该项目由德国研究协会（DFG，何4578/3-1）资助。

A.软骨活检（外科室;步骤1-5在非无菌制剂室） <ol><li>执行最终控制体重的兔子（新西兰白兔，女性，体重3.5-4.0公斤，6个月大），以便能够正确剂量的药物，并监测手术后的重量。 </li><li>兔静脉注射10毫克/公斤的丙泊酚麻醉。 </li><li>插管后，1.5毫克/公斤/分钟异丙酚和0.05毫克/千克静脉注射芬太尼维持麻醉。使用二氧化碳监测，脉搏血氧饱和度和脉率监测麻醉。 </li><li>剃须用电动推剪和真空皮草膝盖上进行操作。 </li><li>剃了膝盖彻底消毒，并用无菌敷料覆盖，其余的兔子。 </li><li>触诊髌骨与髌骨内侧进行皮肤切口。 </li><li>打开膝关节内侧髌骨关节切开，在无菌条件下。尽量避免切割任何小苏perficial血管。 </li><li>横向置换髌骨。 </li><li>检查任何异常的膝关节和宏观软骨病变。 </li><li>小心剥离出滑车ossis的股二头肌的小软骨片（2-3毫米）用无菌手术刀（ 图1）。 </li><li>立即将这些软骨活检到50毫升管与完全培养基（DMEM + 10％新生小牛血清（FCS）的+ 1％青霉素/链霉素（青/链霉素））的操作后，继续在体外培养权利。 </li><li>清洁的缺陷，他们用无菌生理盐水冲洗。 </li><li>重新髌骨内滑车槽。 </li><li>伤口缝合在单纽扣缝线（4-0薇乔）和持续皮肤缝合（4-0 MONOCRYL）层。使用可吸收缝合材料。 </li><li>最后，密封伤口，喷雾敷料可渗透水蒸汽。 </li></ol> B. 在体外培养<ol><li>处理的活检Cartilage件尽快在无菌层流手术后的组织文化引擎盖。 </li><li>在500×g离心3分钟，在RT下，洗净无菌收获的软骨2倍的PBS中，然后离心。 </li><li>剪切软骨件为1毫米3，转移到50ml隼，在振荡器上30分钟，用10 ml的胰蛋白酶-EDTA（0.25％）和10毫升PBS和消化30分钟</li><li> 30分钟后，停止加入完全培养基胰蛋白酶。然后，摇动另一软骨片胶原酶A（0.21 U /毫克）的无血清培养基（DMEM + 1％PEN /链球菌）12小时。 </li><li>离心得到的溶液，在170×g离心3分钟，在RT。 </li><li>在5毫升完全培养基中重悬细胞沉淀。 </li><li>种子的25cm 2的组织培养瓶中，在5毫升的完整的介质中，并保持在潮湿的细胞培养箱在37℃下，5％CO 2的分离软骨细胞。 </li><li>软骨细胞的生长密度为80％。 </li><li>释放的细胞F罗烧瓶洗涤暴露于0.05％胰蛋白酶-EDTA 3分钟前，在PBS中2倍。一旦软骨细胞分离，停止胰蛋白酶，加入10微升的完全培养基。 </li><li>离心悬浮在300 XG（3分钟）和完全培养基重悬沉淀。 </li><li>在组织培养瓶（75厘米2）和1:3的比例（〜5000个细胞/ cm 2），每5天或80％汇合后，分割的种子细胞。 </li><li>台盼蓝染色确定细胞的数量和活力。 </li></ol> C.细胞播种矩阵<ol><li>注入洗涤和释放细胞之前，如上所述。进行细胞计数和5×10 4细胞转移到1.5毫升离心管中。然后在500 XG离心5分钟，在室温。 </li><li>矩阵为一个完整的饱和度的15微升的完全培养基中重悬沉淀。 </li><li>切的双层胶原蛋白支架滑车缺损的确切尺寸的无菌活检一拳。 </li><li>将基体置于适当大小的组织培养皿中（ 如 24孔板）。 </li><li>到多孔的，细胞基体的粘接剂侧的种子细胞。这阻碍了细胞迁移，相差的矩阵。 </li><li>留下种子细胞矩阵没有进一步的细胞培养液中，1小时内的孵化器，，允许完全遵守的软骨细胞。 </li><li>然后，细胞-基质构造成新鲜的完全培养基的培养容器（ 如 24孔板）与。小心不要洗出任何基质细胞脱落，即使预期附着力强。 </li><li>现在，细胞 - 基质的结构可以采取重新植入手术的空间。 </li></ol> D.矩阵辅助自体软骨细胞移植<ol><li>执行（A. 1-8）如上所述对侧膝髌骨关节切开。 </li><li>创建两个隔离软骨缺损用无菌空气业庭在滑车沟G电源钻（直径3.6毫米）。 </li><li>清洁的缺陷，并用无菌生理盐水冲洗。 </li><li>应注意，在任何时候不开骨髓腔。如果有必要，密封的底部与电烙术的缺陷，防止出血。 </li><li>植入与多孔面朝下（ 图2）与培养的软骨细胞接种的矩阵。然后压入钻孔冲洗周围表面。 </li><li>带着一点点的纤维蛋白胶（Tissucol铎S），以确保安全可靠固定密封植入矩阵。 </li><li>凝固后，滑车沟搬迁髌骨内全方位的运动，并应用到膝关节几次。 </li><li>横向置换髌骨再次检查任何事件或固定不稳定的迹象。矩阵必须持有还是很到位的。 </li><li>再次更换髌骨层和喷雾穿衣如上所述做完手术伤口缝合。 </li></ol>

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什么都没有透露。

该协议提供了一个建立了9,12,13和容易复制的技术，人为制造的兔子膝盖软骨缺损随后的增殖和再植入自体软骨细胞隔离。使用自体软骨细胞的重构和修复关节软骨病变已经在临床使用中提供满意的长期效果（6）。 例如骨膜肥厚和钙化的主要问题，移植物剥离，或与第一代和第二代的自体软骨细胞植入14发生的供体部位的发病率。因此，研究人员已经?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom AG</td>
			<td>F 0415</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td>0.21 U/mg</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>PAN Biotech GmbH</td>
			<td>3702-P103009</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2213</td>
			<td>10,000 U/ml/10,000 &mu;g/ml</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KgaA</td>
			<td>410230</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %/0.02 %</td>
			<td>Biochrom AG</td>
			<td>L2163</td>
			<td>in PBS w/o Ca2+, Mg2+</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Delta Select GmBH</td>
			<td>1819340</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>Bbraun</td>
			<td>8333A193</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes 100 mm/150 mm</td>
			<td>TPP AG</td>
			<td>93100/93150</td>
			<td>Growth area 60.1 mm2/147.8 mm2</td>
		</tr>
		<tr>
			<td>Tissue culture flasks 25/75 mm2</td>
			<td>TPP AG</td>
			<td>90025/90075</td>
			<td>25 mm2, 75 mm2</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>L8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
		<tr>
			<td>Chondro-Gide&Ograve;</td>
			<td>Geistlich Pharma AG</td>
			<td>30915.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>pfm medical ag</td>
			<td>48351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissucol Duo S</td>
			<td>Baxter</td>
			<td>3419627</td>
			<td>0.5 ml</td>
		</tr>
	</tbody>
</table>

matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model

关节软骨缺损被认为是一个主要的健康问题，因为关节软骨具有自我再生能力是有限的。未处理的软骨病变导致持续的疼痛，生活质量造成不利影响，并易患骨关节炎。在过去的几十年中，一些外科技术已被开发来治疗这种病变。然而，到现在为止，这是不可能实现了全覆盖肺透明关节软骨缺损或提供令人满意的长期恢复2-4维修。因此，关节软骨损伤仍然再生技术，如组织工程的首要目标。相反，其他外科手术技术，这往往导致纤维或纤维软骨组织的形成，组织工程的目的是充分恢复原来的关节软骨的复杂结构和性能的影响通过使用诱导成软骨细胞的移植细胞的潜力。 ŗ最近几个发展开辟了前景的可能性再生软骨疗法。 第一个单元格为基础的全层软骨或骨软骨病变的治疗方法是在1994年由Lars彼得森和Mats Brittberg的的谁率先临床自体软骨细胞移植（ACI）5。如今，该技术在临床上以及建立大型的膝盖软骨缺损的治疗，植入后6甚至10至20年中保持了良好的临床效果。近年来，自体软骨细胞植入经历了快速进展。使用一个人工的三维胶原基质上的细胞，其后再植变得越来越流行7-9。 MACT包括两个手术:首先，为了收集软骨细胞，软骨活检需要执行从非负重软骨区的t他的膝关节。然后，软骨细胞的提取，纯化，并扩展到足够的细胞数量的影响 。软骨细胞，然后接种到一个三维矩阵，并随后可以被重新注入。当准备植入组织工程，增殖率和分化的能力是至关重要的一个成功的组织再生10。一个三维基质作为细胞载体的使用被认为是支持这些细胞的特征11。 以下协议将总结并展示一种技术，用于隔离的软骨细胞软骨活检，其在体外增殖和他们的播种到一个三维矩阵（ 软骨纪德 ，Geistlich生物材料，Wollhusen，瑞士）。最后，植入到人工创造的兔子的膝关节软骨缺损的细胞和基质的构建进行说明。这种技术可以用作一个实验设定进一步的实验中软骨修复。

手术技术允许一个成功的隔离和自体软骨细胞植入到人工软骨缺损。实验装置的植入导致一个成功的整合到周围的软骨。 12周后， 在体内的 ，均匀的和完整的表面，从而减少了到植入物（ 图4）的剪切应力和损坏软骨缺损修复组织填充。此外，没有肥厚或钙化的植入物被看见了。修复组织呈僵硬和坚实的质量，这是与周围健康软骨组织。这是一个重要的?...

Watch this Scientific Journal Video about Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model at JoVE.com

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

用于治疗在兔子的膝关节软骨缺损的实验技术进行说明。在矩阵上的自体软骨细胞植入是被广泛接受的方法重塑和修复关节软骨病变提供满意的长期效果。基质辅助自体软骨细胞移植（MACT）提供一个标准化的临床植入方法。

基质辅助重塑和修复软骨缺损的自体软骨细胞移植在兔模型

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

matrix-assisted-autologous-chondrocyte-transplantation-for-remodeling

Research

JoVE Journal

Medicine

14.3K 次观看.  Klinikum rechts der Isar der Technischen Universität München. 用于治疗在兔子的膝关节软骨缺损的实验技术进行说明。在矩阵上的自体软骨细胞植入是被广泛接受的方法重塑和修复关节软骨病变提供满意的长期效果。基质辅助自体软骨细胞移植（MACT）提供一个标准化的临床植入方法。

视频: Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

闭塞性细支气管炎在原位肺移植小鼠模型的发展

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

大鼠颈动脉球囊损伤模型试验抗血管重建治疗

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

心脏重构通过大鼠腹主动脉缩窄的模型

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

猪自体移植肝细胞外细胞外细胞基因治疗

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

兔子模型中的跨胸超声心动图检查

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

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 ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.