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该研究协议经低视力学院道德委员会批准, 所有受试者均按照《赫尔辛基宣言》签署书面同意书。本研究已获得拉夫博勒和谢菲尔德大学的伦理认同。
注: 在表 1中描述了与干燥年龄相关的黄斑变性患者接受脉络自体移植的 Limoli 视网膜修复技术 (LRRT) 的纳入和排除标准。
1. 干燥年龄相关黄斑变性患者的诊断
<ol><li>确定用共焦扫描激光检眼镜、SD OCT 和我的诊断。</li>
	<li>评估每组 BCVA 的距离和远近。测量 VA 为近视觉 (特写) 在点 (临时)。测量 BCVA 在时间 0 (T0), 90 (T90) 和180天 (T180) 比较早期治疗糖尿病视网膜病变研究 (EDTRS) 图表在4米在 logMAR。</li>
	<li>记录电子暗、中间和明细胞活动, 或闪光视网膜电, 根据国际视觉临床电生理 (ISCEV)112009年制定的标准。</li>
</ol>2. 麻醉
注: LRRT 麻醉中的金标准为局部麻醉, 经亚榫麻醉和镇静剂的渗透增强。在特定情况下, 一般麻醉是首选。
<ol><li>应用局部麻醉药, 在4% 和罗哌卡因1% 时, 采用局部麻醉剂注入滴状 15-20 分钟, 获得角膜和结膜麻醉。</li>
	<li>将麻醉直接渗入亚结膜和 subtenon 的空间。</li>
	<li>使用局部浸润在腹部区域, 在脂肪组织被提取之前, 并且在亚结膜和子榫的空间, 12 毫米从角膜缘。采用局部麻醉 carbocaine 或 marcain 混合1200内肾上腺素。</li>
	<li>通过麻醉提供术中镇静剂, 通过重复小的口服芬太尼作为麻醉镇痛剂, 可以正确的进行。用量一般为0.025 毫克芬太尼, 每丸1毫克咪达唑仑。</li>
</ol>3. Limoli 视网膜修复技术的制备
注意: 此技术代表佩洛斯的一种变体, 在 subscleral 空间1、6、7、12中移植了眼眶自体脂肪。经手术移植的细胞可在周围组织、脉络膜和视网膜上产生多种神经营养和 angiotrophic 特性的 GFs,18,19,20,21,22 ,23,24,25。在 LRRT 中, 移植自体细胞与脉络膜之间的距离通过深巩膜降低, 茎与脉络膜的接触面积扩大, 促进分泌自体细胞分泌进入脉络膜流9, 10,14。
<ol><li>在脉络膜和巩膜之间进行细胞移植手术前, 对每只眼睛进行适当的消毒, 这一过程称为 Limoli 视网膜恢复技术 (LRRT)15,16,17。</li>
	<li>移植干细胞, 由科尔曼et 等和劳伦斯的技术 (图 1) 从腹部脂肪, 在 SVF 在 sovrachoroidal 空间15,16,17。</li>
	<li>通过以下步骤, 从 PRP 凝胶中提取血小板浸润脂肪蒂。</li>
	<li>离心血液6,12和收集富含血小板的血浆 (PRP)。刺激血小板脱粒导致 GF 释放脂肪蒂6,12。</li>
</ol>4. 技术规格和战略
注: 根据劳伦斯和科尔曼技术17(材料表), 从患者腹部皮层收集和纯化脂肪组织。
<ol><li>根据劳伦斯和科尔曼技术17 (图 2 a/2 b), 从每个病人的腹部皮下手动收获10毫升脂肪组织, 使用3毫米钝套管连接到锁定注射器。</li>
	<li>在20摄氏度 (图 2C) 上, 用离心法将脂肪组织从血液、脂肪、油和液体中分离出来, SVF 1500 x g 的5分钟。SVF 是非常丰富的干细胞17。</li>
	<li>收集8毫升的人外周血与22克针, 并在一个单独的管为 PRP 准备。</li>
	<li>在20摄氏度 (图 2D) 上, 离心收集的血液在 1500 x g 处为5分钟。在 LRRT, 随后的变化导致更好的生存自体脂肪移植, ADSC 增殖, 这有利于增加脉络膜灌注, 并更全面的调节作用的那些因素, 只分泌脂肪7, 11,17。</li>
	<li>建立脉络口袋 (步骤4中的更多细节, 特别是4.4 和 4.5), 以容纳从眼眶脂肪获得的移植物和饱和的剩余体积的干细胞从 SVF 和 PRP, 获得根据劳伦斯和科尔曼技术17。</li>
</ol>5. LRRT 脉络自体移植 (Limoli 视网膜修复技术): 外科手术及技术细节
<ol><li>锚定巩膜与6-0 丝缝合, 近颞缘。</li>
	<li>用 5.5 "保罗·韦斯科特 Tenetomy 弯剪刀" 将结膜和 subtenonian 空间从下颞缘处打开11毫米。</li>
	<li>在这个空间插入 Limoli 巴西莱结膜牵引器, 做一个巩膜手术场。</li>
	<li>用5毫米的新月形刀倾斜, 在8毫米的巩膜上, 从角膜缘处切开一侧的皮瓣。瓣铰链总是径向和在外科医生的左侧。</li>
	<li>在下颞象限, 在8毫米从角膜缘, 打开一个深巩膜门大约5毫米在侧面由径向铰链用一把新月刀, 倾斜的斜角。在适当的深度进行巩膜, 以查看脉络膜的石板色。</li>
	<li>通过去除皮瓣远端部分的小笼盖, 以促进脉络自体移植后的血液循环, 从而产生间隙。</li>
	<li>用眼科钳提取从下斜肌上方间隙的眼眶脂肪。确保提取的脂肪有足够的血管化, 使其能够在植入后存活下来。</li>
	<li>轻轻地将自体脂肪瓣放在脉络膜床上, 在门近端脉络膜 6/0 polyglactin 纤维缝合。</li>
	<li>缝合巩膜瓣以避免在脂肪蒂或其营养血管上受压迫。</li>
	<li>采用30克角 (30°) 套管, 渗透1毫升 PRP 凝胶 (通过血液材料的离心、组分分离和血小板脱粒26) 的脂肪蒂的基质。</li>
	<li>准备的结膜两侧的缝合。然后, 取出结膜牵引器。</li>
	<li>用 6/0 polyglactin 纤维缝合结膜。</li>
	<li>在关闭前, 留出一个空间插入到 subscleral 的空间, 在皮瓣, 脉络膜和自体脉络膜之间, 一个小的灵活的塑料管与自身脂肪移植。</li>
	<li>将自体脂肪移植、脉络膜和巩膜瓣与0.5 毫升的 SVF (丰富的干细胞) 的剩余空间饱和, 以前在步骤3.2 中制备, 由一个小的柔性塑料管插入巩膜口袋。</li>
	<li>在将剩余空间饱和后, 关闭缝合线。</li>
	<li>手术后, 用500毫克阿奇霉素进行三天的抗生素治疗。此外, 还提供了抗生素和类固醇结合, 如氯霉素和倍他米, 约 15-20 天滴眼疗法。 注意: 由脂肪细胞、SVF 干细胞和 PRP 组成的自体移植现在已获得26。通过深巩膜降低移植自体细胞与脉络膜之间的距离, 刺激自体细胞分泌分泌进入脉络膜流。为了同样的目的, 扩大茎和脉络膜之间的接触区域。</li>
</ol>

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提交于 ARVO 2015, 可 3-7-丹佛, 联合美国。

本研究的主要目的是评估脂肪细胞、干细胞在 SVF 和 PRP 中的脉络移植是否能改善长期干性 AMD 影响眼中的 VA 和视网膜敏感度。另一项主要目的是根据最近的文献证明这些细胞可能的治疗效果, 因为一些临床前研究表明, 基于 GF 的治疗方法对于几种疾病的病人护理是有益的。
事实上, 一些研究表明, 自体人类诱导多潜能干细胞 (iPSC) 可以代表一个细胞源的移植, 目的是视网膜色素...

细胞治疗, 包括全身或局部注射在受伤地区的茎/祖细胞治疗多种慢性疾病, 已引起密切注意在过去十年1。自二十世纪九十年代以来, 研究了生长因子 (GFs) 在视网膜萎缩症的潜在治疗作用2。事实上, 许多人类细胞可以生产 GFs, 这是特定的蛋白质, 能够阻止或减缓细胞凋亡,即, 程序性死亡的细胞3。
众所周知, 干燥年龄相关的黄斑变性 (AMD) 是一种萎缩性视网膜疾病, 其中渐进和不可逆转的细胞死亡涉及感光层损伤, 因此, 中央视觉功能丧失4。AMD 是发达国家55岁以上人群失明的主要原因, 占所有黄斑退化的 80%, 至今尚无有效治疗。
有数项研究显示, 有多种来源可供自体飞行情报队获得。这些构成不同的细胞类型, 包括来自眼眶脂肪的脂肪基质细胞, 富含血小板的血浆 (PRP) 的血小板, 以及脂肪来源的干细胞 (干细胞) 包括在脂肪组织的基质血管分数 (SVF) 中5 ,6,7。目前的 GF 集确保视网膜 neuroenhancement, 和菲拉托夫, Meduri, 佩洛斯和 Limoli 进行的研究表明, 自体脂肪移植 (尾部) 是有效的8,9,10。
此外, 一项先前的研究显示, 在干燥的 AMD 受影响的眼睛中, 视网膜电图 (脉络) 的数据有显著的改善, 记录后的自体移植,11。脉络空间的手术移植组织调节视网膜细胞的分泌分泌, 延缓其凋亡6,7,12。考虑外核层厚度, 豚鼠视网膜组织学检查显示, GFs 对视网膜有营养作用。因此, 直接或间接使用 GFs 可以通过分子诱导剂和抑制剂之间的平衡关系带来治疗好处6,7,12。
该方法的目的是评估脂肪细胞, 干细胞在 SVF 和 PRP 的脉络移植是否能提高最佳矫正视力 (BCVA) 和 microperimetry (我) 反应在干燥的 AMD 受影响的眼睛。本研究的目的是在其 GF 生产的基础上, 展示自体移植的治疗效果, 根据引用文献6,7,12,13。

<table><tbody><tr><td>Blunt cannula, 3 mm.&amp;nbsp;</td><td>Mentor, Santa Barbara, CA.</td><td></td><td></td></tr><tr><td>Luer-LokTM syringe.&amp;nbsp;</td><td>BD Biosciences, Franklin Lakes, NJ.</td><td></td><td></td></tr><tr><td>Regen-BCT tube.&amp;nbsp;</td><td>RegenKit; RegenLab, Le Mont-sur-Lausanne, CH.</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>&amp;nbsp;RegenPRP Centri. RegenLab, Le Mont-sur-Lausanne, CH.</td><td></td><td></td></tr><tr><td>BD Venflon Pro Safety 22G x 1.00 inch (0.9 mm x 25 mm).&amp;nbsp;</td><td>BD Biosciences, Franklin Lakes, NJ.</td><td></td><td></td></tr><tr><td>SPSS Statistics Version 19.0</td><td>IBM Corp., Armonk, NY, USA.</td><td></td><td></td></tr><tr><td>Confocal scanning laser ophthalmoscope&amp;nbsp;</td><td>Nidek Inc, Fremont, CA</td><td>Nidek F10&amp;nbsp;</td><td></td></tr><tr><td>Cirrus 5000 Spectral Domain-Optical Coherence Tomography</td><td>Carl Zeiss Meditec AG, Jena, Germany</td><td>&amp;nbsp;SD-OCT&amp;nbsp;</td><td></td></tr><tr><td>Maia 100809 Microperimetry&amp;nbsp;</td><td>CenterVue S.p.A., Padua, Italy</td><td></td><td></td></tr><tr><td>Ocular electrophysiology electromedical system,</td><td>C.S.O., S.r.l., Scandicci, Italy</td><td></td><td>&amp;nbsp;Retimax for ERG&amp;nbsp;</td></tr></tbody></table>

regenerative therapy by suprachoroidal cell autograft in dry age-related macular degeneration: preliminary in vivo report

本研究的目的是检查自体细胞脉络移植是否能提高最佳矫正视力 (BCVA) 和对 microperimetry (我) 的眼睛的反应, 在受干旱年龄相关的黄斑变性 (AMD) 期间通过生长因子 (GFs) 在周围组织的生产和分泌。患者随机分配给每个研究组。所有患者均确诊为干性 AMD, BCVA 等于或大于1对数的最小分辨率 (logMAR)。采用 Limoli 视网膜修复技术 (LRRT) 在 a 组脉络自体移植, 其中包括11例11眼。该技术是通过植入脂肪细胞, 从基质血管分数获得的脂肪干细胞, 以及富含血小板的血浆在脉络空间中的血小板进行的。相反, B 组, 包括14眼14例患者, 被用作对照组。对于每位患者, 通过共焦扫描激光检眼镜和光谱域光学相干层析成像 (SD OCT) 验证诊断。在 A 组, 术后 BCVA 改善0.581 至 0.504 90 天, 0.376 logMAR 180 天 (升 32.20%)。此外, 我的测试在180天内增加了11.44 分贝到12.59 分贝。脉络膜后接枝的不同细胞类型能够保证其在脉络流中的恒定分泌。结果表明, 在六月后, 嫁接组的视力 (VA) 可比对照组增加多。

使用这里提出的程序, 两组的干燥 AMD 受影响的病人, 与 BCVA 等于或大于1对数的最低分辨率 (logMAR), 被登记在该研究。a 组, 包括11眼11例, 接受脉络自体移植 Limoli 视网膜修复技术 (LRRT), 而 B 组, 包括14眼14例患者, 被用作对照组。
学生的 t 检验和卡方测试分别用于比较两个研究组之间的平均年龄和性别分布 (表 2)。LRRT ...

Watch this Scientific Journal Video about Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report at JoVE.com

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

脉络细胞自体移植治疗老年性黄斑变性的再生疗法: 初步体内报告

本研究的目的是评估脉络移植的脂肪干细胞是否包含在基质血管和血小板丰富的血浆 Limoli 视网膜恢复技术, 可以提高视力和老年黄斑变性患者眼内视网膜敏感反应的影响。

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

regenerative-therapy-suprachoroidal-cell-autograft-dry-age-related

Research

JoVE Journal

Medicine

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

10.1K 次观看.  Low Vision Research Centre of Milan. 本研究的目的是评估脉络移植的脂肪干细胞是否包含在基质血管和血小板丰富的血浆 Limoli 视网膜恢复技术, 可以提高视力和老年黄斑变性患者眼内视网膜敏感反应的影响。

视频: Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

科研

医学

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

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

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

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

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

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.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

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 ...

神经科学

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.

一种用于兔视网膜下手术分步协议

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

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.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

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

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.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

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

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.

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

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.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

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

生物学

焕活视力:干细胞疗法治疗视网膜变性

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.

作者聚焦:推进视力恢复 - 基于干细胞的视网膜疾病疗法 

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小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送

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