我们要感谢阿南德·斯瓦鲁普为Nrl-GFP小鼠、乔琴·哈斯提供技术支持，以及辛迪·布姆和埃米利·莱斯曼提供畜牧业。	
	这项工作得到了德国福松斯格梅因沙夫特（DFG）：FZT 111 - 德累斯顿再生疗法中心、CRTD种子赠款计划、SFB 655和普罗雷蒂娜e.V.的支持。 基金会、DIGS-BB德累斯顿研究生课程和特克诺洛尼亚基金会（SFRH/BD/60787/2009）

动物的道德使用和护理声明：
所有动物实验都是严格按照欧盟和德国法律进行的，并遵守了《关于在眼科和视觉研究中使用动物的ARVO声明》。所有动物实验均得到德累斯顿和德累斯顿陆地动物伦理委员会的批准（批准编号：24D-9168.11-1/2008-33）。
1. 在开始细胞分离和细胞排序之前
<ol><li>标记三个 15 毫升反应管：洗涤 （W）、正分 （+） 和负分数 （-）。</li>
</ol>2. 视网膜分离
<ol><li>用剪刀斩首PN 4幼崽。</li>
	<li>不久后用70%的乙醇冲洗幼崽的头部，然后用PBS清洗。</li>
	<li>将头部转移到寒冷的 HBSS 并诱导眼睛（为所有头部重复此步骤）。要进行诱导，打开眼睑，在视神经区域用弯曲的钳子固定眼睛。然后小心地把眼睛从轨道上拉出来。</li>
	<li>在诱导所有眼睛后，隔离视网膜：将封闭的剪刀引入视神经并打开刀片。将 RPE/乔里德剔除。使用弯曲的钳子取出镜片和血管。</li>
	<li>将分离的视网膜转移到含有木瓜溶液的 1.5 毫升反应管中（由帕帕因分离套件提供）。</li>
	<li>在37°C的水浴或摇床（400 rpm）中将视网膜孵化在木瓜溶液中30-60分钟。<ol>
<li>当视网膜在帕潘溶液中孵化时，移液器 1 毫升卵巢状溶液到 15 毫升反应管（标签"1"）。</li>
			<li>将 60μl 的 DNase I （10 毫克/毫升） = 60μl 的卵巢溶液（由套件提供） + 520 μl 的 EBSS 添加到 15 毫升反应管（标签"2"）。</li>
		</ol>
</li>
	<li>在木瓜孵育后，将含有部分消化视网膜的木瓜溶液转移到反应管"2"。</li>
	<li>使用火抛光移液器，执行机械分离（上下 10 倍）。</li>
	<li>派珀特单细胞悬架反应管"1"。尝试通过将细胞悬架轻轻分层到卵巢溶液顶部来生成 2 层。</li>
	<li>离心机在300 x g（约1，300 rpm）下5分钟。</li>
	<li>丢弃超自然物，在 500 μl 的 MACS 缓冲器中重新补充细胞。</li>
</ol>3. 使用磁相关细胞排序 （MACS） 的单元格排序
<ol><li>将 X μl 大鼠抗 CD73 抗体添加到 500 μl 中，以达到 10 μg/ml 的最终浓度。</li>
	<li>在冰上孵化5分钟。</li>
	<li>用 MACS 缓冲器将 15 毫升反应管填充到 10 毫升，以 300 x g 的速度将其离心机进行 5 分钟。</li>
	<li>去除超自然。</li>
	<li>将颗粒重新插入 480μl MACS 缓冲区，并添加 120 μl 山羊抗鼠 IgG 微珠。</li>
	<li>在冰上孵化15分钟;不要搅拌、摇动或混合。</li>
	<li>将 15 毫升反应管填充到 5 毫升，用 MACS 缓冲器和离心机在 300 x g 下进行 5 分钟。</li>
	<li>由于反应管已离心：<ol>
<li>将LS柱调整到磁性支架中。</li>
			<li>将预分离过滤器放在 LS 列的顶部。</li>
			<li>用 3 毫升 MACS 缓冲器将预分离滤镜和 LS 柱水合，并将 MACS 缓冲器收集到洗涤 （W） 管中。</li>
		</ol>
</li>
	<li>去除超自然。</li>
	<li>将颗粒重新喷入 500μl MACS 缓冲区。</li>
	<li>将 500 μl 细胞悬架加载到过滤器中，然后将 1 毫升 MACS 缓冲器添加到过滤器中，并将负分数收集到负分数 （-） 反应管中。</li>
	<li>接下来，在列中添加 3 x 3 毫升 MACS 缓冲器，以洗掉不绑定到列的单元</li>
	<li>一旦这 9 毫升通过 LS 列，从磁性支架上取出列并将其安装在正分 （+） 反应管的顶部。</li>
	<li>快速将 5 毫升 MACS 缓冲器加载到 LS 列中，并将柱塞放入 LS 列中，并按下它，直到整个缓冲区通过柱。</li>
	<li>在 300 x g 下，将包含正分的反应管离心 5 分钟。</li>
	<li>取出超自然，在 500 μl 的 MACS 缓冲器中重新喷出，并保持在 4 °C。</li>
	<li>使用普通的细胞计数设备计算细胞总量。</li>
	<li>准备从MACS缓冲区中含有2 x 105 个细胞/μl的正分拣分数的细胞悬架，并保持在4°C</li>
</ol>4. 将MAC排序的光感受器前体细胞移植到小鼠网膜中
<ol><li>请务必准备好以下所有解决方案：1 毫升无菌 PBS 或 HBSS，10 μl DNAse I，1-2 毫升无菌除离子 H2O，细胞悬浮分数在 4 °C 的别名。</li>
	<li>麻醉成年小鼠（即2-4个月大），注射盐酸甲基二胺（0.01毫克/10克体重），氯胺酮（0.75毫克/10克体重）。然后做皮下注射丁丙诺啡（0.05毫克/公斤体重）以减轻疼痛。</li>
	<li>扩张学生与苯甲肾上腺素下降 2.5% - 热带 0.5% 。</li>
	<li>将鼠标固定在鼠标头架中，放在立体显微镜下。</li>
	<li>涂抹一滴维西迪凝胶，防止眼睛干燥。</li>
	<li>使用无菌的30G 1/2针在硬膜和角膜（分别 奥拉塞拉塔）之间的边界上打一个小洞。</li>
	<li>使用钻石笔将普通盖滑成小块（约 5 mm x 5 mm），将其中一块放在角膜顶部，从而可直接可视视视。</li>
	<li>用除离子水多次冲洗预制的微滑器注射器。</li>
	<li>用1μl的细胞悬浮加载微升注射器，通过结膜和硬膜直接针头切线，并在视网膜的鼻部置于视觉控制之下。</li>
	<li>轻轻将一个孔打入视网膜，直到到达亚视网膜空间，将细胞悬浮注入子视网膜空间。</li>
	<li>观察视网膜的牛皮分离，这应该是均匀的，覆盖大约1/4的视网膜空间，没有任何出血。</li>
	<li>轻轻取出注射器，视网膜孔自动密封。</li>
	<li>用除离子水多次冲洗微滑器注射器。</li>
	<li>从头架上释放鼠标。</li>
	<li>通过注射盐酸甲酰胺唤醒小鼠，以逆转盐酸甲基二甲基二甲烷的作用。</li>
	<li>将鼠标置于温暖的暗室（约 25 °C）中，以唤醒时间。</li>
	<li>移植后2天，布丙诺啡（0.05毫克/千克体重）应在上午和下午进行皮下注射，以缓解疼痛。</li>
</ol>

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+repair+by+transplantation+of+photoreceptor+precursors.">Retinal repair by transplantation of photoreceptor precursors.</a> Nature. 444, 203-207 (2006).</li><li>Eberle, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+segment+formation+of+transplanted+photoreceptor+precursor+cells.">Outer segment formation of transplanted photoreceptor precursor cells.</a> PLoS One. 7, (2012).</li><li>Lakowski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+photoreceptor+transplantation+in+models+of+the+childhood+retinopathy+Leber+congenital+amaurosis+using+flow-sorted+Crx-positive+donor+cells.">Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells.</a> Hum. Mol. Genet. 19, 4545-4559 (2010).</li><li>Barber, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+the+degenerate+retina+by+photoreceptor+transplantation.">Repair of the degenerate retina by photoreceptor transplantation.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 354-359 (2013).</li><li>Pearson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+vision+after+transplantation+of+photoreceptors.">Restoration of vision after transplantation of photoreceptors.</a> Nature. , (2012).</li><li>Singh, M. 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作者声明没有相互竞争的经济利益。

光感受器前体细胞的亚视移植是实现这些光敏细胞在大量1，2中融入宿主视网膜的可靠工具。这可能使建立一个细胞疗法，用于治疗视网膜退行性疾病在未来6。细胞的供体群，目前从PN 4视网膜分离，是不同细胞类型的混合物，只有光感受器前体细胞在亚视网膜注射后整合。通过使用基于CD73的MAC排序，供体细胞悬架中的光感受器比例可以增加到≈90%，与未分类的细胞群8相比，野生型宿主?...

视觉是人类的主要感觉之一。这种意识的损害和失明是工业化国家残疾的主要原因之一。视力障碍或失明的主要原因是视网膜退化，其特征是光感受器细胞丧失，因为它可以在黄斑变性、视网膜炎色素瘤、锥形杆营养不良和其他疾病中观察到。迄今为止，还没有有效的治疗方法来恢复视力丧失。2006年和2008年，两个不同的实验室分别报告，成功将杆光感受器前体细胞移植到成年野生型小鼠视网膜1，2。因此，产生光感受器前体细胞移植的可能性也到退化视网膜，以取代退化的光感受器和恢复视力。事实上，最近已经证明，这种移植的光感受器前体细胞引起成熟野生型光感受器的形态学标准，如正确开发的外段3、靠近内源性双极细胞的突触终端和位于外核层2-4的圆形细胞体，以及功能集成到主动脉电路5-7的能力。这一策略的主要原则之一是使用产后第4天（PN 4，PN0定义为出生日）幼鼠视网膜，从而产生不同细胞类型的混合移植。在未来治疗应用的背景下，这种混合物必须纯化为光感受器前体细胞。CD73被描述为第一个细胞表面标记，专门为年轻的光感受器在网膜8-10。在这里，我们演示了基于此细胞表面标记的光感知器前体细胞净化方法，并采用了磁相关细胞分拣 （MACS） 技术。与荧光激活的细胞分拣技术相比，MACS 可能有优势，因为分拣时间快，对 GMP 条件的调整也更容易。当将富集的人口移植到成人野生型视网膜的亚视网膜空间时，我们可以证明其富足度为 90%，融合率高达 3 倍。因此，基于MACS的光感受器前体细胞富集和亚视网膜移植，是开发视网膜退化治疗再生治疗策略的可靠和有前途的技术。

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Papain Dissociation System</td>
			<td style="width:111px;">Worthington&#160; Biochemical Corporation</td>
			<td style="width:103px;">LK003150</td>
			<td style="width:176px;">supplied DNase I is not used in the method</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">purified rat anti-mouse CD73, clone TY/23</td>
			<td style="width:111px;">BD Pharmingen</td>
			<td style="width:103px;">550738</td>
			<td style="width:176px;">Stock concentration 0.5mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Goat Anti-Rat IgG MicroBeads</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-048-501</td>
			<td style="width:176px;">Total volume of 2ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">PBS</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">10010-015</td>
			<td style="width:176px;">Used to count the total number of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">DNase I</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">D5025-150KU</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">HBSS</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">14025050</td>
			<td style="width:176px;">Used for dissociation of the retinas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Trypan blue</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">Fluka93595</td>
			<td style="width:176px;">Used to count the total number of cells</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:195px;">Vidisic</td>
			<td style="width:111px;">Dr. Mann Pharma / Andreae-Noris Zahn AG</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Domitor</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:103px;">76579</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Ketamin 10%</td>
			<td style="width:111px;">Ratiopharm</td>
			<td style="width:103px;">7538843</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Antisedan</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:103px;">76590</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:195px;">Phenylephrin 2.5%-Tropicamid 0.5%</td>
			<td style="width:111px;">University clinics Dresden pharmacy</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Name of Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:176px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Pre-Separation Filters</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-041-407</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">LS Columns</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-042-401</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">MACS MultiStand</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-042-303</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">QuadroMACS Separator</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-090-976</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">fire polish glass pasteur pipette</td>
			<td style="width:111px;">Brand</td>
			<td style="width:103px;">74777 20</td>
			<td style="width:176px;">The pipette&#8217;s tips need to be fire-polished and autoclaved.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">MACS 15ml tube rack</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-091-052</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Cell count chamber</td>
			<td style="width:111px;">Carl Roth</td>
			<td style="width:103px;">T728.1</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Sterile 15ml tubes</td>
			<td style="width:111px;">Greiner Bio-one</td>
			<td style="width:103px;">188271</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Leica M651 MSD</td>
			<td style="width:111px;">Leica</td>
			<td style="width:103px;">M651 MSD</td>
			<td style="width:176px;">can be used instead of Olympus SZX10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Olympus SZX10</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:103px;">SZX10</td>
			<td style="width:176px;">can be used instead of Leica M651 MSD</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Olympus inverted stereo microscope CKX41</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:103px;">CKX41</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Cell culture hood Thermo Scientific MSC-Advance</td>
			<td style="width:111px;">Thermo scientific</td>
			<td style="width:103px;">51025411</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">1.5ml reaction tube</td>
			<td style="width:111px;">Sarstedt</td>
			<td style="width:103px;">727706400</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">2ml reaction tube</td>
			<td style="width:111px;">Sarstedt</td>
			<td style="width:103px;">72695</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Eppendorf Centrifuge 5702</td>
			<td style="width:111px;">VWR (Eppendorf)</td>
			<td style="width:103px;">521-0733</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Mouse head holder</td>
			<td style="width:111px;">myNeurolab</td>
			<td style="width:103px;">471030</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">BD Microlance 3 30G 1/2&#8221;</td>
			<td style="width:111px;">BD Pharmingen</td>
			<td style="width:103px;">304000</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Hamilton microliter syringe 5&#181;l, 75RN</td>
			<td style="width:111px;">Hamilton</td>
			<td style="width:103px;">065-7634-01</td>
			<td style="width:176px;">delivered without needles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Hamilton RN special needle GA34</td>
			<td style="width:111px;">Hamilton</td>
			<td style="width:103px;">065-207434</td>
			<td style="width:176px;">Blunt, 12mm length</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Vannas-T&#252;bingen Spring Scissors - 5mm Blades Straight</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">15003-08</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Dumont #7 Forceps - Titanium Biologie</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">11272-40</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Diamond pen</td>
			<td style="width:111px;">Tools-tech</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">15x15mm Cover slips</td>
			<td style="width:111px;">Sparks</td>
			<td style="width:103px;">MIC3366</td>
			<td style="width:176px;">-</td>
		</tr>
	</tbody>
</table>

subretinal transplantation of macs purified photoreceptor precursor cells into the adult mouse retina

视网膜光感细胞（ 即 光感受器）的丧失导致视力障碍和失明，是工业化国家残疾的主要原因。通过细胞移植取代退化的光感受器是未来临床应用中一种可能的治疗选择。事实上，最近的临床前研究表明，在产后第4天从新生儿小鼠视网膜中分离出来的不成熟的光感受器，在亚视网膜移植后有可能融入成年小鼠视网膜。供体细胞产生成熟的光感受器形态，包括内段和外段、位于外核层的圆形细胞体和靠近内源性双极细胞的突触终端。事实上，最近的报告显示，供体光感受器在功能上融入宿主小鼠的神经回路。对于这种细胞替代方法的未来临床应用，必须生成选择细胞的纯化悬浮，并放置在正确的位置，以便正确地融入眼睛。对于光感受器前体的丰富，排序应基于特定的细胞表面抗原，以避免遗传报告器对供体细胞进行改造。在这里，我们显示磁相关细胞分拣 （MACS） - 丰富可移植棒光感受器前体分离出的新生儿网膜的光感受器特异性记者小鼠基于细胞表面标记CD73。与抗CD73抗体的孵化，以及微珠结合的二次抗体，使MACS的棒光感受器前体的丰富度接近90%。与流动细胞学相比，MACS 的优势在于它可以更容易地应用于 GMP 标准，并且可以在相对较短的时间内对大量细胞进行排序。将富集细胞悬浮液注入成年野生型小鼠的亚视空间，与未分类的细胞悬浮相比，整合率高3倍。

为了评估棒光感受器集成到小鼠网状网膜的能力，使用了鼠标报告线，其中GFP由神经网状网膜白素拉链（Nrl，Nrl-GFP）促进器11驱动。Nrl 是杆光感受器的最早标记，在整个成年期间从 E12.5 开始表达，允许对供体杆光感受器细胞进行特定标记。
PN 4 Nrl-GFP幼崽被斩首，眼睛被引诱。然后，使用上述方法隔离和分离视网膜。然后使用基于 CD73 的 MACS（图 1和<strong...

Watch this Scientific Journal Video about Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina at JoVE.com

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

细胞移植是治疗视网膜退化的一种策略，其特征是光感受器丧失。在这里，我们描述了一种丰富可移植光感受器及其亚视移植到成年小鼠的方法。

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 ...

subretinal-transplantation-macs-purified-photoreceptor-precursor

Research

JoVE Journal

Medicine

10.6K 次观看.  Technische Universität Dresden. 细胞移植是治疗视网膜退化的一种策略，其特征是光感受器丧失。在这里，我们描述了一种丰富可移植光感受器及其亚视移植到成年小鼠的方法。

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

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.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

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 ...

科研

医学

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

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

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

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

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

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

鼠肾移植：移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

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 ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

原位后肢移植中的鼠标

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

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 ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

腹部小鼠心脏移植模型中的血液回路重建

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...