Name: performing subretinal injections in rodents to deliver retinal pigment epithelium cells in suspension
Uploaded: 2015-01-23T13:00:00.000+00:00
Duration: 6 min 4 s
Description: Watch this Scientific Journal Video about Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension at JoVE.com

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.

참고 : 모든 동물이 스크립스 연구소에 의해 설립 된 윤리 지침에 따라 처리 하였다. 사출 용 재료 1. 준비 (~ 20 분) <ol style="margin-left: 40px;"><li> 사전 따뜻한 세포 분리 솔루션 (바람직하게는 혈청 희석을 통해 비활성화되어 하나), 멸균 PBS, 문화 미디어 (표 1). </li><li> 그것을 분해하고 15 분 동안 물에 부품을 끓여 무딘 바늘과 주사기를 소독. </li></ol> 사출에 대한 RPE 세포의 2. 준비 (~ 30 분 내지 1) <ol style="margin-left: 40px;"><li> 37 ℃에서 5-8 분 동안 예열 세포 분리 용액을 사용하여 RPE 세포를 분리. </li><li> 여전히 연결되어 있는지 어떤을 해제 부드럽게 세포를 긁어. </li><li> 배양액 대용량 (15ml의 튜브를 기입) 해리 용액을 비활성화하고 카운트하기로 세포를 희석. </li><li> 펠릿 세포 5 분 동안 800 XG에 원심 분리기. </li><li> 멸균 미리 예열 PBS에서 / μL 200,000 세포 (0.5 μL 볼륨에서 10 만 셀을 제공하기 위해)에서 세포를 재현 탁하고 1.5 ml의 마이크로 원심 튜브로 전송합니다. </li><li> 선택적으로, 살아있는 세포 과도 형광 마커를 추가하고 30 ~ 45 분 동안 37 ℃에서 배양한다. </li><li> 세포의 0.5 μL와 무딘 바늘과 주사기를로드합니다. 가능한 빨리 세포 주입한다. </li></ol> 3. 하위 망막 사출 (~ 사출 당 5 분) 참고 : 가능하면 윤부 선박 시각화 훨씬 용이하기 때문에, 성인 흰쥐와 기술을 배웁니다. 보다 쉽게​​ 주사 부위의 시각화를 용이하게하기 위해 (세포를 주입하기 전에) 배울 때 빠른 그린 용액을 주입한다. <ol style="margin-left: 40px;"><li> 쥐를 마취. 100 ㎎ / ㎖ 케타민 10 ㎎ / ㎖ 자일 라진 (xylazine) (20 μL / 10g 본체 w의 복강 내 주사를 사용하여이소 플루오 란 흡입을 통해 여덟)는 설치류를 기동 흡입기의 주둥이와 눈에 주입하기 어렵 기 때문에. <ol><li> 동물 깊이 그것의 발 중 하나를 집어 마취되어 있는지 확인합니다. 이 flinches 경우, 몇 분 이상 기다린 후 망막 주입을 시작하기 전에 다시 시도하십시오. </li></ol></li><li> 주입되는 눈이 천장을 향하도록 옆에 쥐를 놓습니다. </li><li> 눈 소켓 (임시 안구 돌출)에서 튕겨 나올 그냥 귀 위의 턱으로 가볍게 눈꺼풀에 피부 평행 스트레칭 두 손가락으로 머리를 잡고 더 접근하기가되도록 해부 현미경 부드럽게 피부를 스트레칭 그래서 눈이 약간 소켓에서 팝업됩니다 (그림 1C 참조). 목에 너무 가까이 쥐를 잡지 마십시오. </li><li> 30 G 날카로운 일회용 사전 멸균 바늘, 선박이 충돌하는 경우, 출혈입니다 b (즉시 윤부 아래에 구멍을중요한 전자와 나중에 구멍을 찾기 어려울 수)과 각도로 바늘 (그림 1D)으로 렌즈에 손이 닿지 않도록. 발생 날카로​​운 (또는 무딘) 바늘 또는 즉시 백내장으로 렌즈를 만지지 마십시오. 참고 : 주사는 두 사람이 잘 작동. 이런 식으로 한 사람은 구멍이 어디에 초점을 유지하기 위해 날카로운 일회용 바늘로 첫 번째 구멍을 만든 후 주입 작업을하는 사람이 무딘 바늘 주사기를 전달할 수 있습니다. </li><li> 머리에 그립을 유지하면서 눈에서 일회용 날카로운 바늘을 집어 넣으십시오. 구멍이 정확히 기억하십시오. </li><li> 중 미세 조작기에 무딘 바늘로 사전로드 된 주사기를 장착하거나 손을 잡고, 렌즈를 만지지 조심스럽게 눈을 통해 밀어 다시는주의하면서 구멍을 통해 무딘 바늘 주사기의 끝 부분을 삽입 한 후 아주 부드럽게 느낌 저항 (그림 1D)까지. </li><li> 케이최소한으로 모든 움직임을 eeping, 조심스럽게 천천히 망막 하 공간에 RPE 세포를 주입. 참고 : RPE / 망막 박리가 유도 될 것이다; 이 불가피하다. 그러나, 청소기 주입 박리를 최소화 크게 재 부착 (도 1E)의 기회를 향상시킨다. 모든 과장된 움직임은 망막에 다시 바늘을 이동 할 수 있으며, 옆으로 움직임이 망막에 손상을 줄 수 있습니다. 분사 펌프의 사용은 선택적이지만, 매우 정확한 전달을 허용한다. </li><li> 천천히 주사기를 집어 넣으십시오. 눈 보습을 적용하는 것은 수화 눈을 떼지 삭제합니다. </li><li> 이 흉골 드러 누움을 회복 할 때까지 동물을 모니터링하기 위해 계속합니다. 무인 동물을 떠나거나 흉골 드러 누움을 회복 할 때까지 다른 경고 동물 케이지에 반환하지 않습니다. </li></ol>

None of the authors have any commercial disclosures to declare.

이 문서에서 우리는 쥐와 생쥐에서 부유 망막 색소 상피 세포의 망막 주사를 수행하기위한 비교적 간단한 방법을 설명합니다. 이 프로토콜은 적은 외상에 번역 할 기술로 쉽게 배울 수 많은 경험이다 (그림 3, 이것은 더 나은 주사 중 하나를 나타냅니다) 미세 조작기는 (그림 1A)를 사용하는 경우에는 특히. 모든 외상은 cSLO 간섭 단층 시스템 (그림 2) 가능한 경?...

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.

<table><tbody><tr><td>2-Mercaptoethanol (55 mM)</td><td>Gibco&amp;nbsp;</td><td>21985-023</td><td>50 ml x 1&amp;nbsp;</td></tr><tr><td>Cell Scapers</td><td>VWR</td><td>89260-222</td><td>Case x 1</td></tr><tr><td>CellTracker Green CMFDA</td><td>Molecular Probes</td><td>C34552</td><td>50 &amp;micro;g x 20</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190-144</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258</td><td>25 g x 1&amp;nbsp;</td></tr><tr><td>Genteal Geldrops Moderate to Severe Lubricant Eye Drops&amp;nbsp;</td><td>Walmart</td><td>4060941</td><td>25 ml x 1</td></tr><tr><td>Hamilton Model 62 RN SYR</td><td>Hamilton</td><td>87942</td><td>Syringe x 1&amp;nbsp;</td></tr><tr><td>Hamilton Needle 33 G, 0.5&quot;, point 3 (304 stainless steel)</td><td>Hamilton</td><td>7803-05</td><td>Needles x 6</td></tr><tr><td>Knockout DMEM</td><td>Gibco</td><td>10829-018</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>10828-028</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>L-Glutamine 200 mM</td><td>Gibco</td><td>25030-081</td><td>100 ml x 1</td></tr><tr><td>Magnetic Stand</td><td>Leica Biosystems</td><td>39430216</td><td>Stand x 1</td></tr><tr><td>MEM Non-Essential Amino Acids Solution 100X&amp;nbsp;</td><td>Gibco</td><td>11140-050</td><td>100 ml x 1</td></tr><tr><td>Micromanipulator</td><td>Leica Biosystems</td><td>3943001</td><td>Manipulator x 1</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td>100 ml x 1</td></tr><tr><td>Slip Tip Syringes without Needles BD&amp;nbsp; (3 ml)&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>BD309656</td><td>Pack x 1</td></tr><tr><td>Specialty-Use Needles BD&amp;nbsp; (30 G, 1&quot;)</td><td>VWR</td><td>BD305128</td><td>Box x 1</td></tr><tr><td>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

전기 자극으로 광 변환 외측 망막에서 발생 콘로드와 ​​감광체와 망막 색소 상피 (RPE) 세포에 의해 주로 수행된다. RPE는 감광체 및 사망 또는 RPE 세포의 기능 장애 연령 관련 황반 변성 (AMD), 명 55 세 이상에서 영구 실명의 주요 원인의 중요 특성 인 지원을 제공한다. AMD에 대한 치료법이 발견되지 않은 반면, 병변 RPE 건강한 눈 주입은 효과적인 치료를 증명할 수 있고, RPE 세포의 다수 용이 다 능성 줄기 세포로부터 생성 될 수있다. RPE 세포 전달의 안전성과 효능에 관한 몇 가지 흥미로운 질문 여전히 동물 모델에서 시험 될 수 있고, RPE를 주입하기 위해 사용되는 잘 용인 프로토콜이 개발되었다. 여기에 기재된 기술은 다양한 여러 연구 그룹에 의해 사용 된과 제 날카로운 바늘의 눈 구멍을 만드는 것을 포함한다. 블루와 다음 주사기세포로드 NT 바늘 구멍을 통해 삽입하고 부드럽게 RPE에 닿을 때까지 유리체를 통과한다. 비교적 간단하며 최소한의 설비를 필요이 주입 방법을 사용하여, 우리는 동물 모델에서 광 수용체 변성증의 상당량을 방지 호스트 RPE 사이에서 줄기 세포 유래 RPE 세포를 효율적이고 일관성있게 통합을 달성. 실제 프로토콜의 일부 있지만, 우리는 또한 주입하는 방법과 세포가 생체 내 이미징을에 사용하여 망막 하 공간에 주입되었는지 확인하는 방법에 의해 유도 된 외상의 범위를 결정하는 방법에 대해 설명합니다. 마지막으로,이 프로토콜의 사용은 RPE 세포에 한정되지 않는다; 이는 망막 하 공간에 임의의 화합물 또는 세포를 주입하는 데에 이용 될 수있다.

우리는이 논문에서 기재된 기술을 사용하여 신속하고 지속적으로 설치류 망막 하 공간 내로 RPE 세포의 현탁액을 제공 할 수있다. 필요한 것은 아니지만, 외상은 크게 그림 1A &amp; B의 미세 조작기와 같은 설정을 사용하여 최소화 할 수 있습니다. 일시적인 안구 돌출 그림 1C에서와 같이 쥐를 잡으십시오. 미세 조작기 또는 손에 의해 수행되는 경우에 단계는 동일; 이 ...

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.

서스펜션에 망막 색소 상피 세포를 제공하기 위해 설치류에서 망막 주사를 수행

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

Research

JoVE Journal

Medicine

17.3K 조회수.  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.

비디오: 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.

마우스 눈에 유전자 치료 벡터와 줄기 세포의 Subretinal 주입

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 ...

연구

의학

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.

마우스 망막 색소 상피의 유전자 치료에 대한 바이러스 벡터의 윤부 접근 - 망막 주입

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 ...

신경과학

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

망막 하 공간에 액세스하기위한 대안의 검증 사출 방법<i&gt;를 통해</i&gt;를 Transcleral 후방 접근

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

쥐의 눈으로 인간 망막 안료 상피 세포의 트랜스 scleral Subretinal 이식에 대 한 세련 된 프로토콜의 개발

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

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.

초고 해상도 마우스 광학 일관성 단층 촬영 망막 유전자 치료 연구에서 안 구내 주사를 돕기 위해

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 Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.

Minipigs의 캐리어에 RPE의 망막하 이식: 수술 전 준비, 수술 기술 및 수술 후 관리에 대한 지침

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

생체공학

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

망막 변성 마우스 모델에서 망막하 이식편의 Transpupillary-Guided Trans-Scleral Transplantation

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Development of a Surgical Technique for Subretinal Implants in Rats

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

지리적 위축의 눈이 큰 모델에 인간 배아 줄기 세포 유래 망막 색소 상피 세포의 subretinal 이식

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

생물학

시력 회복: 망막 변성을 위한 줄기세포 치료

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.

저자 스포트라이트: 시력 회복의 발전 - 망막 질환을 위한 줄기세포 기반 치료 

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.

rd10 마우스에서 인간 배아 줄기 세포 유래 광수용체 전구체의 망막하 전달

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