Name: mesenchymal stem cell isolation from pulp tissue and co-culture with cancer cells to study their interactions
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions at JoVE.com

这项研究得到了叶迪特佩大学的支持。本文中使用的所有数据和数字都是以前公布的。

经机构道德委员会批准后, 获得了患者的书面知情同意。
1. dpsc 隔离和培养
<ol><li>从17至20至20至15毫升的年轻成年人身上获得的智齿含有完整的 dulbecco 的修饰鹰培养基 (dmem) [低葡萄糖 dmem 培养基, 辅以10% 的胎牛血清 (fbs) 和1% 青霉素/两性霉素 (psa) 溶液], 切除后8小时内。在转移过程中保持组织材料的低温 (4°c), 以避免潜在的细胞死亡。</li>
	<li>通过无菌拔模从牙齿中心小心地?...

<ol>
	<li>Camberlain, 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=Mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25 (11), 2739-2749 (2007).</li><li>Demirci, S., Do&#287;an, A., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dental Stem Cells. , 109-124 (2016).</li><li>Fox, J. M., Chamberlain, G., Ashton, B. 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H., Gupta, A., Spaeth, E., Andreeff, M., Marini, F. <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:+dissecting+a+discrepancy+in+the+literature:+do+mesenchymal+stem+cells+support+or+suppress+tumor+growth.">Concise review: dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth.</a> Stem cells. 29 (1), 11-19 (2011).</li><li>Albini, A., Sporn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumour+microenvironment+as+a+target+for+chemoprevention.">The tumour microenvironment as a target for chemoprevention.</a> Nature Reviews Cancer. 7 (2), 139 (2007).</li><li>Quante, M., 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=Bone+marrow-derived+myofibroblasts+contribute+to+the+mesenchymal+stem+cell+niche+and+promote+tumor+growth.">Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth.</a> Cancer cell. 19 (2), 257-272 (2011).</li><li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proceedings of the National Academy of Sciences. 97 (25), 13625-13630 (2000).</li><li>Mori, G., 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=Dental+pulp+stem+cells:+osteogenic+differentiation+and+gene+expression.">Dental pulp stem cells: osteogenic differentiation and gene expression.</a> Annals of the new York Academy of Sciences. 1237 (1), 47-52 (2011).</li><li>Mori, G., 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+properties+of+human+dental+pulp+stem+cells.">Osteogenic properties of human dental pulp stem cells.</a> Journal of biological regulators and homeostatic agents. 24 (2), 167-175 (2010).</li><li>Kerkis, I., 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=Isolation+and+characterization+of+a+population+of+immature+dental+pulp+stem+cells+expressing+OCT-4+and+other+embryonic+stem+cell+markers.">Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers.</a> Cells Tissues Organs. 184 (3-4), 105-116 (2006).</li><li>Potdar, P. D., Jethmalani, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells:+applications+in+future+regenerative+medicine.">Human dental pulp stem cells: applications in future regenerative medicine.</a> World journal of stem cells. 7 (5), 839 (2015).</li><li>Ahmed, N. E. -. M. B., Murakami, M., Hirose, Y., Nakashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+dental+pulp+stem+cell+secretome+for+Alzheimer's+disease+treatment:+an+in+vitro+study.">Therapeutic potential of dental pulp stem cell secretome for Alzheimer's disease treatment: an in vitro study.</a> Stem cells international. 2016, (2016).</li><li>Gorin, C., 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=Priming+dental+pulp+stem+cells+with+fibroblast+growth+factor-2+increases+angiogenesis+of+implanted+tissue-engineered+constructs+through+hepatocyte+growth+factor+and+vascular+endothelial+growth+factor+secretion.">Priming dental pulp stem cells with fibroblast growth factor-2 increases angiogenesis of implanted tissue-engineered constructs through hepatocyte growth factor and vascular endothelial growth factor secretion.</a> Stem cells translational medicine. 5 (3), 392-404 (2016).</li><li>Wakayama, 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=Factors+secreted+from+dental+pulp+stem+cells+show+multifaceted+benefits+for+treating+acute+lung+injury+in+mice.">Factors secreted from dental pulp stem cells show multifaceted benefits for treating acute lung injury in mice.</a> Cytotherapy. 17 (8), 1119-1129 (2015).</li><li>Do&#287;an, A., 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=Differentiation+of+human+stem+cells+is+promoted+by+amphiphilic+pluronic+block+copolymers.">Differentiation of human stem cells is promoted by amphiphilic pluronic block copolymers.</a> International Journal of Nanomedicine. 7, 4849 (2012).</li><li>Ta&#351;l&#305;, P. N., Do&#287;an, A., Demirci, S., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boron+enhances+odontogenic+and+osteogenic+differentiation+of+human+tooth+germ+stem+cells+(hTGSCs)+in+vitro.">Boron enhances odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs) in vitro.</a> Biological trace element research. 153 (1-3), 419-427 (2013).</li><li>Yalvac, M., 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=Isolation+and+characterization+of+stem+cells+derived+from+human+third+molar+tooth+germs+of+young+adults:+implications+in+neo-vascularization,+osteo-,+adipo-and+neurogenesis.">Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo-and neurogenesis.</a> The pharmacogenomics journal. 10 (2), 105 (2010).</li><li>Do&#287;an, A., 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=Sodium+pentaborate+pentahydrate+and+pluronic+containing+hydrogel+increases+cutaneous+wound+healing+in+vitro+and+in+vivo.">Sodium pentaborate pentahydrate and pluronic containing hydrogel increases cutaneous wound healing in vitro and in vivo.</a> Biological trace element research. 162 (1-3), 72-79 (2014).</li><li>Do&#287;an, A., Yalva&#231;, M. E., Y&#305;lmaz, A., Rizvanov, A., &#350;ahin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+F68+on+cryopreservation+of+mesenchymal+stem+cells+derived+from+human+tooth+germ.">Effect of F68 on cryopreservation of mesenchymal stem cells derived from human tooth germ.</a> Applied biochemistry and biotechnology. 171 (7), 1819-1831 (2013).</li><li>Rizvanov, A. A., 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=Interaction+and+self-organization+of+human+mesenchymal+stem+cells+and+neuro-blastoma+SH-SY5Y+cells+under+co-culture+conditions:+A+novel+system+for+modeling+cancer+cell+micro-environment.">Interaction and self-organization of human mesenchymal stem cells and neuro-blastoma SH-SY5Y cells under co-culture conditions: A novel system for modeling cancer cell micro-environment.</a> European Journal of Pharmaceutics and Biopharmaceutics. 76 (2), 253-259 (2010).</li><li>Troiano, 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=Multiparametric+analysis+of+cells+with+different+mitochondrial+membrane+potential+during+apoptosis+by+polychromatic+flow+cytometry.">Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry.</a> Nature protocols. 2 (11), 2719 (2007).</li><li>Melzer, C., von der Ohe, J., Lehnert, H., Ungefroren, H., Hass, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+niche+models+and+contribution+by+mesenchymal+stroma/stem+cells.">Cancer stem cell niche models and contribution by mesenchymal stroma/stem cells.</a> Molecular cancer. 16 (1), 28 (2017).</li><li>Aguirre, A., Planell, J., Engel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+bone+marrow-derived+endothelial+progenitor+cell/mesenchymal+stem+cell+interaction+in+co-culture+and+its+implications+in+angiogenesis.">Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis.</a> Biochemical and biophysical research communications. 400 (2), 284-291 (2010).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomimetics and Stem Cells. , 29-36 (2013).</li><li>Plotnikov, E., 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=Cell-to-cell+cross-talk+between+mesenchymal+stem+cells+and+cardiomyocytes+in+co-culture.">Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.</a> Journal of cellular and molecular. 12 (5a), 1622-1631 (2008).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cell-cell+communication+in+co-culture.">Studying cell-cell communication in co-culture.</a> Biotechnology journal. 8 (4), 395-396 (2013).</li><li>Brunetti, G., 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=High+expression+of+TRAIL+by+osteoblastic+differentiated+dental+pulp+stem+cells+affects+myeloma+cell+viability.">High expression of TRAIL by osteoblastic differentiated dental pulp stem cells affects myeloma cell viability.</a> Oncology reports. 39 (4), 2031-2039 (2018).</li><li>Do&#287;an, A., Demirci, S., Apdik, H., Apdik, E. 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骨髓间充质干细胞对肿瘤环境的贡献受到多种相互作用的调节, 包括干细胞与癌细胞之间的混合细胞融合、昆虫病或细胞因子和趋化因子活性.结构组织、细胞相互作用和分泌因素决定了癌细胞在肿瘤促进、进展和转移到周围组织方面的行为。需要适当的体外模型系统来研究住院细胞群体相互作用背后的机制, 以了解细胞通信促进癌症进展和转移。
<p class="jove_conten...

间充质干细胞 (mscs) 具有分化能力, 有助于骨髓、软骨、肌肉、韧带、肌腱和脂肪等间充质组织的再生, 几乎从成人体内的所有组织中分离出 1,2. 除了在慢性炎症或损伤的情况下通过产生常驻细胞来提供组织稳态外, 它们还产生重要的细胞因子和生长因子, 以协调血管生成、免疫系统和组织重塑3。骨髓间充质干细胞与肿瘤组织的相互作用尚不清楚, 但积累的证据表明, 骨髓间充质干细胞可能促进肿瘤的发生、进展和转移4。
骨髓间充质干细胞对受伤或长期发炎区域的归巢能力使其成为干细胞治疗的宝贵候选。然而, 癌症组织, "永远不会愈合伤口", 也释放炎性细胞因子, 亲血管生成分子, 和重要的生长因子, 吸引骨髓间充质干细胞到癌变区 5.虽然有有限的报告显示骨髓间充质干细胞对癌症生长的抑制作用 6,7, 他们的癌症进展和转移促进的影响已被广泛报道8。骨髓间充质干细胞以不同的方式直接或间接地影响致癌, 包括抑制免疫细胞、分泌支持癌细胞增殖和迁移的生长因子细胞因子、增强血管生成活性和调节上皮间充质转变 (emt)9,10。肿瘤环境由多种细胞组成, 包括与癌症相关的成纤维细胞 (caf) 和肌成纤维细胞、内皮细胞、脂肪细胞和免疫细胞11。其中, caft 是肿瘤区最丰富的细胞类型, 分泌各种促进癌症生长和转移的趋化因子8。研究表明, 骨髓源性骨髓间充质干细胞可在肿瘤基质12中分化为 caf。
牙髓干细胞 (dpscs), 被描述为第一个牙科组织衍生的骨髓间充质干细胞由 gronthos等人.13在 2000年, 然后被广泛调查的其他 14,15, 表达多能标记, 如oct4, sox2, 和nanog16 , 并可以分化为各种细胞链17。基因和蛋白质表达分析证明, dpsc 产生的生长因子/细胞因子水平与其他骨髓间充质干细胞相当, 如血管内皮生长因子 (vegf)、血管生成素、成纤维细胞生长因子 2 (fgf2)、白细胞介素-4 (il-4)、il-6、il-10、和干细胞因子 (scf), 以及 fms 样酪氨酸激酶-3 配体 (flt-3l), 可能促进血管生成, 调节免疫细胞, 并支持癌细胞增殖和迁移18,19,20.虽然骨髓间充质干细胞与癌症环境的相互作用已在文献中得到了充分的记录, 但 dpscs 与癌细胞之间的关系尚未得到评估。在本研究中, 我们建立了高度转移性前列腺癌细胞系 pc-3 和 dpscs 的共培养和条件培养基治疗策略, 提出了牙科骨髓间充质干细胞机制在癌症进展和转移中的潜在作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11885084</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>PSA</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>For cell dissociation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>10010023</td>
			<td>For washes</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Component of differentiation media</td>
		</tr>
		<tr>
			<td>&#946;-Glycerophosphate</td>
			<td>Sigma</td>
			<td>G9422</td>
			<td>Component of osteogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td>Component of osteo- and chondro-genic differentiation medium</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS &#8722;G)</td>
			<td>Invitrogen</td>
			<td>41400045</td>
			<td>Component of chondrogenic differentiation medium</td>
		</tr>
		<tr>
			<td>TGF-&#946;</td>
			<td>Sigma</td>
			<td>SRP3171</td>
			<td>Component of chondrogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Component of adipogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I7018</td>
			<td>Component of adipogenic differentiation medium</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma</td>
			<td>I7378</td>
			<td>Component of adipogenic differentiation medium</td>
		</tr>
		<tr>
			<td>MTS Reagent</td>
			<td>Promega</td>
			<td>G3582</td>
			<td>Cell viability analyses</td>
		</tr>
		<tr>
			<td>TUNEL Assay</td>
			<td>Sigma</td>
			<td>11684795910</td>
			<td>Apoptotic analyses</td>
		</tr>
		<tr>
			<td>24-well plate inserts</td>
			<td>Corning</td>
			<td>3396</td>
			<td>For trans-well migration assay</td>
		</tr>
		<tr>
			<td>PKH67</td>
			<td>Sigma</td>
			<td>PKH67GL</td>
			<td>For co-culture cell staining</td>
		</tr>
		<tr>
			<td>PKH26</td>
			<td>Sigma</td>
			<td>PKH26GL</td>
			<td>For co-culture cell staining</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>For cell fixation</td>
		</tr>
		<tr>
			<td>von Kossa Kit</td>
			<td>BioOptica</td>
			<td>04-170801.A</td>
			<td>For cell staining (differentiation)</td>
		</tr>
		<tr>
			<td>Alcian blue</td>
			<td>Sigma</td>
			<td>A2899</td>
			<td>For cell staining (differentiation)</td>
		</tr>
	</tbody>
</table>

mesenchymal stem cell isolation from pulp tissue and co-culture with cancer cells to study their interactions

癌症作为一个多步骤的过程和复杂的疾病, 不仅受单个细胞增殖和生长的调节, 还受肿瘤环境和细胞相互作用的控制。癌症和干细胞相互作用的识别, 包括细胞外环境的变化、身体的相互作用和分泌的因素, 可能有助于发现新的治疗方案。我们结合已知的共培养技术, 建立了间充质干细胞 (mscs) 和癌细胞相互作用的模型系统。在目前的研究中, 牙髓干细胞 (dpscs) 和 pc-3 前列腺癌细胞相互作用的直接和间接共培养技术进行了研究。利用从 dpscc 和0.4μm 孔径反井膜中获得的条件介质 (cm) 研究了副井活性。对不同细胞类型的共培养进行了研究, 以研究细胞直接相互作用。结果表明, cm 能增加前列腺癌细胞培养中细胞增殖, 减少细胞凋亡。cm 和经井系统都提高了 pc-3 细胞的细胞迁移能力。用不同的膜染料染色的细胞被播种到同一培养容器中, dpscs 在这种直接共培养条件下参与了与 pc-3 细胞的自组织结构。总体而言, 结果表明, 共培养技术可用于癌症和 msc 相互作用作为一个模型系统。

图 1描述了在培养条件下 dpscs 的一般 msc 特征。dpsc 在电镀后发挥成纤维细胞样细胞形态 (图 1b)。msc 表面抗原 (cd29、cd73、cd90、cd105 和 cd166) 表达强烈, 而造血标志物 (cd34、cd45 和 cd14) 为阴性 (图 1c)。在 dpsc 培养中观察到与骨、软骨和脂肪基因分化有关的形态和分子水平的变化, 然后是分化鸡尾酒的应用<stron...

Watch this Scientific Journal Video about Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions at JoVE.com

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

我们提供了基于直接和间接共培养方法评估从牙髓和前列腺癌细胞相互作用中分离出的间充质干细胞的方案。条件介质和反井膜适用于分析间接副胺活性。一起播种不同染色细胞是直接细胞相互作用的合适模型。

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

这种方法有助于回答干细胞领域有关成人干细胞在调节癌细胞行为中的作用的关键问题。这项技术的主要优点是，它允许评估癌细胞和干细胞在体外相互作用，而无需使用生物体。这项技术具有癌症转移的潜力，因为它模型成人干细胞在癌症转移到硬组织如骨骼的促进作用。手术的演示将由我实验室的博士后HuseyinApddik进行。首先使用无菌提取钳子从17至20岁的年轻人获得的智齿中心去除纸浆组织。将组织放入10厘米组织培养皿中，用手术刀将样品切碎成两到三毫米的碎片。将每道菜中的碎片转移到经过六孔处理的组织培养的单个孔中，用200微升完整的DMEM覆盖每孔的组织。允许组织附着在井底，在加湿的37摄氏度和5%的CO2细胞培养箱中至少孵育两个小时。在孵育结束时，用2至2.5毫升的新鲜完整培养剂喂养组织，再培养8至9天的细胞。当培养达到80%的汇合时，用两毫升PBS洗净每井，然后用两毫升的三辛在37摄氏度下分体细胞分离。两分钟后，停止反应与两毫升完整的DMEM每井和通过离心收集细胞。然后，每两口细胞将颗粒重新用15毫升新鲜完整培养，将细胞进入T75烧瓶中，以进行后续培养。在至少八段之后，通过光显微镜对细胞进行可视化。牙髓干细胞应附着在培养皿上，并表现出主轴状细胞形态。对于表面标记分析，在证明三辛化后，在室温下用4%半甲醛将细胞修复20分钟，在1.5毫升管中，然后每次洗涤500微升PBS中三次清洗。上次洗涤后，将细胞与感兴趣的适当抗体标记在100微升PBS每管4摄氏度下一小时。在孵育结束时，将细胞在PBS中清洗三次，如证明，并在每管100微升PBS中用适当的二级抗体标记细胞，在4摄氏度下30分钟。然后在PBS中清洗样品三次，然后根据标准方案通过流式细胞测量分析细胞。为了对牙髓干细胞进行分化，在500微升完整DMEM中将10倍至4个细胞种子种子放入24孔板的单个孔中，在细胞培养培养箱中24小时孵化。第二天，用适当的分化介质替换每井的培养物，将细胞返回到培养箱，每周两次刷新该培养物，为期两周。根据标准协议，冯·科萨和阿尔西安蓝色染色可以确认差异。经过两到四个通道，当培养物达到80%汇合时，从培养的牙髓干细胞中收集有条件的中超细胞。然后，将收集的介质离心，以清除任何废物组织材料和细胞碎屑，并在负20摄氏度下储存介质，直到使用。对于癌细胞治疗，将10倍至5个肿瘤细胞播种到12孔板的单个孔中，在37摄氏度和5%CO2下进行隔夜孵育。第二天早上，使用无菌的200微升尖端，使每个井的划痕损伤，并立即更换每一个井的上能与含有各种浓度的调节介质的新鲜介质。然后，立即观察倒置显微镜下的划痕，并定期获取受伤培养物的图像。在分析结束时，在 ImageJ 中测量一段时间的划痕闭合。为了评估牙髓干细胞的迁移，首先将三倍10至四个牙髓干细胞的种子到单独的24孔板插入物上，在250微升DMEM中用0.4微米孔隙，在37摄氏度下过夜孵育这些插入物。接下来，将四个肿瘤细胞的5倍10种子放入500微升DMEM的24孔板的单个孔中，在37摄氏度下进行夜间孵育。第二天早上，划伤肿瘤细胞，如证明。用500微升的新鲜DMEM代替超级钠，并将一个牙髓干细胞种子插入到每个用新鲜培养基喂养的井中。然后立即在倒置显微镜上观察细胞，并定期对细胞进行成像，以评估牙髓干细胞迁移。为了评估牙髓干细胞与癌细胞的直接相互作用，trypsin化每个标记培养，以获得单细胞悬浮液，并通过离心收集分离细胞。将颗粒重新填充在稀释剂 C 缓冲液中，根据制造商的说明提供，并孵育细胞室温 10 分钟。用100微升胎儿牛血清终止染色反应，通过离心收集细胞。然后将具有完整生长介质的细胞离心，然后将五倍10电镀到每井的四个牙髓干细胞和肿瘤细胞，在两毫升全培养的两毫升中以一比一的比例。通过离心在24或48小时的孵育后收集细胞，然后用PBS清洗。然后，在5毫升的圆底流细胞学管和涡流中重新填充300微升荧光活性细胞分选缓冲液中的细胞，以分散任何细胞聚集体，然后根据标准流量细胞位分析方案分析细胞。牙髓干细胞在电镀后表现出成纤维细胞状细胞形态，并表达中层干细胞表面抗原，而不是造血细胞标记。在适当的分化鸡尾酒应用之后，在牙髓干细胞培养物中也观察到与骨质、软骨和致生分化相关的形态和分子水平的变化。与控制性中等治疗的肿瘤细胞培养物相比，以10%和20%的调控性培养物治疗的划痕性肿瘤细胞培养物显著增加划痕闭合。与控制细胞共培养物相比，牙髓干细胞插入共培养物的分泌分子也增加了受伤肿瘤细胞的划痕闭合。荧光显微镜分析牙髓茎和肿瘤细胞在体外细胞培养中的相互作用，揭示了一个组织良好的结构，其中肿瘤细胞在48小时后迅速增殖。在尝试此程序时，重要的是要记住将两个组织样本留在冷介质中，并立即将样品运送到实验室，以尽量减少细胞死亡。该技术开发后，为干细胞领域的研究人员探索成人干细胞癌在人类中的相互作用铺平了道路。按照这个程序，其他方法，如免疫细胞跟踪各种干细胞和癌症类型的差异标记细胞，以回答有关成人干细胞如何与癌细胞相互作用和控制癌症转移的其他问题。不要忘记，使用人体样本可能很危险，在执行此程序时，应始终实施诸如分析传染病患者样本和获得患者书面同意等预防措施。

Research

JoVE Journal

Cancer Research

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

科研

癌症研究

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

复杂核型细胞周期捕获细胞的生成与分离

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

从妇科和乳腺癌肿瘤获得癌症干细胞球

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

肝癌的仿生模型，用于研究具有可调生物物理特性的3D环境中的肿瘤-斯特罗马相互作用

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

研究TGF-β信号和TGF-β引起的乳腺癌和正常细胞的上皮到中位过渡

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...