by multiple independent groups, including our own, to significantly slow retinal degeneration in rat and mouse lines with spontaneous retinal degeneration16,18,21,22,25,28,29. This combination of clinical and preclinical supporting evidence is so compelling that several clinical trials to prevent retinal degeneration using stem cell-derived RPE cells are now ongoing30,31. 
RPE can be readily derived from hES and/or hiPS and implanted in the subretinal space of rodents using various derivation and injection techniques32,33. (See Westenskow et al. for a methods paper in multimedia format demonstrating the directed differentiation protocol we employ)34. There are critical remaining questions regarding the safety, survival, and functional capacity of exogenously delivered RPE cells upon implantation, therefore the ability to perform subretinal injections in rodents is a critical skill16,18,21,29,36,37. The delivery of RPE is not trivial, and the field is divided on the most effective injection technique. The protocol we describe here is a simple and effective way to deliver of bolus of RPE cells subretinally, and was used in the first clinical trial for stem cell-derived RPE transplantation31. (The reader may also refer to another JoVE article by Eberle et al. for an alternative depiction of subretinal injections in rodents.38) 
The technique outlined in this manuscript cannot be visualized and trauma is unavoidable (as with any subretinal injection technique). It is performed by making a hole just under the limbus vessels and inserting a blunt needle along a transscleral route to inject a bolus of cells under the diametrically opposed retina. The person doing the injection will feel resistance as the blunt needle touches the retina. The cells may be directly visualized after the injection, however, and the degree of the induced retinal detachment can be determined by labeling the RPE cells with a transient fluorescent marker and detecting them with a confocal scanning ophthalmoscope (cSLO). An optical coherence tomography (OCT) system can also be used to monitor the trauma and easily identify the injection site.

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			<td height="20" style="height:20px;width:419px;">Name of Material/ Equipment (A-Z)</td>
			<td style="width:145px;">Company</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="20" style="height:20px;">2-Mercaptoethanol (55 mM)</td>
			<td>Gibco&#160;</td>
			<td>21985-023</td>
			<td>50 mL x 1&#160;</td>
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			<td height="20" style="height:20px;">Cell Scapers</td>
			<td>VWR</td>
			<td>89260-222</td>
			<td>Case x 1</td>
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			<td height="20" style="height:20px;">CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C34552</td>
			<td>50 ug x 20</td>
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			<td height="20" style="height:20px;">DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>500 mL x 1&#160;</td>
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			<td height="20" style="height:20px;">Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7258</td>
			<td>25 g x 1&#160;</td>
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			<td height="20" style="height:20px;">Genteal Geldrops Moderate to Severe Lubricant Eye Drops&#160;</td>
			<td>Walmart</td>
			<td>4060941</td>
			<td>25 mL x 1</td>
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			<td height="20" style="height:20px;">Hamilton Model 62 RN SYR</td>
			<td>Hamilton</td>
			<td>87942</td>
			<td>Syringe x 1&#160;</td>
		</tr>
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			<td height="21" style="height:21px;width:419px;">Hamilton Needle 33 gauge, 0.5&#34;, point 3 (304 stainless steel)</td>
			<td style="width:145px;">Hamilton</td>
			<td style="width:119px;">7803-05</td>
			<td>Needles x 6</td>
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			<td height="20" style="height:20px;">Knockout DMEM</td>
			<td>Gibco</td>
			<td>10829-018</td>
			<td>500 mL x 1&#160;</td>
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			<td height="20" style="height:20px;">KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>500 mL x 1&#160;</td>
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			<td height="20" style="height:20px;">L-Glutamine 200 mM</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Magnetic Stand</td>
			<td style="width:145px;">Leica Biosystems</td>
			<td style="width:119px;">39430216</td>
			<td>Stand x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids Solution 100X&#160;</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Micromanipulator</td>
			<td style="width:145px;">Leica Biosystems</td>
			<td style="width:119px;">3943001</td>
			<td>Manipulator x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slip Tip Syringes without Needles BD&#160; (3 mL)&#160;&#160;</td>
			<td>VWR</td>
			<td>BD309656</td>
			<td>Pack x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specialty-Use Needles BD&#160; (30 gauge, 1&#34;)</td>
			<td>VWR</td>
			<td>BD305128</td>
			<td>Box x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express Enzyme (1X), no phenol red</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>100 mL x 1</td>
		</tr>
	</tbody>
</table>

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.

We can deliver a suspension of RPE cells into the subretinal space of rodents quickly and consistently using the technique described in this manuscript. While not required, traumas can be greatly minimized using the setup shown with a micromanipulator in Figure 1A&#38;B. Hold the rodent as shown in Figure 1C for temporary proptosis. The steps are the same if performed with the micromanipulator or by hand; these are depicted in the cartoon in Figure 1D. When performed cle...

Watch this Scientific Journal Video about Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension at JoVE.com

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

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 ...

performing-subretinal-injections-rodents-to-deliver-retinal-pigment

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17.2K Views.  The Scripps Research Institute. 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.

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

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

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 ...

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.

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 ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.