Name: isolation and cultivation of mandibular bone marrow mesenchymal stem cells in rats
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Description: Watch this Scientific Journal Video about Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats at JoVE.com

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>
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A., Bekchanova, E. S., Belyavskii, A. V., Sukhikh, G. 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L., 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=Osteogenic+potential+of+mandibular+vs.+long-bone+marrow+stromal+cells.">Osteogenic potential of mandibular vs. long-bone marrow stromal cells.</a> Journal of Dental Research. 89 (11), 1293-1298 (2010).</li><li>Kaigler, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapy+for+craniofacial+bone+regeneration:+a+randomized,+controlled+feasibility+trial.">Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial.</a> Cell Transplantation. 22 (5), 767-777 (2013).</li><li>Li, C., Wang, F., Zhang, R., Qiao, P., Liu, H. <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+proliferation+and+osteogenic+differentiation+potential+of+rat+mandibular+and+femoral+bone+marrow+mesenchymal+stem+cells+in+vitro.">Comparison of proliferation and osteogenic differentiation potential of rat mandibular and femoral bone marrow mesenchymal stem cells in vitro.</a> Stem Cells Development. 2020, 1941629 (2020).</li><li>Matsubara, T., 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=Alveolar+bone+marrow+as+a+cell+source+for+regenerative+medicine:+differences+between+alveolar+and+iliac+bone+marrow+stromal+cells.">Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells.</a> Journal of Bone and Mineral Research. 20 (3), 399-409 (2005).</li><li>Chan, C. 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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. 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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?...

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?...

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 ...

Research

JoVE Journal

Medicine

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

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

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for 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

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

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

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

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

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

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

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 ...