我们感谢上海第九人民医院颅面异常数字化口腔学实验室和研究中心的帮助。本稿件由中国国家自然科学基金委员会（NSFC）资助 [81570950，81870740，81800949]， 上海峰会-高原学科，上海精密医学研究所、上海市第九人民医院、上海交通大学医学院（JC201809）的SHIPM亩基金，上海交通大学医学院高层次创新团队激励项目。L.J.是上海"医学人才之星"青年发展计划和上海交通大学"陈星"项目的学者。

本论文所有动物实验程序均经上海市第九人民医院动物护理委员会、上海交通大学医学院批准。
1. 准备
<ol><li>使用两只5周大的雄性斯普拉格道利大鼠进行实验。</li>
	<li>消毒所有仪器，包括针架、钳子和剪刀在高温下或浸入75%乙醇10分钟。 注意:乙醇浸入不应太长，以避免细胞损伤。</li>
	<li>事先准备文化媒体，其组成在 表1中提供。补充下文所述的每个介质。<ol>
<li>准备α-MEM培养介质（含10%FBS）:补充最低基本中等α（α-MEM）与10%胎儿牛血清和1%青霉素和链霉素。</li>
		<li>骨质分化介质的制备:补充骨质分化基础介质，10%胎儿牛血清，1%谷氨酰胺，1%青霉素-链霉素，0.20%抗皮质酸，1%β甘油磷酸盐和0.01%脱氧酮。</li>
		<li>骨质诱导介质的制备:混合 70% α-MEM 培养介质（10% FBS）和 30% 骨质分化介质。</li>
		<li>制备脂肪分化介质A:补充脂肪分化基础介质，10%胎儿牛血清，1%谷氨酰胺，1%青霉素-链霉素，0.20%胰岛素，0.10%IBMX，0.10%罗西格利塔酮和0.01%德西美酮。</li>
		<li>制备脂肪分化介质 B:补充脂肪分化基础介质，含10%胎儿牛血清、1%谷氨酰胺、1%青霉素-链霉素和0.20%胰岛素。</li>
		<li>制备致胆分化介质:补充致胆分化基底介质，含0.01%德萨美松、0.30%抗酸、1%ITS、0.10%丙酸钠、0.10%蛋白酶和1%TGF-β3。 注意:骨质分化介质必须在配置后一个月内用完。配置的软骨分化介质的有效周期为 12 h。</li>
	</ol>
</li>
</ol>2. 大鼠MBMSC的隔离和栽培
注:所有实验操作应尽可能在冰上进行，以保持细胞的生存能力。
<ol><li>收获老鼠可食用物<ol>
<li>通过二氧化碳 窒息对两只5周大的雄性斯普拉格·道利大鼠实施安乐死。在进行实验之前，确保动物的呼吸停止。</li>
		<li>在含有75%乙醇的烧瓶中冲洗这些老鼠3分钟。</li>
		<li>将老鼠放在一个干净的烟罩里，以收获可食用的。 注意:使用前通过紫外线辐射对烟罩消毒30分钟。</li>
		<li>将鼠放在一个苏平的位置。将皮肤和布奇纳托肌肉从双边角臂或角肌结合到可切割物的后部区域，这是唯一具有较低切口的可移动骨骼。 注意:建议在角角的每一侧切口2厘米，以获得一个清晰的操作场。</li>
		<li>用两个大拇指将最大细管和下垂切口向相反的方向向前推开大鼠的嘴，以露出附着在可操纵的齿上的下垂牙齿。</li>
		<li>完全断开胸肌和肌腱连接到线骨和下部边界的可操纵。 注意:由于缺乏肌肉张力，在此步骤中观察到可操纵性增强。</li>
		<li>按下下下角的齿，向后旋转，直到两侧的圆锥体明显暴露。 注意:此步骤是为了人为地打开大鼠的嘴，尽可能使宽体脱位。可操纵物通过双面圆锥体与头骨相连，因此孔迪的暴露导致头骨和可操纵性之间的脱节。</li>
		<li>将可操纵的身体与头骨分开。</li>
		<li>使用湿纱布清洁骨表面的粘附软组织。</li>
		<li>将骨骼放在一个10厘米的无菌玻璃盘中，里面装满了预冷的最小基本中α（α-MEM）或磷酸盐缓冲盐水（PBS），以保持生存能力。</li>
	</ol></li>
	<li>MBMS 的隔离和培养<ol>
<li>沿着第一个摩尔的中缘从曼迪布勒身体切断前骨。沿着第三摩尔的解剖边缘取出包括共生体和康赛尔在内的曼迪布拉拉穆斯，以暴露骨髓腔。</li>
		<li>用 10 毫升的α-MEM（10% FBS）填充 10 厘米无菌玻璃盘。</li>
		<li>使用 1 mL 注射器吸气α-MEM 培养介质。将注射针插入骨髓腔，反复将骨髓冲入盘中。分别从骨的中度和解剖面冲洗骨腔至少3次，直到骨变白。 注:这是最关键的一步，因为大鼠的骨髓腔不像长骨那么明显，插入针头的正确点需要经验确定。以上所有实验样品应储存在冰上，以保持细胞的生存能力，因此操作时间不得超过2小时。</li>
		<li>将含有冲洗细胞的介质转移到 15 mL 离心管和离心机中，在 4 °C 中以 800 x g 的速度传输 5 分钟。丢弃超自然人。</li>
		<li>用3毫升的α-MEM（10%FBS）来补充细胞。将细胞板入新的 10 厘米培养皿中，并在 5% CO2 孵化器中以 37 °C 孵化。</li>
		<li>在培养的第三天检查这些细胞的形态变化和生长。用不相干的细胞和组织片段去除培养介质。轻轻添加10毫升的新鲜α-MEM（带10%FBS）。</li>
		<li>经过7天的培养，P0 mBMSC达到70%至80%的交汇点。</li>
	</ol></li>
	<li>流单元分拣 注:P0 mBMSC 通过流细胞分类进行表型识别和主要纯化。<ol>
<li>吸气并丢弃培养介质，然后用 PBS 清洗盘子。丢弃PBS。</li>
		<li>加入 1 mL 的 0.25% 三氯辛与 0.02% EDTA 在菜中。在37°C下消化细胞5分钟，然后加入2毫升的α-MEM（10%FBS）来停止反应。</li>
		<li>将细胞悬浮液转移到 15 mL 离心管和离心机中，800 x g 5 分钟。</li>
		<li>离心后，将细胞在 120μL 的 PBS（10% FBS）中重新供用。</li>
		<li>在 4 °C 下用 1μL 抗体阻断这些细胞悬架，以 15 分钟的速度对抗 CD16/CD32。</li>
		<li>将细胞悬浮液的 100 μL 传输到新的微中微富管中， 在黑暗的13、15中，用植物红素（PE）结合抗体对CD45、氟辛-异氰酸酯（FITC）-共聚抗体对CD90和阿洛菲科恰宁（APC）-抗体对CD29的抗体在4°C上对CD29进行污渍，在13、15的黑暗中持续1小时。本实验中使用的抗体浓度显示在表2中。将细胞悬浮的其他 20 μL 用作未染色的负控制。</li>
		<li>然后以 800 x g 的速度将管子离心 5 分钟，丢弃悬架，并在 0.5 mL PBS（10% FBS）中重新注入细胞。</li>
		<li>在分析前 10 分钟添加 10 微升 0.01 毫克/mL DAPI。</li>
		<li>使用放置在离心管上的 40 纳米滤镜来过滤细胞。</li>
		<li>分析荧光激活细胞排序器上的细胞。将面板设置如下。首先，通过在选定的细胞中作为目标 MBMSC，将 DAPI（细胞，然后门 CD29+CD90+CD45） 从总细胞计数中去除死细胞。</li>
		<li>将排序的 CD29+CD90+CD45- 将电池放入 15 mL 离心机管中，预制 3 mL 的α-MEM（FBS 为 10%）。</li>
		<li>以800 x g 的离心管5分钟。取出收集缓冲，并添加 1 mL 的新鲜α-MEM（带 10% FBS）以补充细胞。然后用6厘米的文化菜盘。</li>
	</ol></li>
</ol>3. 殖民地形成能力
注:执行此步骤是为了检查 MBMSC 的划分能力。15
<ol><li>为此实验选择第二通道 mBMSC （P2）。当细胞汇合达到80%至90%时，在6个井板中连续梯度地重新给细胞进行种子，以评估其克隆增殖能力。 注意:建议将梯度从每口井1×102 到1×10 3 细胞设置，但人类或动物起源的不同细胞系的最终稀释是经验确定的。</li>
	<li>培养α-MEM中的细胞（FBS为10%）大约两周，直到在光显微镜下可以看到大量的菌落形成单元。每 3 天刷新一次文化介质。</li>
	<li>吸气并丢弃培养介质，每次用 PBS 洗两次油井 5 分钟。接下来，为每口井添加4%的甲醛溶液，并修复至少10分钟。</li>
	<li>去除甲醛，用PBS冲洗两次井。</li>
	<li>用水晶紫罗兰色溶液染色细胞，在37°C中孵育约10分钟。 注意:染色时间适当调整，直到达到所需的阴影。</li>
	<li>取出染色溶液，用蒸馏水清洗样品，以阻止反应。</li>
	<li>立体显微镜下的图像，并计算由至少50个细胞组成的分散细胞群落。</li>
</ol>4. MBMSC 的多线分化
注:P2 mBMSC 用于后续实验，除非另有说明。
<ol><li>mBMSC 的骨源诱导<ol>
<li>摘要上述 mBMSC。种子细胞的密度为2.5 x 105/cm2在12井板补充1 mL α-MEM（10%FBS）。</li>
		<li>当细胞汇合达到60-70%时，将介质更改为30%的骨感应介质。培养具有α-MEM（10%FBS）作为负控的细胞，每 2 天更换一次介质。 注:在骨质疏松期间出现大量钙结核后，建议每2天将中等交换模式改为半体积中等交换，以防止骨细胞漂浮。</li>
		<li>经过7天的培养，评估这些细胞的钙化通过碱性磷酸污渍16，17。</li>
		<li>取出培养介质，用4%的副甲醛固定细胞10分钟。使用每口井1mL PBS冲洗细胞3分钟两次。</li>
		<li>然后用碱性磷酸盐染色液染色细胞，在37°C孵育10-30分钟。 注:可以在室温下进行夜间孵化，以获得所需的颜色。</li>
		<li>用蒸馏水清洗水井，以阻止反应。在光显微镜下拍照。红色色素颗粒代表碱性磷酸酶 （ALP） 活性。</li>
		<li>在再培养剩余的细胞7天后，进行阿利扎林红色染色，以评估细胞的矿化能力。</li>
		<li>在细胞固定10分钟16，18分钟后，用0.5%的阿利扎林红色染色溶液染色细胞。3-5分钟后停止蒸馏水的染色反应。 注意:反应时间可以根据开发的颜色延长。</li>
		<li>最后，将培养板放在光显微镜下观察骨质污渍的作用:红色结核表示这些细胞的钙沉积。</li>
	</ol></li>
	<li>mBMSC 的辅助诱导<ol>
<li>种子 P2 mBMSC 在 12 井板如上所述。</li>
		<li>井成为90%的汇合物后，培养具有脂肪分化介质A的细胞，使用在α-MEM培养介质培养的细胞（10%FBS）作为负控制。</li>
		<li>上岗2天后，从井中吸气介质A，并在每口井中加入1mL的增生分化介质B。</li>
		<li>一天后，取出中B，然后用中等A重新加入井中重新开始循环。培养细胞交替使用中等A和B。</li>
		<li>将分化介质A和B交替应用三个周期，通过油红色O染色16、18检测这些细胞的脂肪生成。 注意:通过用分化介质B培养这些细胞，可以观察到大量的圆形和大脂质液滴，再延长2至3天。</li>
		<li>取出介质，用4%的副甲醛固定细胞10分钟。用 PBS 洗掉细胞。</li>
		<li>用油红色 O 染色溶液染色细胞，在 37 °C 孵育约 3 分钟。</li>
		<li>取出染色溶液，用蒸馏水清洗井。</li>
		<li>在光显微镜下观察脂肪生成。</li>
	</ol></li>
	<li>MBMS 的琼德罗根西斯诱导<ol>
<li>种子P2 mBMSC在15mL离心管的密度为5×105/厘米2。</li>
		<li>将管子在300 x g 下离心5分钟。丢弃超自然人。</li>
		<li>用0.5 mL的软骨分化介质补充细胞。以300 x g 的离心力将细胞分化5分钟。</li>
		<li>松开离心管的盖子，在 5% 的 CO2 孵化器中以 37 °C 孵育。每 2 天更新一次介质。 注:细胞在24小时后开始打动。建议不要将离心机管移动 48 小时。改变介质时，要小心不要吸气细胞颗粒。</li>
		<li>在21天的感应后，用4%的甲醛固定细胞颗粒，然后脱水、石蜡嵌入和分割。</li>
		<li>去水化和水化后，用阿尔西亚蓝色溶液将滑梯染色至少15分钟18、19、20、21，然后用蒸馏水清洗以阻止反应。在光显微镜下拍摄照片。</li>
		<li>对于胶原蛋白 II 型的免疫荧光染色，执行以下步骤。<ol>
<li>脱水和水化步骤后，在 37 °C 时用 5μg/mL 蛋白酶 K 孵化幻灯片 15 分钟。</li>
			<li>在 37 °C 时将幻灯片孵化在阻塞缓冲区中 1 小时。</li>
			<li>在 200 μL 阻塞缓冲器中应用 1μL 胶原蛋白 II 型抗体，并在 4 °C 下过夜孵化。</li>
			<li>第二天，用PBS洗两次幻灯片，在37°C下用二次抗体孵育1小时。</li>
			<li>用 40，6-迪亚米迪诺-2-苯林多 （DAPI） 孵化切片 10 分钟。</li>
		</ol>
</li>
		<li>在荧光显微镜下拍摄每张幻灯片的照片。</li>
	</ol></li>
</ol>5. 实时 PCR
<ol><li>使用基于商业可用试剂的异氰酸钛从 mBMSCs 中提取总RNA。</li>
	<li>使用市售工具包将RNA反向转录为补充性DNA。反向转录的反应条件如下:5 分钟 65 °C，15 分钟 37 °C，15 秒 85 °C。</li>
	<li>使用 表 3 中列出的引物执行实时 PCR 以检测骨质生成和脂肪生成特定基因。PCR 放大过程如下: 95 °C 5 分钟 95 °C 5 秒， 60 °C 30°C 40 周期。</li>
</ol>

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M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay+of+cells+in+vitro.">Clonogenic assay of cells in vitro.</a> Nature Protocol. 1 (5), 2315-2319 (2006).</li><li>Xu, H., 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=Icariin+prevents+oestrogen+deficiency-induced+alveolar+bone+loss+through+promoting+osteogenesis+via+STAT3.">Icariin prevents oestrogen deficiency-induced alveolar bone loss through promoting osteogenesis via STAT3.</a> Cell Proliferation. 53 (2), 12743 (2020).</li><li>Zhang, P., Wu, Y., Jiang, Z., Jiang, L., Fang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+response+of+mesenchymal+stem+cells+to+continuous+mechanical+strain+is+dependent+on+ERK1/2-Runx2+signaling.">Osteogenic response of mesenchymal stem cells to continuous mechanical strain is dependent on ERK1/2-Runx2 signaling.</a> Internation Journal of Molecular Medicine. 29 (6), 1083-1089 (2012).</li><li>Zhu, H., 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=A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+compact+bone.">A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone.</a> Nature Protocol. 5 (3), 550-560 (2010).</li><li>Lach, M. S., 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=Chondrogenic+Differentiation+of+Pluripotent+Stem+Cells+under+Controllable+Serum-Free+Conditions.">Chondrogenic Differentiation of Pluripotent Stem Cells under Controllable Serum-Free Conditions.</a> International Journal of Molecular Sciences. 20 (11), 2711 (2019).</li><li>Huang, X., Zhong, L., Hendriks, J., Post, J. N., Karperien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+the+WNT-Signaling+Modulators+BIO+and+PKF118-310+on+the+Chondrogenic+Differentiation+of+Human+Mesenchymal+Stem+Cells.">The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.</a> International Journal of Molecular Sciences. 19 (2), 561 (2018).</li><li>Gale, A. L., Linardi, R. L., McClung, G., Mammone, R. M., Ortved, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+Chondrogenic+Differentiation+Potential+of+Equine+Synovial+Membrane-Derived+and+Bone+Marrow-Derived+Mesenchymal+Stem+Cells.">Comparison of the Chondrogenic Differentiation Potential of Equine Synovial Membrane-Derived and Bone Marrow-Derived Mesenchymal Stem Cells.</a> Frontiers in Veterinary Sciences. 6, 178 (2019).</li><li>Li, X., Zhang, Y., Qi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+isolation+methods+and+culture+conditions+for+rat+bone+marrow+mesenchymal+stem+cells.">Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells.</a> Cytotechnology. 65 (3), 323-334 (2013).</li><li>Kagami, H., Agata, H., Tojo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+(bone+marrow-derived+multipotent+mesenchymal+stromal+cells)+for+bone+tissue+engineering:+basic+science+to+clinical+translation.">Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation.</a> International Journal of Biochemistry and Cell Biology. 43 (3), 286-289 (2011).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25 (11), 2739-2749 (2007).</li><li>Abdallah, B. 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所有作者都表示，他们没有利益冲突。

该协议描述了一种通过结合整个骨髓的粘合性和荧光细胞分拣，将BMSC与体外大鼠的可支配物分离的方法，这是获得具有很强分化能力的增殖性MBMSC的简单可靠的方法。这种方法可以通过流细胞分拣初步净化mBMSC，但如果对细胞均质性有更高的要求，可能需要更精确的纯化方法。
目前，用于分离MBMSC的有四种主要技术，包括全骨髓依从性、密度梯度离心、荧光细胞分拣和磁激活细胞分拣等22项。全骨髓粘性和密度梯度离心是在短时间内获取MBMSC的最常见、最简单的方法，然而，收获的mBMSC的低纯度是其主要缺点。最后两种方法可以通过免疫学技术分离高度纯化的mbMSC，但具有成本高、时间长、细胞生存能力受损等缺点。在这项研究中，结合了整个骨髓粘合和荧光细胞分拣方法的优点，在短时间内获得足够数量的增殖mbMSC。
毫无疑问，本协议中最关键的步骤之一是解剖可操纵的老鼠，这与轴向骨骼和近视骨骼的解剖非常不同。必须了解大鼠的解剖结构，以便获得完整的样本。与人类类似，大鼠可在最大齿下方，将下牙保持到位，并通过双面圆锥体连接到头骨。由于可硬骨是唯一可以在头骨中移动的骨骼，因此有许多肌肉附着在下部，控制着它的运动。只有完全去除这些软组织，最大限度地张开嘴，才能暴露与头骨相连的圆锥体。同样值得一提的是，结膜颈部是可操纵的身体弱点，容易骨折。如果在向下旋转时发现过度阻力，则意味着乳胶肌可能不会完全去除。观察到此情况时，不要约束旋转，否则很容易折断结体颈部，从而导致细胞污染。mBMSC分离和培养的其他困难包括骨髓含量低、细胞活性细腻、纯度低、细胞频率低以及造血细胞18、23的污染。要获得具有良好增长和相对高分化潜力的MBMSC，确保MBMSC的活动至关重要。有几个关键步骤，包括使用四周大的老鼠进行这个和随后的实验，因为年轻的老鼠更倾向于保持良好的生存能力。许多研究已经证实，mBMSC的活动与实验动物的年龄有关。这些来自老年捐赠者的mBMSC可能导致低扩散活性、分化潜力和寿命2。细胞收获期间的所有操作都需要在冰上完成，操作时间应尽可能短，最好在 2 小时内完成。此外，保持肌素消化时间不超过3分钟。最后，本议定书的另一个挑战是收获MBMSC的过程。从骨腔中冲洗mBMSC可能很麻烦，因为大鼠的腔非常小，所以熟悉解剖结构是非常重要的。微型CT图像在这方面有很大的帮助。此外，值得注意的是，由于幼骨细长而脆，断裂会导致污染。
提到免疫酚型特征，BMSC表示几个表型，但没有一个是专门针对他们24。人们普遍认为，BMSC不表达CD11b、CD14、CD34或CD45，但它们具有高表达性，即Sca-1、CD29、CD90和CD105。这项研究选择了CD29、CD90和CD45等广为接受的标记物进行荧光电池分拣13、14、25。研究发现，CD29+CD90+CD45−细胞恰当地占细胞总数的80%，这足以进行后续的细胞培养和研究。
几十年来，干细胞疗法广泛应用于各种疾病的治疗，如免疫系统疾病、血液系统疾病、癌症或创伤。毫无疑问，mBMSC作为BMSC的替代品，由于其优越的特性，可以用作干细胞治疗中更安全、更强大的工具。因此，细胞培养和mBMSC的扩展对于获得足够数量的细胞进行治疗变得尤为重要。
总之，这项研究展示了一个很有前途和可靠的协议，在短时间内收获大量具有高同质性和多分化能力的MBMSC。

骨髓间质干细胞（BMSCs）是来自骨髓的非造血干细胞，具有很强的增殖能力和多系分化潜力。事实上，自发现以来，BMSC一直被认为是骨组织工程和再生的理想候选者。多年来，头骨和股骨等骨骼或长骨一直是BMSC颅面再生的最常见来源。然而，或社会BMSC，如曼迪布细胞BMSC（MBMSC），显示与长骨BMSC的一些差异，如不同的胚胎起源和发展模式。可溶性产生于神经结肠细胞的神经顶细胞，并经历膜内骨化，而轴向和近视骨骼则来自中皮，并经历内皮骨化。此外，临床观察和实验动物研究一直表明，5号、6号、7号、8号兽体和腹腔之间存在功能差异。报告显示，来自颅面骨（如可硬骨、最大细骨和血管骨）的BMSC比轴向骨和近足骨骼的BMSC表现出优越的增殖、寿命和分化能力。因此，mBMSC被认为是未来颅面疾病治疗应用的首选资源，如颅骨病、下颚肿瘤、下颚骨骨骨质疏松症和牙周组织缺陷10、11、12。为了了解在细胞前实验中的治疗潜力，必须建立一种在体外快速隔离和培养mBMSC的方法。
在这项研究中，目的是通过整个骨髓的粘性和流动细胞分拣获得纯化的MBMSC。通过微计算断层扫描（Micro-CT）和组织学部分清楚地观察到的可解剖形态表明，可硬骨的血管骨在切口髓空间和肺骨之间。从血管骨的骨髓被冲洗，以获得男性骨髓细胞，但以这种方式培养的细胞不是纯MBMC，并可能包括多种类型的细胞与不确定的效力和不同的血统，如细胞从骨，脂肪和内皮细胞13，14。细胞净化的下一步尤为重要。流动细胞学通过识别细胞表面蛋白质的组合来过滤细胞，并在中性干细胞的富集中被广泛采用。细胞同质性是流动细胞学的主要优点，但这个过程不能决定细胞的生存能力，并可能导致细胞产量有限。在这项研究中，从整个骨髓粘性中获得的P0 mBMSC通过流动细胞学进行排序，以获得纯度高、增殖能力强的MBMS。
本研究引入了一个可重复和可靠的协议，用于使用整个骨髓粘性和流动细胞分拣相结合的大鼠男性BMSC的隔离、培养和分化。它是相关领域研究人员使用可靠、方便的方法。

<table>
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA&#65288;1X&#65289;</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>10cm culture dish</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>acutenaculum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adipogenic differentiation medium</td>
			<td>Cyagen biosciences inc.</td>
			<td>MUBMX-90031</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Beyotime Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Color Development Kit</td>
			<td>Beyotime Biotechnology</td>
			<td>C3206</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha-Minimum essential medium</td>
			<td>GE Healthcare HyClone Cell Culture</td>
			<td>SH30265.01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti -CollagenII Rabbit pAb</td>
			<td>Abcam</td>
			<td>ab34712</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies against CD16/CD32</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antifade Mounting Medium with DAPI</td>
			<td>Beyotime Biotechnology</td>
			<td>P0131</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse/rat CD29 Antibody</td>
			<td>biolegend inc</td>
			<td>102215</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Esco</td>
			<td>AC2-4S8-CN</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (OX1), PE, eBioscience</td>
			<td>Invitrogen</td>
			<td>12-0461-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), FITC, eBioscience</td>
			<td>Invitrogen</td>
			<td>11-0900-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>cence</td>
			<td>L500</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondrogenesis differentiation medium</td>
			<td>cyagen biosciences inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td>LSM880</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet Staining Solution</td>
			<td>Beyotime Biotechnology</td>
			<td>C0121</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GE Healthcare HyClone Cell Culture</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Esco</td>
			<td>CCL-170B-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Magzol reagent(Trizol reagent)</td>
			<td>Magen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>micropipettor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osteogenic differentiation medium</td>
			<td>cyagen biosciences inc.</td>
			<td>MUBMX-90021</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline&#65288;1X&#65289;</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Kit</td>
			<td>TakaRa Bio Inc</td>
			<td>RR036A</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P6556</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickBlock Blocking Buffer</td>
			<td>Beyotime Biotechnology</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR1 Premix</td>
			<td>TakaRa Bio Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Beyotime Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cultivation of mandibular bone marrow mesenchymal stem cells in rats

在这里，我们提出了一种有效的方法，在体外分离和培养男性骨髓骨髓中性干细胞（mBMSC），以迅速获得许多高质量的细胞的实验要求。mBMSC由于具有出色的自我更新能力和多系分化潜力，将来可广泛应用于组织工程细胞的治疗应用，以防颅面疾病和颅面再生。因此，大量获取 MBMSC 非常重要。
在这项研究中，骨髓从可溶性中冲洗，通过整个骨髓粘附培养分离出原发性mBMSC。此外，CD29+CD90+CD45− mBMSC 通过荧光电池分类进行了纯化。第二代纯化mBMSC用于进一步研究，并显示出区分成骨细胞、脂肪细胞和软骨细胞的潜力。利用这种体外模型，可以获得大量的增殖MBMSC，从而有助于研究MBMSC的生物特征、对微环境的后续反应以及其他应用。

使用此协议，在初始培养后的第三天，很大一部分细胞粘附在板上。通常，经过额外的3-4天的培养，细胞汇合达到70至80%（图1B）。通过荧光细胞分类，DAPI-CD29+CD90+CD45− mBMSC 被纯化18，22，其中约占 81.1% 的 P0 细胞 （图 1C）.
在6口井板每口井的100个细胞上播种P2 mBMSC一周后，观察到大量的菌落形成单元，这表明MBMSCs（图1D）具有显著的菌落形成能力。
为了评估多系分化能力，MBMSC分别被诱导到12个井板中的骨系、软骨系和脂肪系。mBMSC表现出很强的骨质分化能力。ALP活性增加，红钙结核在阿利扎林红染色下偶尔分布，骨原特异基因Runx2、Alp、Bsp和Ocn（图2）的表达增加，表明雌激素诱导。对于由油红O染色识别的脂肪生成，在9天的感应后，许多富含脂质的真空体是显而易见的。同样，Pparγ1和Cebpa的脂肪特异性基因的表达也显著增加（图3）。对于致痛分化滑梯的微观观察，样本显示阿尔西恩蓝色呈阳性污渍。此外，用抗II型胶原蛋白抗体进行免疫，显示软骨基质积累增强（图4）。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>培养基</td>
			<td>元件</td>
			<td>最终浓度</td>
		</tr>
<tr>
<td>1.α-MEM 文化介质（带 10% FBS）</td>
			<td>α最低基本介质</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>胎儿牛血清</td>
			<td>10%</td>
		</tr>
<tr>
<td></td>
			<td>青霉素和链霉素</td>
			<td>1%</td>
		</tr>
<tr>
<td>2.骨源性感应介质</td>
			<td>α-MEM 文化介质（带 10% FBS）</td>
			<td>70%</td>
		</tr>
<tr>
<td></td>
			<td>骨质分化分化介质</td>
			<td>30%</td>
		</tr>
<tr>
<td>3.骨源分化介质</td>
			<td>骨质分化基底介质</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>胎儿牛血清</td>
			<td>10%</td>
		</tr>
<tr>
<td></td>
			<td>谷氨酰胺</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>青霉素-链霉素</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>抗坏血酸</td>
			<td>0.20%</td>
		</tr>
<tr>
<td></td>
			<td>β-甘油磷酸盐</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>地塞米松</td>
			<td>0.01%</td>
		</tr>
<tr>
<td>4.辅助分化介质A</td>
			<td>脂肪分化基底介质</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>胎儿牛血清</td>
			<td>10%</td>
		</tr>
<tr>
<td></td>
			<td>谷氨酰胺</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>青霉素-链霉素</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>胰岛素</td>
			<td>0.20%</td>
		</tr>
<tr>
<td></td>
			<td>IBMX</td>
			<td>0.10%</td>
		</tr>
<tr>
<td></td>
			<td>罗西格利塔佐内</td>
			<td>0.10%</td>
		</tr>
<tr>
<td></td>
			<td>地塞米松</td>
			<td>0.01%</td>
		</tr>
<tr>
<td>5.生化分化介质 B:</td>
			<td>脂肪分化基底介质</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>胎儿牛血清</td>
			<td>10%</td>
		</tr>
<tr>
<td></td>
			<td>谷氨酰胺</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>青霉素-链霉素</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>胰岛素</td>
			<td>0.20%</td>
		</tr>
<tr>
<td>6.致癌分化介质:</td>
			<td>琼德罗根化分化基础介质</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>地塞米松</td>
			<td>0.01%</td>
		</tr>
<tr>
<td></td>
			<td>抗坏血酸</td>
			<td>0.30%</td>
		</tr>
<tr>
<td></td>
			<td>其</td>
			<td>1%</td>
		</tr>
<tr>
<td></td>
			<td>苯丙酸钠</td>
			<td>0.10%</td>
		</tr>
<tr>
<td></td>
			<td>脯氨酸</td>
			<td>0.10%</td>
		</tr>
<tr>
<td></td>
			<td>TGF-+3</td>
			<td>1%</td>
		</tr>
</tbody></table>表1:文化媒介和分化介质的组成部分。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>抗体</td>
			<td>浓度</td>
		</tr>
<tr>
<td>CD90.1 （Thy-1.1） 单克隆抗体</td>
			<td>0.5毫克/升</td>
		</tr>
<tr>
<td>CD45 单克隆抗体</td>
			<td>0.2毫克/升</td>
		</tr>
<tr>
<td>CD29 抗体</td>
			<td>0.2毫克/升</td>
		</tr>
<tr>
<td>胶原蛋白II兔多克隆抗体</td>
			<td>5毫克/升</td>
		</tr>
<tr>
<td>山羊抗兔IgG H&L （亚历克萨氟® 488）</td>
			<td>1毫克/升</td>
		</tr>
</tbody></table>表2:本研究中使用的抗体浓度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆</td>
			<td colspan="2">序列（5'到3'）</td>
		</tr>
<tr>
<td>盖普德</td>
			<td colspan="2">福沃德: 克卡格卡卡格塔格</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">反向: 阿加卡特卡</td>
		</tr>
<tr>
<td>伦克斯2</td>
			<td colspan="2">福沃德: 海合会</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">反向:特加克特</td>
		</tr>
<tr>
<td>阿尔卑斯山</td>
			<td colspan="2">福沃德: 阿阿克特克塔特克茨克</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">反向:特格特加特</td>
		</tr>
<tr>
<td>英国石油公司</td>
			<td colspan="2">福沃德: 加格加塔</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">反向: 阿特加特克塔格特</td>
		</tr>
<tr>
<td>奥克恩</td>
			<td colspan="2">转发:中国民航总局</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">反向:格卡卡卡茨塔克</td>
		</tr>
<tr>
<td>塞巴</td>
			<td colspan="2">福沃德: 阿格特格特加塔加卡阿卡阿奇</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">反向: C 格特卡特克特卡特</td>
		</tr>
<tr>
<td>Pparγ1</td>
			<td colspan="2">福沃德: 卡塔加加卡特卡加卡</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">反向: GTCT 技术</td>
		</tr>
</tbody></table>表3:实时 PCR 中使用的引物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61532/61532fig01.jpg"> 图1:MBMS的隔离和文化。（A） 协议的示意图图。mBMSC 在第 0 天被隔离和镀层，并孵育出α-MEM 文化介质。第7天，P0 mBMSC通过流动细胞测量分类进行净化，分拣的细胞被镀在一个新的培养皿上。第14天，P1 mBMSC被收集并镀在12井板上。第15天，P2 mBMSC在相应的诱导介质下被诱导到骨细胞、脂肪细胞和软骨细胞中。（B） 大鼠骨髓的原理图模型和P0 MBMSC的微观观察。（C） 大鼠mBMSC的流动细胞学分类。流动细胞学分析表明，这些细胞对CD29和CD90呈阳性，但对CD45呈阴性，这与BMSC特性一致。其中，1.4 x 106 个细胞被分类，占细胞总数的80%。（D） 水晶紫罗兰染色 P2 MBMSC 克隆的代表图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61532/61532fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61532/61532fig02.jpg"> 图2:mBMSC的骨质分化潜力。（A） 经过7天的骨感应，ALP活性的变化被可视化。大量矿化结核在骨质分化诱导后14天被染上阿利扎林红色污渍。（B ， C）使用图像 J 软件评估了 ALP 和阿利扎林红色染色的正面积。（C-G）骨细胞特异性标记 Runx2、阿尔卑斯、Bsp 和 Ocn 的 mRNA 表达在骨质疏松 7 天后显著增加。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61532/61532fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61532/61532fig03.jpg"> 图3:MBMSC在上岗九天后的副生分化潜力。（A） 大量的脂质液滴形成，脂肪细胞被油红-O染色。（B ， C）脂肪标记 Cebpa 和 Pparγ1 的mRNA表达在经过9天的脂肪生成后显著增加。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61532/61532fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61532/61532fig04.jpg"> 图4:致胆分化效果的立体镜视图。（A） mBMSC 在 21 天后软化感应显示对阿尔恰恩蓝色染色呈阳性。（B） 带有抗II型胶原蛋白的软体聚合物的免疫荧光图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61532/61532fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats at JoVE.com

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

本文提出了一种结合整个骨髓粘附性和流动细胞测量分拣的方法，用于从大鼠的骨髓中分离、培养、分拣和识别骨髓中甲基干细胞。

大鼠骨髓骨髓的隔离和培养

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

isolation-cultivation-mandibular-bone-marrow-mesenchymal-stem-cells

Nanyang Technological University

Research

JoVE Journal

Medicine

5.1K 次观看.  National Clinical Research center of Stomatology. 本文提出了一种结合整个骨髓粘附性和流动细胞测量分拣的方法，用于从大鼠的骨髓中分离、培养、分拣和识别骨髓中甲基干细胞。

视频: Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

成年大鼠心肌细胞的分离与培养

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

环境毒物暴露大鼠下颌切牙釉质器官的显微解剖

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.