本研究由上海市科技委员会 (CN) 赞助的上海创新基金 (1402H294300) 资助, Dr.。感谢 Dr. 广东周 (中国国家组织工程中心) 对本论文的技术支持和科学建议。我们还要感谢 Mr. 矿泉夏 (上海第九人民医院) 在动物福利方面的帮助。

涉及动物的程序由当地机构动物保育和伦理委员会批准, 以尽量减少动物的痛苦。本协议由上海市交通大学医学院附属机构动物护理与使用委员会 (IACUC) 批准, 并与本协议编号 [2017] 063. 
 1. 手术诱发大鼠膝骨关节炎模型的建立 <ol> <li> 为此外科手术, 使用 8-12 周老雄性大 (SD) 大鼠, 体重介于250和300克之间。
	<li> 麻醉40毫克/千克 tiletamine 和40毫克/千克唑通过肌肉注射的动物. </li>
	<li> 将动物置于处于加热阶段的左侧面位置。用剃刀把右膝彻底剃掉。用 10% providone 碘溶液和70% 乙醇进行消毒, 再重复两次, 再涂上 10% providone 碘溶液, 用手术垫覆盖非手术区. </li>
	<li> 使用外科手术刀, 沿髌腱侧向2厘米切口以暴露 jointcapsules. </li>
	<li> 使用外科手术刀将内侧副韧带横断面。反映股骨半月板. </li>
	<li> 在手术过程中, 在关节软骨表面滴入无菌0.9% 氯化钠溶液, 防止软骨干燥. </li>
	<li> 用镊子抓住内侧半月板, 用手术刀从胫骨附件的最窄点切开半月板 ( 图 1A )。避免损伤软骨. </li>
	<li> 用 4-0 的可吸收缝合线关闭关节囊. </li>
	<li> 监控手术鼠, 直到他们恢复知觉. </li>
	<li> 注射20毫克/千克庆大霉素, 以防止感染和0.05 毫克/千克丁丙诺啡通过肌肉注射的途径, 以减轻疼痛后手术. </li>
	<li> 在一个温度控制的动物设施中, 在12小时的光照/暗循环下维持 3-8 周的大鼠 KOA。动物继续接受止痛药, 包括但不限于丁丙诺啡 (0.05 毫克/千克, 贝聿铭) 和庆大霉素 (20 毫克/千克, 贝聿铭), 如果疼痛症状持续. </li>
</ol> 2。大鼠 KOA 模型的组织学评价 <ol> <li> 牺牲过量 CO 的大鼠 2 吸入 (在腔室中增加每分钟容积约30% 的填充率与二氧化碳到现有空气中, 使动物失去知觉在呼吸停止后2-3 分钟内维持 CO2 流至少1分钟。在指定的时间点 (代表性的结果显示在8周的 KOA 诱导范围从3到8周) 后, 手术. 和 #160; </li> <li> 解剖膝盖关节周围的皮肤和肌肉, 用手术刀切断膝关节. </li>
	<li> 将4% 甲醛 (粉煤灰) 的膝关节固定在室温下3天。然后, decalcify 20% 乙酸乙酸 (edta) 中的样品, 在4和 #176 ° C 下进行约4周, 每3天更换一次 edta. 
	警告: 甲醛是有毒的, 穿戴适当的个人防护设备 (PPE), 如手套和眼镜. </li>
	<li> 使用自动组织处理器脱水样品。设置菜单开始一个冲洗周期如下: 70% 乙醇为 1 h;80% 乙醇和95% 乙醇分别为 1 h;其次2循环100% 乙醇为 1 h;随后, 二甲苯每次两次1小时, 石蜡3次在58和 #176; C 每次1小时. 
	注意: 二甲苯是有毒的, 所以佩戴适当的 PPE, 如手套和眼镜. 
	注: (可选) 使用 Coplin 罐进行手动脱水, 其设置与自动组织处理器相同. </li>
	<li> 将完整的关节面的内侧部分向下放置, 并将其嵌入到中心的石蜡块中. 
	注: 可选择使用镊子手动将样品嵌入石蜡块中. </li>
	<li> 开始切割5和 #181; 从关节内侧缘的矢状段到30和 #181; m 间隔, 由旋转切片2个连续部分. </li>
	<li> 在玻璃幻灯片上安装剖面并 deparaffinize 二甲苯中的幻灯片。在2个连续部分之间, 染色一张幻灯片与苏木精和 #38; 曙红 (H 和 #38; E) 染色: 0.1% 苏木精为 15 min, 1% 乙酸溶液为十年代和0.5% 次曙红为 10 min。和染色的其他幻灯片与红 o/快速绿色染色: 0.1% 苏木精为15分钟, 0.02% 快速绿色为3分钟, 1% 醋酸溶液为十年代和0.1% 红 O 为 3 min. 
	注意: 二甲苯有毒, 佩戴适当的 PPE, 如手套、眼镜. </li>
	<li> 使用骨关节炎研究协会国际 (OARSI) 大鼠组织病理学评估系统对彩色幻灯片进行评分 6 . </li>
</ol> 3。人骨髓间充质干细胞与荧光染料的标签做了 <ol> <li> 准备在100% 乙醇浓度为1毫米的细胞标记溶液。保护细胞标签的解决方案从光和存储在室温下6月. </li>
	<li> 在 Dulbecco 和 #39 中使用标准的单元格培养程序扩展 haMSCs; s 修饰的鹰和 #39; s 培养基 (DMEM), 辅以1% 青霉素和链霉素和10% 胎小牛血清 (FCS) 在10厘米的 petri 皿中. </li>
	<li> 分离 haMSCs 与2毫升0.25% 胰蛋白酶与1毫米 EDTA 在大约80% 汇合之前. </li>
	<li> 中和胰蛋白酶与4毫升培养基和旋转的细胞在 800 x g 在室温下3分钟. </li>
	<li> 在5毫升无血清培养基中, 以 1 x 10 6 细胞/毫升的密度将细胞挂起. </li>
	<li> 通过添加50和 #181 对单元格进行标记; 我在37和 #176 的5毫升无血清培养基中做了细胞标记液; C 为 50 min. </li>
	<li> 将标记的单元格在 800 x g 处旋转3分钟. </li>
	<li> 用5毫升磷酸酯缓冲盐水 (PBS) 在 800 x g 上再清洗两次电池, 每次3分钟. </li>
	<li> 使用 Cy5 过滤器设置的荧光显微镜检查标签 haMSCs. </li>
	<li> 将台盼蓝染色的单元格计数并获得 2.5 x 10 6 100 和 #181 中的活细胞; L PBS 注射. </li>
</ol> 4。 在体内 荧光成像跟踪 haMSCs <ol> <li> 手术后八周 (步骤 1), 麻醉 KOA 鼠肌肉注射25毫克/千克 tiletamine 和25毫克/千克唑. </li>
	<li> 用剃刀彻底剃掉右膝, 露出关节区域。用70% 乙醇对关节区域进行消毒. </li>
	<li> 使用 26 G 注射器针, 注入 2.5 x 10 6 标记的单元格在100和 #181 中挂起; L PBS 位于由髌韧带内侧侧、股骨内侧髁和胫骨内侧髁形成的三角形区域中心 ( 图 1B ). </li>
	<li> 打开图像软件并初始化 在体内的 生物发光成像系统. </li>
	<li> 将大鼠置于成像系统中央阶段的仰卧位, 并关闭室的门. </li>
	<li> 检查 #34; 荧光 #34; 复选框, 然后选择 640 nm 的荧光滤光片进行励磁, 680 nm 发射 ( 图 2A ). </li>
	<li> 选择视野到 d. 设置像素分到中 (8), F/停止到2和曝光时间自动。保持最佳在整个研究中获得可比较结果的曝光时间 ( 图 2A ). </li>
	<li> 将动物从舞台上移除, 并从麻醉中恢复. 
	注意: 将动物放在覆盖着2-3 层毛巾的加热垫上. </li>
	<li> 选择荧光测量的辐射效率单位 ( 图 2B )。选择感兴趣的区域 (ROI) 分析和量化每个图像的荧光信号 ( 图 2C ). </li>
	<li> 重复此过程 在体内 依次对每只老鼠进行成像, 以研究 haMSCs 在关节中的纵向保留. 
	注: 和 #160; 我们建议成像动物和 #39; 荧光信号的活体成像系统每两天一次注射后第一周和每周一次, 直到信号无法检测. </li>
</ol>

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孟里、明浩和文王是目前的员工和细胞生物医学组的股票期权持有者 (纳斯达克: CBMG)。其他作者声明没有利益冲突。

我们迫切需要干细胞治疗的安全标准和分布研究, 才能将再生干细胞治疗 KOA 从长凳到床边。然而, 疾病的病理环境在移植 haMSCs 的持续和分布中起着重要作用10。最近, 我们的小组表明, 关节内注射 haMSCs 在病理 KOA 环境中的持续时间比正常情况下注射在8。炎症和退行性微环境可能会招募骨髓间充质干细胞进行修复, 随后有利于保留注入的 haMSCs1<s...

膝关节骨关节炎 (KOA) 是一种退行性疾病, 由关节软骨丧失和渐进炎症, 这已成为一个主要的慢性病在世界各地的老年人1。然而, 目前的治疗使用抗炎药物, 物理补充剂和手术程序可能只提供暂时缓解症状性疼痛2。
人脂肪源性间充质干细胞 (haMSCs) 已成为一种有希望的再生治疗膝关节骨性关节炎, 由于其多能分化潜能的软骨再生和免疫调节特性3, 4。与药理学途径, 以研究的作用机制的行为在体内, 跟踪活 haMSCs 在小 KOA 动物模型目前是有益的建立的理由和可行性 haMSC 治疗前的临床应用。在临床前测试中, 内侧切除 (MM) 破坏关节的机械负荷诱导大鼠 KOA, 提供了一个相对可行的模型, 具有一致的重现性5。KOA 诱发的早于前十字韧带横断, 或与部分内侧切除6相结合。因此, 在由 MM7、8引起的大鼠中, 通常会对注射 haMSCs 与 KOA 的病理微环境之间的长期相互作用进行评估。
虽然 haMSCs 的治疗效果已被广泛报道, 有关知识的体内持续的植入 haMSCs 通过关节内 (IA) 注射是稀缺的9,10。因此, 已经开发了各种细胞标记方法, 包括 immunohistology11、荧光12、绿色荧光蛋白13转染、氧化铁标记为磁共振成像 (MRI)14和众多荧光细胞染料8、15、16。与 labor-intensive 组织学分析相比,在体内非侵入性成像采用光学器件来检测标记有荧光信号的细胞的 real-time 分布和动态,1017。对于功能性活细胞成像, cytocompatible 荧光标记是一种先进的无放射性跟踪技术, 以揭示细胞活动后, 干细胞移植18。此外, 多色荧光亲油性染料具有相对于氨基活性亲水染料或荧光蛋白的优势, 包括其改进的细胞通透性和增强的荧光量子产率19。
因此, 这里所包含的协议使用红色激光激发带有亲油性 carbocyanines 的细胞 (DilC18(5)), 这是一个远红色荧光 Dil (dialkylcarbocyanines) 模拟20。红移激发和发射光谱的确有避免 autofluorescent 干扰, 并允许深层成像在很长一段时间的活动物8。这种跟踪细胞在体内的方法是有效的监测移植的干细胞, 如 haMSCs, 在动物模型, 这是必不可少的了解和改善现有的干细胞再生疗法。

<table><tbody><tr><td>Matrx VMR animal anesthesia system</td><td>Midmark</td><td>VIP3000</td><td></td></tr><tr><td>4-0 suture</td><td>Shanghai Jinhuan</td><td>KC439</td><td></td></tr><tr><td>Razor</td><td>Pritech</td><td>LD-9987</td><td></td></tr><tr><td>Gentamicin</td><td>Zhejiang Jindakang Animal Health Product Co., Ltd.</td><td>None</td><td></td></tr><tr><td>0.9% Sodium chloride solution</td><td>Hunan Kelun Pharmaceutical Co., Ltd.</td><td>H43020455</td><td></td></tr><tr><td>Penicillin</td><td>Shanghai Kangfu chemical pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Buprenorphine</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>16005</td><td>Dilute to final concentration of 10% in PBS</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td>Dilute to final concentration of 20% in PBS</td></tr><tr><td>0.1% Hematoxylin Solution, Mayer&amp;rsquo;s</td><td>Sigma-Aldrich</td><td>MHS16</td><td></td></tr><tr><td>0.5% Eosin Y solution, alcoholic</td><td>Sigma-Aldrich</td><td>HT110116</td><td></td></tr><tr><td>Safranin O</td><td>Sigma-Aldrich</td><td>S8884</td><td></td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258</td><td></td></tr><tr><td>Shandon Excelsior ESTM Tissue Processor</td><td>Thermo Fisher</td><td>A78400006</td><td></td></tr><tr><td>Shandon Histocentre&amp;trade; 3 Tissue Embedding Center</td><td>Thermo Fisher</td><td>B64100010</td><td></td></tr><tr><td>Fully Automated Rotary Microtome</td><td>Leica</td><td>RM2255</td><td></td></tr><tr><td>DiD</td><td>Molecular Probes, Life&lt;br/&gt; Technologies</td><td>V-22887</td><td></td></tr><tr><td>D-MEM High Glucose</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>PBS</td><td>GIBCO, Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-114</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>Corning</td><td>V118877</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>Optima MAX-TL</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>0.4% Trypan Blue solution</td><td>Sigma-Aldrich</td><td>93595</td><td></td></tr><tr><td>Titetamme</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Zolazepam</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Sterile hyposermic syringe for single use 26G</td><td>Shanghai Misawa Medical Industry</td><td>None</td><td></td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>PerkinElmer</td><td>124262</td><td></td></tr><tr><td>Living Imaging 4.0 software</td><td>PerkinElmer</td><td>None</td><td></td></tr></tbody></table>

in vivo tracking of human adipose-derived mesenchymal stem cells in a rat knee osteoarthritis model with fluorescent lipophilic membrane dye

为了支持人脂肪源性间充质干细胞 (haMSC) 治疗膝骨关节炎 (KOA) 的临床应用, 我们研究了 haMSCs 在动物模型中的细胞持久性和分布的有效性。我们展示了一种用亲油性荧光染料标记 haMSCs 细胞膜的方法。随后, 由体内成像系统动态监测经手术诱导的 KOA 大鼠的关节内注射标记细胞。我们雇用的亲油性 carbocyanines 做了 (DilC18 (5)), 一个远红色荧光 Dil (dialkylcarbocyanines) 模拟, 它利用红色激光, 以避免激发自然绿色自发荧光从周围的组织。此外, 红移发射光谱的确有允许深层成像在活体动物和标记程序没有造成细胞毒作用或功能损害 haMSCs。这种方法已被证明是一种有效的跟踪方法的 haMSCs 在大鼠 KOA 模型。该方法的应用也可用于确定临床前研究中其他来源的骨髓间充质干细胞的最佳用药路线和剂量。

为了诱导 KOA 模型, MM 在 SD 大鼠的右膝关节中进行 (图 3)。术后八周, 对大鼠进行了牺牲, 并用 H 和 #38 对膝关节的连续段进行了评价; E 和红 O/快速绿色染色 (图 4)。对于 H 和 #38; E 染色, 关节软骨的表面在手术膝部比正常关节表现出更粗糙的边界而不进行手术。对于红/快速绿色染色, 我们观察到在手术关节中, 与正常对照组相比, ?...

Watch this Scientific Journal Video about In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye at JoVE.com

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

该协议描述了一种有效的方法来监测人脂肪来源的骨髓间充质干细胞 (haMSCs) 的细胞持久性和分布的远红色荧光标记的大鼠膝骨关节炎 (KOA) 模型通过关节内 (IA) 注射。

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

in-vivo-tracking-human-adipose-derived-mesenchymal-stem-cells-rat

Research

JoVE Journal

Developmental Biology

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

8.9K 次观看.  Cellular Biomedicine Group, China. 该协议描述了一种有效的方法来监测人脂肪来源的骨髓间充质干细胞 (haMSCs) 的细胞持久性和分布的远红色荧光标记的大鼠膝骨关节炎 (KOA) 模型通过关节内 (IA) 注射。

视频: In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

医学

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

免疫与感染

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

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 ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

期新生儿大鼠干细胞的内切内培养

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

<meta charset="utf8"></head><body>人间充质干细胞的椎管腔内注射并追踪它们迁移到大鼠大脑中

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

人间充质干细胞的椎管腔内注射并追踪它们迁移到大鼠大脑中

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain ...

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

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