The study was supported in part by grants awarded to B. R. by the National Institutes of Health (R01EY019320), the Department of Veterans Affairs (RX000444 and BX003050), and the South Carolina SmartState Endowment.

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Medical University of South Carolina Animal Care and Use Committee under protocol ID 00399.
1. Cell Culture
<ol>
	<li>Generate human retinal pigment epithelial cells (ARPE-19) cell line stably expressing the gene of choice according to published protocols14,15.</li>
	<li>Maintain cells in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2.</li>
	<li>Replace the medium every 2&#8211;3 days.</li>
	<li>Passage the cells after reaching 70%&#8211;80% confluence using standard tissue culture procedures.</li>
</ol>
2. Cell Encapsulation
<ol>
	<li>Mix sodium alginate with deionized (DI) water for a final concentration of 2% w/v and purify by filtration with a 0.2 &#181;m sterile syringe filter.</li>
	<li>Prepare the cells to be mixed with the alginate solution by trypsinizing, centrifuging, and washing them with 10 mM HEPES buffered saline solution (pH = 7.4). Using a hemocytometer, count the cells and adjust the final cell concentration to 1 x 106 in alginate solution. 
	NOTE: The encapsulation process should be run inside a sterilized hood.</li>
	<li>Load ~300 &#181;L aliquots of alginate and cells mixture into a 3 mL syringe and attach it to a syringe pump. The solution will be pumped through a 30 G blunt tip needle to a sterile gelling bath placed in a sterile 50 mL beaker below the syringe tip at 7 mm for a needle to bath spraying distance.</li>
	<li>The gelling bath contains a volume of 40 mL of 10 mM HEPES buffered saline containing 100 mM calcium chloride (CaCl2) and 0.5% w/v poly-L-ornithine (PLO). The PLO is a secondary polymer coating that can be omitted or changed according to the needs of the investigator.</li>
	<li>Adjust the voltage and flow rate and keep them both constant during the encapsulation process at 60 mm/h flow rate and 6.0 kV initial voltage to produce microcapsules size of ~150 &#181;m.</li>
	<li>Connect the clip of anode wire (red) of a high voltage generator needle tip to the needle and connect the ground clip (black) to the copper wire that is halfway submerged in the gelling bath. One batch of the alginate + cell mixture (1 mL) takes approximately 30 min to prepare the encapsulated cells (approximately 25,000 microcapsules).</li>
	<li>Wash the formed microcapsules containing cells with washing solution (10 mM HEPES buffered saline containing 1.5 mM CaCl2, pH = 7.4) twice. Do not use PBS for washing.</li>
	<li>Incubate the encapsulated cells with 10% FBS supplemented DMEM media in a humidified incubator at 37 &#176;C and 5% CO2. 
	NOTE: Encapsulation process instruments are depicted in Figure 1.</li>
</ol>
3. Confirmation that Encapsulation Does Not Affect Cell Viability
<ol>
	<li>After incubating the encapsulated cells for 24 h in media, prepare a small sample of 500 &#181;L (~30 microcapsules) for staining.</li>
	<li>Wash the microcapsules 2x using washing solution (10 mM HEPES buffered saline containing 1.5 mM CaCl2) and stain them for live-dead viability using a live/dead assay kit.</li>
	<li>Prepare a staining mixture of calcein AM (acetoxymethyl) and ethidium homodimer-1 at final concentrations of 2 &#181;M and 4 &#181;M, respectively.</li>
	<li>Add 2 mL of the staining mixture to the encapsulated cells and incubate for 30&#8211;45 min in the dark at room temperature (RT).</li>
	<li>With careful aspiration, aspirate the staining solution and wash the microcapsules 2x with the washing solution. Use a fluorescent microscope system to observe and image the encapsulated cells. 
	NOTE: Documentation of encapsulation is depicted in Figure 2.</li>
</ol>
4. Confirmation That Capsules Are the Appropriate Size for Delivery and Delivery of the Biologic
<ol>
	<li>Intravitreal injections in a mouse eye are typically performed with a 27 G blunt-tip needle (inner diameter of 210 &#181;m) attached to a 2.5 &#181;L Hamilton syringe.</li>
	<li>Generate capsules ranging from 100 to 200 &#181;m, dilute them in serum-free media, and carefully pull them up into the Hamilton syringe. PBS is not a suitable vehicle, as alginate capsules will dissolve in PBS.</li>
	<li>Slowly eject 1 &#181;L drops containing capsules onto a microscope slide and determine their integrity using an upright bright-field microscope.</li>
	<li>Adjust the capsule size (see step 2.3) by adjusting voltage and flow rate accordingly. Smaller microcapsules are produced in a nonlinear fashion by increasing voltage and slightly decreasing flow rate8. In our hands, capsules of 150 &#181;m in diameter proved to be most suitable. Adjustment of capsule size is also dependent on keeping the alginate concentration constant while changing the other parameters.</li>
	<li>Maintain a separate set of cells in capsules of the appropriate size in serum-free medium to determine the amount of secretion of the desired biologic. Use sensitive ELISAs or western blotting to determine the concentration of the biologics in the supernatant.</li>
	<li>Determine the required amount of capsules that need to be injected based on the known PK/PD (pharmacokinetics/pharmacodynamics) of the therapeutic. In our hands, 10 capsules per mouse eye proved to be most efficacious.</li>
</ol>
5. Capsule Delivery into Mouse Vitreous
<ol>
	<li>Perform intravitreal injections using a dissecting microscope. See Figure 3 for surgical set-up and materials used during the procedure. A detailed protocol for intravitreous injections can be found elsewhere16.</li>
	<li>Anesthetize mice by intraperitoneal injection of xylazine and ketamine (20 mg/kg and 80 mg/kg) or other preferred anesthetics approved by the specific institution&#8217;s Animal Care and Use Committee. Ensure the appropriate depth of anesthesia using a toe pinch17.</li>
	<li>Dilate the mouse pupils with phenylephrine HCL (2.5%) and atropine sulfate (1%) to allow for good visibility of the vitreous chamber and apply a lubricant eye gel to the eyes to keep them hydrated during the procedure.</li>
	<li>Puncture the sclera at the limbus with a 26 G needle as the guide hole, making sure that 1) the needle is at a 45&#176; angle with the eye and table and 2) the beveled tip is pointed upwards to avoid puncturing the lens.</li>
	<li>Carefully inject the capsules using a 27 G blunt-tip needle attached to a Hamilton syringe at a 45&#176; angle under visual inspection, making sure to avoid touching the lens with the needle. Injury of the lens will lead to cataract formation. The capsules should be visible in the vitreous using the dissecting microscope (Figure 4A).</li>
	<li>After retraction of the needle, treat the injection site with antibiotic ointments neomycin and polymyxin B sulfates in addition to dexamethasone ophthalmic antibiotic ointment.</li>
	<li>Apply goniotaire hypromellose demulcent ophthalmic solution (2.5%) to both eyes to prevent the corneas from drying out during the recovery period.</li>
	<li>Place the mouse on a heating pad held at 37&#176;C and monitor until fully awake.</li>
	<li>A successful injection of encapsulated ARPE-19 cells should reveal the presence of intact capsules in the vitreous chamber of the mouse with only minor amounts of debris when imaging the eye by optical coherence tomography or other methods (Figure 4B).</li>
	<li>The injected mouse eye, after a few days of recovery due to the surgery, is now ready for the experimental paradigm at hand. 
	NOTE: The surgical set-up and documentation of capsules in the eye are depicted in Figure 3 and Figure 4, respectively.</li>
</ol>

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The authors declare no competing financial interests.

This cell encapsulation technique is relatively quick and easy to perform; however, certain points must be kept in mind to obtain accurate downstream results. Cells should be maintained in culture in a Petri dish prior to encapsulation and held at proper confluency. Encapsulation should be performed in a proper ventilation hood with regulated air flow, if possible. Too strong of an air current can affect capsule formation, especially in long-term experiments. Sterile utensils and solutions are critical for long-term main...

Cell-based therapies represent revolutionary biological techniques that have been applied widely in medicine. Recently, they have been successfully applied in the treatment of neurodegenerative diseases, eye diseases, and cancer. Cell therapies cover a wide range of fields from cell replacement to drug delivery, and this protocol focuses on the latter. Biodegradable alginate microcapsules (MC) have shown effectiveness as a delivery system, and they are becoming widely used in the biomedical field. Alginate has been used in microencapsulation due to its simple gelling process, biodegradability, excellent biocompatibility, and stability under in vivo conditions1,2,3,4.
The electrospray method, as a microencapsulation technique, has been successfully utilized to encapsulate peptides and proteins using alginate (base polymer) and poly-l-ornithine (secondary coating polymer). Both polymers are naturally found and used for their biocompatibility5,6,7. However, the main challenge in cell-based therapies is suppression of the host immune system to avoid side effects caused by immunosuppressive drugs. The permeability of alginate microcapsules is considered a suitable property for cell encapsulation, which allows diffusion of nutrients, waste, and therapeutic factors into and out of cells while masking them from the host immune response8,9,10.
In the eye, encapsulated cells have been used in clinical trials for the constant delivery of biologics (i.e., growth factors11,12 and growth factor antagonists13) for the treatment of retinitis pigmentosa or age-related macular degeneration. Other targets such as complement inhibitors14 are also currently being explored in preclinical settings.

<table><tbody><tr><td>3 mL Syringe</td><td>BD</td><td>309656</td><td></td></tr><tr><td>30 G 1&quot; Blunt needle</td><td>SAI Infusion technology</td><td>B30-100</td><td></td></tr><tr><td>Alginic acid sodium salt, from brown algae</td><td>Sigma</td><td>A0682</td><td></td></tr><tr><td>Atropine Sulfate Ophthalmolic solution (1%)</td><td>Akorn</td><td>NDC 17478-215-15</td><td>for pupil dilation</td></tr><tr><td>BD 1 mL Syringe 26 G x 3/8 (0.45 mm x 10 mm)</td><td>Becton, Dickinson and Company</td><td>DG518105 500029609 REF 309625</td><td>to generate the guide hole</td></tr><tr><td>Calcium chloride, Anhydrous, granular</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>GenTeal Tears</td><td>Alcon</td><td>NDC 0078-0429-47</td><td>to lubricate the eyes during anesthesia</td></tr><tr><td>Goniotaire: Hypromellose (2.5%) Ophthalmolic Demulcent Solution (Sterile)</td><td>Altaire Pharmaceuticals Inc.</td><td>NDC 59390-182-13</td><td>to lubricate the eyes during anesthesia</td></tr><tr><td>Hamilton Needle/syringe Tip: 27 G, Small Hub RN NDL, custum length (12 mm), point style 3, 6/PK</td><td>Hamilton</td><td>7803-01</td><td>for intravitreal delivery of capsules</td></tr><tr><td>Hamilton Syringe: 2.5 &amp;micro;L, Model 62 RN SYR, NDL Sold Separately</td><td>Hamilton</td><td>7632-01</td><td>for intravitreal delivery of capsules</td></tr><tr><td>HEPES buffer, 1 M</td><td>Fisher Bioreagents</td><td>BP299100</td><td></td></tr><tr><td>High voltage generator</td><td>ESD EMC Technology</td><td>ES813-D20</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit</td><td>Thermofisher Scientific</td><td>L3224</td><td></td></tr><tr><td>L-Ornithine hydrochloride, 99%</td><td>Alfa Aesar</td><td>A12111</td><td></td></tr><tr><td>Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmolic Ointment</td><td>SANDOZ</td><td>NDC 61314-631-36</td><td>antibiotic to prevent infection after intravitreal injection</td></tr><tr><td>Phenolephrine Hydrochloride Ophthalmolic Solution (2.5%)</td><td>Akorn</td><td>NDC 17478-201-15</td><td>for pupil dilation</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S-5886</td><td></td></tr><tr><td>Sterile syringe filters, 0.2 &amp;mu;m</td><td>VWR</td><td>28143-312</td><td></td></tr><tr><td>Syringe pump</td><td>GRASEBY</td><td>MS16A</td><td></td></tr></tbody></table>

encapsulated cell technology for the delivery of biologics to the mouse eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

ARPE-19 cells are a spontaneously immortalized human RPE cell line that has been shown to be amenable to encapsulation and long-term survival upon implantation of capsules into the eye. The tools for alginate encapsulation are shown in Figure 1. In this study, it was demonstrated that upon encapsulation in alginate, the cells in alginate capsules were confirmed by bright-field imaging (Figure 2A). Live-dead assays were performed on the cells inside the capsules,...

Watch this Scientific Journal Video about Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye at JoVE.com

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...

encapsulated-cell-technology-for-delivery-biologics-to-mouse

Research

JoVE Journal

Bioengineering

6.2K Views.  University of South Carolina. Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Video: Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Medicine

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neuroscience

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Revitalizing Sight: Stem Cell Therapy for Retinal Degeneration

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

Convection-Enhanced Delivery of Antibodies into a Mouse Brain

Source: Beffinger, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59675">Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery.</a> J. Vis. Exp. (2019).
This video demonstrates a convection-enhanced delivery (CED) technique for antibody delivery into the mouse brain. The skull of an anesthetized mouse is exposed, and a burr hole is created at the target area for antibody delivery. A step catheter containing antibodies is inserted at the target site, and via an infusion pump, a pressure gradient is created to deliver antibodies at a controlled rate.

Konwekcyjne dostarczanie przeciwciał do mózgu myszy

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

A Murine Model of Primed Mycobacterial Uveitis to Study Post-Infectious Uveitis

Source: John, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a> J. Vis. Exp. (2021).
This video demonstrates a method to generate a mouse model of primed mycobacterial uveitis (PMU) for studying post-infectious uveitis. Upon induction of PMU by intravitreal injection of heat-killed Mycobacterium tuberculosis in a mouse previously immunized with the same immunogen, the progression of chronic inflammation in the eye is analyzed using imaging and histopathological examination

Mysi model prątkowego zapalenia błony naczyniowej oka do badania poinfekcyjnego zapalenia błony naczyniowej oka

Source: John, S., et al. Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis. J. Vis. Exp. (2021).
This video demonstrates a method ...

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

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Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

In vivo Electroporation of Developing Mouse Retina

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

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Konwekcyjne dostarczanie przeciwciał do mózgu myszy

Mysi model prątkowego zapalenia błony naczyniowej oka do badania poinfekcyjnego zapalenia błony naczyniowej oka