Name: intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model
Uploaded: 2020-09-20T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

这项研究由韩国教育部资助的韩国国家研究基金会（NRF-2018R1D1A1A02049346）基础科学研究计划支持。

所有动物研究程序都是根据《实验室动物福利法》、《实验室动物照料和使用指南》以及韩国天主教大学医学院机构动物护理和使用委员会（IACUC）提供的啮齿动物实验指南和政策提供的。
1. MSC 和注射明胶水凝胶的准备
<ol><li>100 毫米培养皿中的培养 MSC，37 °C 和 5% CO2。当 MSC 增长达到 80% 的汇合时，用 DPBS 洗两次菜，并在 37 °C 下加入 1 mL 的三蛋白替代剂，持续 3 分钟。 注:MSC在常规程序16后从骨髓中分离出，在杜尔贝科的改良鹰介质（DMEM）中培养，含有10%的胎儿牛血清（FBS）和1%的抗生素-抗霉菌溶液，并在7-u20129通道之间用于这项研究。</li>
	<li>加入9 mL的文化介质和离心机在500 x g 3分钟。接下来，丢弃由此产生的超常剂，在PBS的1mL中重新悬浮细胞，并在冰上保持细胞悬浮。</li>
	<li>用 10 μL 的 Trypan 蓝色稀释 10 μL 的细胞悬架，并使用自动电池计数器获得细胞浓度。</li>
	<li>以 1 x 107 细胞/mL 的密度将 MSC 重新悬浮并传输到 1 mL 管中。</li>
	<li>在PBS中准备 6.25 wt% 的 GH 结合解决方案，并分离成 2 瓶。接下来，将 GH 解决方案与 HRP （GH 解决方案 A） 的 6 微克/mL 或 H2O 2（GH 溶液 B） 的 0.07 wt% 混合。 注:根据已公布的协议12、15，准备明胶-羟基丙烯酸（GH）结合。<ol>
<li>将GH结合溶液与HRP（GH溶液A）的容积比保持在9:1，将GH结合溶液分别保持在H 2 O2（GH溶液B）。</li>
	</ol>
</li>
	<li>在将 MSC 与 GH 溶液 A 混合之前，将电池悬架短暂离心至 1，000 x g， 并小心地吸气由此产生的超纳坦。随后，将含有MSC的颗粒与GH溶液A混合。</li>
</ol>2. 原位MSC加载和三维体外培养
<ol><li>将GH溶液A（包含MSC）和GH溶液B加载到双注射器的两侧。板 300μL 的组合 GH 解决方案与 MSC 在最终密度为 5 x 106 细胞/mL 到八井室幻灯片。</li>
	<li>通过酶交叉连接进行原位水凝胶形成和随后的MSC封装后，加入含有10%FBS和1%抗生素-抗霉溶液的700μL DMEM。</li>
	<li>在 37 °C 和 5% CO2 下孵化幻灯片，每 2+u20123 天更换一次培养介质。</li>
</ol>3. 确认GH水凝胶内MSC的体外增殖和生存
<ol><li>要确定 GH 水凝胶中 3D 培养的 MSC 的可行性，请在预定的潜伏时间后使用活/死细胞染色检测。</li>
	<li>在 GH 水凝胶中孵化封装的 MSC 3、5、7 或 14 天后，吸气介质，用 PBS 两次清洗油井。</li>
	<li>在 10 mL 的 DPBS 中准备含有 5 μL 钙蛋白 AM 和 20 μL 同质-1 （EthD-1） 的染色溶液。</li>
	<li>将 200μL 的染色溶液添加到井中，并在室温下在黑暗中孵育 30 分钟。</li>
	<li>吸气染色溶液，用PBS洗两次井。</li>
	<li>小心地将腔室与滑梯分开，并在 GH 水凝胶上放置一个完整的盖片。使用共生显微镜可视化封装 MSC 的增殖程度和形态变化。 注:荧光图像是在 200 倍放大倍数下获得的，图像的激发/发射波长为 470/540 nm 用于钙素，EthD-1 获得 516/607 nm。</li>
</ol>4. 小鼠心肌梗塞的诱导
<ol><li>麻醉7周大的雄性C57BL/6小鼠（20+u201222g），在盐水中注射Zoletil（30毫克/千克）和龙朋（10毫克/千克）的混合物。</li>
	<li>手术前，使用脱毛霜去除小鼠胸部，用碘对皮肤进行消毒。</li>
	<li>将鼠标放在手术台上，通过将导管插入气管以通过机械通风提供补充氧气进行插管。</li>
	<li>用手术剪刀轻轻切开皮肤，然后用微剪刀穿透间切肌肉。使用 5-0 丝缝将第二根和第三根左肋分开，以保持胸腔开放。</li>
	<li>使用 8 - 0 的针架小心地解放左前下降 （LAD） 冠状动脉聚丙烯缝合，并使用电宫切割缝合。</li>
	<li>观察前左心室壁的即时颜色变化。</li>
</ol>5. MSC装载GH水凝胶的心肌内移植
<ol><li>在通过 LAD 结扎诱导心肌梗塞后，使用配备 26G 针头的双注射器将 10μL 的 MSC 加载 GH 溶液注入到梗塞边界区的两个不同点（总计:2 x10 5 MSC/20 μL）。<ol>
<li>按照步骤 1 中描述的相同程序，准备并将 MSC 加载 GH 解决方案转移到双注射器中。 注:为了评估在梗塞区域内雕刻的MSC装载GH水凝胶，MSC和GH结合体分别用PHK26和荧光异氰酸酯（FITC）预先标记。</li>
	</ol>
</li>
	<li>恢复开放的胸腔，并关闭肌肉和皮肤使用5-0缝合。 注:在关闭胸部之前，使用导管注射器取出空气。</li>
	<li>取出气管，在恢复过程中将鼠标放在红外灯下的笼子里。</li>
	<li>对于术后镇痛，至少72小时进行皮下酮洛芬注射（每天5毫克/千克）。所有小鼠都应在适当时间受到密切监测，以确保手术后恢复良好，并进行适当的疼痛治疗。</li>
</ol>6. 回声心动图
<ol><li>移植后四周，先用5%的异氟化麻醉小鼠，然后将异氟浓度调整到1%。</li>
	<li>使用脱毛霜脱毛胸部，并将鼠标放在加热垫上。将超声波传感器凝胶涂在胸前。</li>
	<li>获取二维寄生短轴视图，并在肌水平记录 M 模式跟踪。 注:将线性阵列传感器（7+u201215 MHz）放在左副星线中，查看解剖结构。</li>
	<li>测量LVAW、LVID和LVPW的相应线条，以获得心脏壁厚度、腔室尺寸和部分缩短。 注:比较心脏功能，包括弹出分数（ EF ）、分数缩短（ FS ）和肌水平的末代收缩体积（ ESV ），以确保在同一解剖位置进行适当的评估。</li>
</ol>7. 病理学评价
<ol><li>在将MSC装载的GH水凝胶移植到梗塞的心脏后预定时间，在CO2室中对小鼠实施安乐死，并收集心脏进行组织学分析15。</li>
	<li>对于血氧素和欧辛（H&E）和马森的三叶虫（MT）染色，修复解剖的心脏组织在4%的甲醛（PFA），并嵌入石蜡。接下来，根据标准协议17，将石蜡嵌入的心脏块切割成4个μm串行部分，并用微切除物和MT污渍染色。</li>
	<li>以 20 倍的放大率在幻灯片扫描仪上获取图像，并计算治疗组的梗死大小。 梗死大小（%）=总梗塞周长/总LV周长x 100</li>
	<li>按中线长度测量计算两个周长。对于LV中线周长，测量心内膜和震中表面之间的中线长度。对于中线梗塞周长，测量梗塞的长度，包括超过50%的肌心18的整体厚度。 注:所有图像分析都是使用 ImageJ 软件进行的。</li>
	<li>测量肌肉水平疤痕的壁厚度。</li>
	<li>计算胶原蛋白面积的分数。 胶原蛋白面积 （%） = 间质纤维化/肌细胞面积总面积 x 100</li>
</ol>

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作者对这部作品没有利益冲突。

可注射的GH水凝胶具有巨大的潜力，在体内应用，因为他们的能力，均匀地纳入不同的治疗剂就地。此外，它们的物理和生化特性很容易根据疾病依赖性要求进行操纵。在这方面，已建议注射水凝胶，以解决目前心脏干细胞治疗的主要局限性，阻碍不良生存和细胞保留（即+10%在24小时内移植后）在受伤的心脏19，20。为了克服这一不良结果，本文所述的协议提供了一种简单可行的方法，用于使用心肌移植后可就地交叉连接的GH水凝胶来改善细胞的保留和存活，这显示了在Murine MI模型中对心脏结构和功能的有利影响。
该技术的主要优点是其广泛的活性适用性与任何类型的细胞和生物分子，这可以通过简单地与注射前的预凝胶GH溶液混合加载。此外，为了全面了解捐赠者与宿主之间的相互作用，可以调整GH结合体和/或封装生物分子的简单标记方法，以跟踪其体内稳定性、宿主集成和吸收动力学的变化。据我们所知，使用注射明胶为基础的水凝胶与治疗干细胞相结合，是首次验证心脏组织在体外和体内的恢复性潜力。
在本研究的当前阶段，装载 MSC、注射和异地交叉链接的 GH 水凝胶被用作概念证明，以评估其在 Murine MI 模型中的适用性。虽然这种方法似乎改善了移植心脏组织的MSC雕刻和保留，但应考虑注射期间的详细条件，以优化治疗效果，例如注射部位的位置（即腹腔梗塞区或梗塞区）、注射体积和数量以及水凝胶的刚度（即难以注射或容易泄漏）。
最后，我们演示了LAD结扎具有代表性的Murine MI模型的协议，以及利用原位交叉连接水凝胶对干细胞进行心肌内移植的实用方法，以提高移植的MSC的保留和雕刻。这些技术为MSC装载注射水凝胶的心内移植提供了有效的方法，突出了其在大动物和临床翻译中应用的巨大潜力。

心脏干细胞治疗已成为心肌修复和再生的潜在方法1，2。尽管最近动物模型和临床试验取得了积极成果，但由于在梗塞的心脏组织3、4的留存率低和注射细胞存活率低，干细胞治疗心肌修复的应用有限。因此，对细胞组织工程的使用进行了深入的研究，包括注射生物材料5、心脏贴片6和细胞片7，以改善宿主心肌内的细胞保留和整合。
在生物工程心脏组织修复的各种潜在方法中，注射式水凝胶与适当的细胞类型相结合，如间质干细胞（MSCs）、胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs），是有效将细胞输送到心肌区域8、9的一个有吸引力的选择。明胶是一种众所周知的天然聚合物，与用于生物医学应用的各种生物材料相比，由于其巨大的生物相容性、可显著的生物降解性和免疫原性降低，可以用作注射基质。虽然基于明胶的注射平台有很大的潜力，但其在体内的适用性仍然有限，因为其机械刚度低，在生理环境中容易降解。
为了克服这些局限性，提出了一种由羟基丙酸组成的明胶基水凝胶的全新简单设计，用于体内应用。明胶-羟基丙烯酸（GH）结合物可在酶、马萝卜过氧化酶（HRP）存在的情况下就地交叉连接，随后在水凝胶内封装各种药物、生物分子或细胞，表明在组织工程应用10、11、12、13、14方面具有巨大潜力。此外，我们最近调查了含有封装MSC的GH水凝胶的治疗效果，并证明它们在MI后在Murine模型15中成功用于心脏修复和再生。在此协议中，我们描述了在 GH 水凝胶内封装和体外三维 （3D） 增殖的简单技术。我们还引入了一个外科手术，旨在通过冠状动脉结结和MSC加载GH水凝胶的心内移植到梗塞的心脏产生一个Murine MI模型。

<table><tbody><tr><td>4 % paraformaldehyde (PFA)</td><td>Intron</td><td>IBS-BP031-2</td><td></td></tr><tr><td>5-0 silk suture</td><td>AILEE</td><td>SK534</td><td></td></tr><tr><td>8-0 polypropylene suture</td><td>ETHICON</td><td>M8732H</td><td></td></tr><tr><td>8-well chamber slide</td><td>Nunc LAB-TEK</td><td>154534</td><td></td></tr><tr><td>Angiocath Plus (22GA) catheter</td><td>BD Angiocath Plus</td><td>REF382423</td><td></td></tr><tr><td>Antibiotic-antimyocotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Centrifuge</td><td>GYROGEN</td><td>1582MGR</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 510</td><td></td></tr><tr><td>Cover slipe</td><td>MARIENFELD</td><td>101242</td><td></td></tr><tr><td>Deluxe High Temperature Cautery kit</td><td>Bovie</td><td>QTY1</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14040-133</td><td></td></tr><tr><td>Dual-syringe</td><td></td><td></td><td></td></tr><tr><td>EOSIN</td><td>SIGMA-ALDRICH</td><td>HT110116</td><td></td></tr><tr><td>Ethanol</td><td>EMSURE</td><td>K49350783 739</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Fechtner conjunctiva forceps titanium</td><td>WORLD PRECISISON INSTRUMENTS</td><td>WP1820</td><td></td></tr><tr><td>Fluorescein isothiocyanate isomer I (FITC)</td><td>SIGMA-ALDRICH</td><td>F7250</td><td></td></tr><tr><td>Forcep</td><td>HEBU</td><td>HB0458</td><td></td></tr><tr><td>Hair removal cream</td><td>Ildong Pharmaceutical</td><td></td><td></td></tr><tr><td>Heating pad</td><td>Stoelting</td><td>50300</td><td>Homeothermic Blanket System</td></tr><tr><td></td><td></td><td>50301</td><td>Replacement Heating Pad for 50300 (10 X 12.5cm)</td></tr><tr><td>Hematoxylin</td><td>SIGMA-ALDRICH</td><td>HHS80</td><td></td></tr><tr><td>Horseradish peroxide (HRP; 250-330 U/mg)</td><td>SIGMA-ALDRICH</td><td>P8375</td><td></td></tr><tr><td>Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;; 30 wt % in H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>SIGMA-ALDRICH</td><td>216763</td><td></td></tr><tr><td>Iodine</td><td>Green Pharmaceutical</td><td></td><td></td></tr><tr><td>LIVE/DEAD cell staining kit</td><td>Thermo Fisher</td><td>R37601</td><td></td></tr><tr><td>Mechanical ventilator</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td>HANIL</td><td>Micro 12</td><td></td></tr><tr><td>Micro needle holder</td><td>KASCO</td><td>37-1452</td><td></td></tr><tr><td>Micro scissor</td><td>HEBU</td><td>HB7381</td><td></td></tr><tr><td>Microscope</td><td>OLYMPUS</td><td>SZ61</td><td></td></tr><tr><td>MT staining kit</td><td>SIGMA-ALDRICH</td><td>HT1079-1SET</td><td>Weigert&amp;rsquo;s iron hematoxylin solution</td></tr><tr><td></td><td></td><td>HT15-1KT</td><td>Trichrome Stain (Masson) Kit</td></tr><tr><td>Paraffin</td><td>LK LABKOREA</td><td>H06-660-107</td><td></td></tr><tr><td>PBS buffer</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>PHK26 staining kit</td><td>SIGMA-ALDRICH</td><td>MINI26</td><td></td></tr><tr><td>Slide scanner</td><td>Leica</td><td>SCN400</td><td></td></tr><tr><td>Surgical scissor</td><td>HEBU</td><td>HB7454</td><td></td></tr><tr><td>Surgical tape</td><td>3M micopore</td><td>1530-1</td><td></td></tr><tr><td>Tissue cassette</td><td>Scilab Korea</td><td>Cas3003</td><td></td></tr><tr><td>Transducer gel</td><td>SUNGHEUNG</td><td>SH102</td><td></td></tr><tr><td>Trout-Barraquer needle holder curved</td><td>KASCO</td><td>50-3710c</td><td></td></tr><tr><td>Ultrasound system</td><td>Philips</td><td>Affiniti 50</td><td></td></tr><tr><td>Xylene</td><td>JUNSEI</td><td>25175-0430</td><td></td></tr></tbody></table>

intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model

目前用于预防后发性心力衰竭的心脏干细胞疗法面临的主要问题之一是受伤心肌内移植细胞的保留率和存活率低，限制了其治疗效果。最近，脚手架生物材料的使用已引起人们的关注，以改善和最大限度地提高干细胞治疗。该协议的目的是引入一种简单明了的技术，使用注射羟基丙酸（GH）水凝胶移植骨髓源性间质干细胞（MSCs）:水凝胶是心脏组织工程应用的细胞输送平台，具有现场交叉连接和高生物相容性的能力。我们提出了一个简单的方法来制造MSC加载GH水凝胶（MSC/水凝胶），并评估其生存和增殖的三维（3D）体外培养。此外，我们演示了在小鼠体内移植MSC/水凝胶的技术，描述了通过左前下降（LAD）冠状动脉结结和随后的MSC/水凝胶移植诱导心肌梗塞（MI）的外科手术。

为了有效地将 MSC 输送到梗死的心肌，本协议中使用了 图 1 中描述的 MSC 现场装载的可交叉链接水凝胶。在体内移植之前，GH水凝胶中MSC的增殖和存活通过3D体外活/死细胞染色检测（活:绿色;死:红色）证实。如图 2所示，具有代表性的图像显示了足够的 MSC 增殖，显示了 GH 水凝胶内的分支网络。此外，在第14天清楚地观察到MSC广泛的多细胞3D结构，表明GH水凝胶可以为封装细胞提供适当的微环境。
在通过LAD结扎引入MI后，MSC装载的GH水凝胶被心肌内移植到近心病区（图3A）。如图 3B所示，MSC 和凝胶在梗塞区域内得到适当的维持。MSC，染色PHK26（红色），很好地融入GH水凝胶，染色FITC（绿色），呈现成功的雕刻和保留在梗塞的心脏在体内应用。
为了验证MSC加载GH水凝胶在Murine MI模型中的治疗效果，在移植后的第28天通过回声心动图和组织学分析评估了心脏功能和结构的变化，并在不同的治疗组中进行了比较。与其他组（图4）相比，代表性的超声心动图显示MSC/凝胶治疗组的心脏功能改善，包括FS、EF和ESV。此外，组织学分析显示，与其他组相比，MSC/凝胶处理组的纤维化、梗塞壁更厚、梗死尺寸更小，表明此方案通过显著减少LV改造（图5）而产生了有益的效果。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61752/61752fig01.jpg"> 图1:利用注射式水凝胶改善干细胞保留和雕刻过程方案。通过心肌梗塞注射将含有骨髓源性MSC的原位交叉链接GH水凝胶制备并移植到梗塞的心脏中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61752/61752fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61752/61752fig02.jpg"> 图2:GH水凝胶内体外3D MSC增殖。活体（绿色）/死亡（红色）MSC在孵化3天、5天、7天和14天后通过共生/死细胞染色后通过共生/死细胞染色（200倍放大倍数;刻度=100μm）获得的代表性图像。这些图像和视频是部分改编的许可，从金等人15。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61752/61752fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61752/61752fig03.jpg"> 图3:MSC/水凝胶的体内移植。（A） 显示MI.（B ）移植MSC和GH水凝胶的代表性图像后，分别标有PKH26（红色）和FITC（绿色）的心肌内移植的示意图图。小鼠在移植1天、3天、5天或7天后被牺牲，然后切除它们的心脏，以评估MSC和GH水凝胶雕刻的程度。切除的心脏是低温固定的，准备成串行部分，并通过共生微拷贝（200倍放大倍度;刻度=100微米）进行成像。这些图像部分是经金等人许可改编的。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61752/61752fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61752/61752fig04.jpg"> 图4:MSC/水凝胶移植后心脏功能的改善。（A） 回声心动图的代表视频。（B） 具有测量的代表性短轴M模式图像，包括左心室前壁厚度在硅藻（LVAWd）和音节（LVAW），内部直径在硅藻（LVID）和音节（LVIDs），后壁厚度在硅石（LVPWd）和音节（LVPW）。（C\u2012E）所有治疗组移植28天后，弹射分数 （EF）、分数缩短 （FS） 和末期收缩体积 （ESV） 的功能改进。数据表示平均±标准偏差 （*p < 0.05， **p < 0.001， ***p < 0.0001;n = 每组 9\u201212）。这些视频和结果是部分改编的许可，从金等人15。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61752/61752fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61752/61752fig05.jpg"> 图5:MSC/水凝胶移植后心脏结构的改善。（A） 组织学评价的代表性图像。（B\u2012D）在梗塞大小中观察到结构改进，以及较少的梗塞墙变薄和纤维化。刻度 = 1 毫米。数据表示的平均±标准偏差（*p <0.05，**p <0.001，***p <0.0001: n = 每组 4+20127）。这些图像和结果部分地经过了金等人的许可。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61752/61752fig05large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

干细胞治疗已成为修复心肌梗塞后受伤的心脏组织的有效策略。我们使用明胶水凝胶为干细胞移植提供最佳的体内应用，这些水凝胶能够酶交叉连接。

Murine 模型心肌梗塞后 MSC 加载注射水凝胶的心内移植

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

intramyocardial-transplantation-msc-loading-injectable-hydrogels

Nanyang Technological University

Research

JoVE Journal

Medicine

4.4K 次观看.  The Catholic University of Korea. 干细胞治疗已成为修复心肌梗塞后受伤的心脏组织的有效策略。我们使用明胶水凝胶为干细胞移植提供最佳的体内应用，这些水凝胶能够酶交叉连接。

视频: Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

结扎冠状动脉内注射在小鼠模型梗死

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

科研

医学

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

生物化学

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

心肌内细胞交货：观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

法援署结扎：有心肌梗死的小鼠模型

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

生物学

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

水凝胶构造和纤维蛋白基胶方法来提供治疗的小鼠心肌梗死模型。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心肌梗塞穆林模型中的 3D 生物打印贴片移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

生物工程

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

建立干细胞治疗心肌梗塞心力衰竭的猪模型

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

在穆林模型中缺血再灌注损伤后干细胞的心肌内传递延迟

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...