大鼠骨髓骨髓的隔离和培养
1Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial Science, Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology,National Clinical Research center of Stomatology, 2The 2nd Dental Center, Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology,National Clinical Research center of Stomatology
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 使用两只5周大的雄性斯普拉格道利大鼠进行实验。 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。 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…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.25% Trypsin-EDTA（1X） | Gibco | 25200072 | |
| 10cm culture dish | Corning | ||
| acutenaculum | |||
| Adipogenic differentiation medium | Cyagen biosciences inc. | MUBMX-90031 | |
| Alcian Blue | Beyotime Biotechnology | ||
| Alizarin red | Sigma-Aldrich | A5533 | |
| Alkaline Phosphatase Color Development Kit | Beyotime Biotechnology | C3206 | |
| alpha-Minimum essential medium | GE Healthcare HyClone Cell Culture | SH30265.01B | |
| Anti -CollagenII Rabbit pAb | Abcam | ab34712 | |
| Antibodies against CD16/CD32 | |||
| Antifade Mounting Medium with DAPI | Beyotime Biotechnology | P0131 | |
| APC anti-mouse/rat CD29 Antibody | biolegend inc | 102215 | |
| Biosafety cabinet | Esco | AC2-4S8-CN | |
| CD45 Monoclonal Antibody (OX1), PE, eBioscience | Invitrogen | 12-0461-82 | |
| CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), FITC, eBioscience | Invitrogen | 11-0900-85 | |
| Centrifuge | cence | L500 | |
| Chondrogenesis differentiation medium | cyagen biosciences inc. | ||
| Confocal laser scanning microscope | Zeiss | LSM880 | |
| Countess II FL Automated Cell Counter | Invitrogen | AMQAF1000 | |
| Crystal Violet Staining Solution | Beyotime Biotechnology | C0121 | |
| Fetal Bovine Serum | GE Healthcare HyClone Cell Culture | SH30084.03 | |
| Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) | abcam | ab150077 | |
| Incubator | Esco | CCL-170B-8 | |
| Inverted microscope | olympus | CKX53 | |
| Magzol reagent(Trizol reagent) | Magen | ||
| micropipettor | Eppendorf | ||
| Oil Red O | |||
| Osteogenic differentiation medium | cyagen biosciences inc. | MUBMX-90021 | |
| Penicillin-Streptomycin | Gibco | 15070063 | |
| Phosphate-buffered saline（1X） | Gibco | 20012027 | |
| PrimeScript RT Master Kit | TakaRa Bio Inc | RR036A | |
| Proteinase K | Sigma-Aldrich | P6556 | |
| QuickBlock Blocking Buffer | Beyotime Biotechnology | P0260 | |
| scissor | |||
| SYBR1 Premix | TakaRa Bio Inc | ||
| Toluidine Blue | Beyotime Biotechnology | ||
| Trypan Blue Solution, 0.4% | Gibco | 15250061 |
References
- Jin, C., et al. Stem cell education for medical students at Tongji University: Primary cell culture and directional differentiation of rat bone marrow mesenchymal stem cells. Biochemistry and Molecular Biology Education. 46 (2), 151-154 (2018).
- Chu, D. T., et al. An Update on the Progress of Isolation, Culture, Storage, and Clinical Application of Human Bone Marrow Mesenchymal Stem/Stromal Cells. International Journal of Molecular Sciences. 21 (3), 708 (2020).
- Jin, Z., Chen, J., Shu, B., Xiao, Y., Tang, D. Bone mesenchymal stem cell therapy for ovariectomized osteoporotic rats: a systematic review and meta-analysis. BMC Musculoskeleton Disorder. 20 (1), 556 (2019).
- Jiang, Y., et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 418 (6893), 41-49 (2002).
- Musina, R. A., Bekchanova, E. S., Belyavskii, A. V., Sukhikh, G. T. Differentiation potential of mesenchymal stem cells of different origin. Bulletin of Experimental Biology and Medicine. 141 (1), 147-151 (2006).
- Lloyd, B., et al. Similarities and differences between porcine mandibular and limb bone marrow mesenchymal stem cells. Archives in Oral Biology. 77, 1-11 (2017).
- Zhang, W., et al. Comparison of the use of adipose tissue-derived and bone marrow-derived stem cells for rapid bone regeneration. Journal of Dental Research. 92 (12), 1136-1141 (2013).
- Stefanik, D., et al. Disparate osteogenic response of mandible and iliac crest bone marrow stromal cells to pamidronate. Oral Disorders. 14 (5), 465-471 (2008).
- Aghaloo, T. L., et al. Osteogenic potential of mandibular vs. long-bone marrow stromal cells. Journal of Dental Research. 89 (11), 1293-1298 (2010).
- Kaigler, D., et al. Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial. Cell Transplantation. 22 (5), 767-777 (2013).
- Li, C., Wang, F., Zhang, R., Qiao, P., Liu, H. Comparison of proliferation and osteogenic differentiation potential of rat mandibular and femoral bone marrow mesenchymal stem cells in vitro. Stem Cells Development. 2020, 1941629 (2020).
- Matsubara, T., et al. Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells. Journal of Bone and Mineral Research. 20 (3), 399-409 (2005).
- Chan, C. K. F., et al. Identification of the Human Skeletal Stem Cell. Cell. 175 (1), 43-56 (2018).
- Bianco, P., Riminucci, M., Gronthos, S., Robey, P. G. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells. 19 (3), 180-192 (2001).
- Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. Clonogenic assay of cells in vitro. Nature Protocol. 1 (5), 2315-2319 (2006).
- Xu, H., et al. Icariin prevents oestrogen deficiency-induced alveolar bone loss through promoting osteogenesis via STAT3. Cell Proliferation. 53 (2), 12743 (2020).
- Zhang, P., Wu, Y., Jiang, Z., Jiang, L., Fang, B. Osteogenic response of mesenchymal stem cells to continuous mechanical strain is dependent on ERK1/2-Runx2 signaling. Internation Journal of Molecular Medicine. 29 (6), 1083-1089 (2012).
- Zhu, H., et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nature Protocol. 5 (3), 550-560 (2010).
- Lach, M. S., et al. Chondrogenic Differentiation of Pluripotent Stem Cells under Controllable Serum-Free Conditions. International Journal of Molecular Sciences. 20 (11), 2711 (2019).
- Huang, X., Zhong, L., Hendriks, J., Post, J. N., Karperien, M. The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells. International Journal of Molecular Sciences. 19 (2), 561 (2018).
- Gale, A. L., Linardi, R. L., McClung, G., Mammone, R. M., Ortved, K. F. Comparison of the Chondrogenic Differentiation Potential of Equine Synovial Membrane-Derived and Bone Marrow-Derived Mesenchymal Stem Cells. Frontiers in Veterinary Sciences. 6, 178 (2019).
- Li, X., Zhang, Y., Qi, G. Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells. Cytotechnology. 65 (3), 323-334 (2013).
- Kagami, H., Agata, H., Tojo, A. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. International Journal of Biochemistry and Cell Biology. 43 (3), 286-289 (2011).
- Chamberlain, G., Fox, J., Ashton, B., Middleton, J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 25 (11), 2739-2749 (2007).
- Abdallah, B. M., Alzahrani, A. M., Abdel-Moneim, A. M., Ditzel, N., Kassem, M. A simple and reliable protocol for long-term culture of murine bone marrow stromal(mesenchymal) stem cells that retained their in vitro and in vivo stemness in long-term culture. Biology Proceedings Online. 21, 3 (2019).
Cite This Article
Hong, Y., Xu, H., Yang, Y., Zhou, S., Jin, A., Huang, X., Dai, Q., Jiang, L. Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats. J. Vis. Exp. (162), e61532, doi:10.3791/61532 (2020).