这项工作是由卫生部，国家癌症研究所（1R21CA141039），BD生物科学的研究资助奖，美国癌症协会的机构研究资助（IRG＃58-006-53），在普渡大学癌症中心的国家研究院​​的赞助研究，健康研究和阿尔伯塔癌症委员会研究倡议计划的加拿大学院。 MP是由美国国家癌症研究所癌症预防实习计划的国家卫生研究院，支持（R25CA128770; PI:D. Teegarden）的肿瘤学科学中心和发现式学习研究中心在美国普渡大学管理。

1。提前准备<ol><li>保持无菌的血清学移液器和移液器吸头，在4°C。 故障排除:凝胶基质胶在室温（RT），采用冷移液器和技巧将防止基底膜从文化在安装过程中胶凝。 </li><li>解冻一瓶基底膜在4℃过夜。 </li></ol> 2。试剂的制备<ol><li>制备的纤维结合素储备液:使纤连蛋白的1毫克/毫升的储备溶液以在100毫升蒸馏水中溶解100毫克的人纤连蛋白。一旦纤连蛋白溶解，无菌过滤，分装，并在4℃下储存长达6个月。 故障排除:如果纤连蛋白不溶解很快孵育数分钟在水浴集的溶液在37℃下</li><li> 10倍PBS制备:溶解80克氯化钠氯化钠，14.4克Na 2 HPO 4，2克氯化钾和2.4克KH 2 PO <suB&gt; 4在1升的蒸馏水中。将pH设定为7.4，无菌过滤，并在4℃下贮存长达6个月。 </li><li>的中和缓冲液20的制备:使含有100mM HEPES在2x磷酸盐缓冲盐水（PBS），使用1M的HEPES和10倍的PBS中的储备溶液中的溶液。调节溶液的pH至7.0。储存在4℃下2-3个月。 </li><li> 2 mg / ml的溶液鼠尾胶原类型I的制备:使中和缓冲液（步骤2.3）2毫克/毫升的胶原蛋白I的解决方案。涡的解决方案在低转速和移液器向上和向下轻轻混匀。由于这胶原蛋白是非常粘稠，避免气泡的产生，同时吸取。建议在每次准备新鲜胶原蛋白I的解决方案。保持溶液置于冰上，直到使用。 </li><li>骨内涂层（雷德）的制备:将1毫克/毫升纤维连接蛋白分别与股票2 mg / ml的I型胶原股票（步骤2.1和2.4），以77μg/ ml的终浓度为29微克/毫升，在1x＆＃160;无菌PBS。混合并在4℃下储存长达1个月。 </li><li>准备重建骨基质（RBM）的: <ol><li>预冷的1.5 ml离心管置于冰上。置于冰上的所有矩阵解决方案，在任何时候。 </li><li>混合4份基质胶（从很多到很多基质胶浓度变化，但变化被认为是可以忽略不计），2.5份1毫克/毫升纤维连接蛋白和1份2毫克/毫升的胶原蛋白I冰首先吹打成基底膜使用冷管移液器吸头，然后添加纤维连接蛋白和胶原基质胶一开始凝固温度高于4℃，所以工作迅速而吹打基底膜。混合矩阵轻轻吹打向上和向下，避免引入气泡。保持管上的冰，而混合RBM。 </li></ol></li><li>骨髓生长培养基（BMGM）的制备:使500毫升BMGM含6.2×10 -4 M 氯化钙 ，10 -6 M琥珀酸钠，10 -6 M氢化可的松，20％的人血浆（正常或恶性来自健康供体或患者收集的），和1％青霉素/链霉素的RPMI-1640。的CaCl 2，琥珀酸钠和氢化可的松补充剂可以储存在-80℃下&gt; 6个月为1000×储备溶液。当生长的细胞系，胎牛血清（FBS）的可取代的人血浆。 </li><li>的10％中性缓冲福尔马林（NBF）的制备:加入37％的甲醛原液1倍PBS中的10％V / V的搅拌均匀，在室温下存放于避光2-3周。 </li><li> 1％牛血清白蛋白（BSA）的制备:称取BSA适量和溶解在1×PBS中。储存在4℃下，1周之内使用。 BSA溶液往往受到感染，如果冷藏保存更长的时间。或者，BSA溶液可以等分并冷冻在-20℃下长期贮存。避免反复冻融。 </li><li>细胞恢复解决方案（CRS）准备:5毫米EDTA，1 mM的钒酸钠（钠&lt;子&gt; 3 VO 4）和1.5mM氟化钠（NaF）溶液中的1×PBS。无菌过滤并储存于4℃至6个月。 </li></ol> 3。非恶性和恶性人类骨髓细胞内含3-D文化<ol><li>通过按照制造商的指示，聚蔗糖 - 斑块梯度离心纯化初级骨髓单个核细胞。 可选步骤 :细胞可以被标记，用0.25微米羧基二乙酸琥珀酰亚胺酯（CFSE）按照生产商的说明来跟随细胞增殖整个培养期间。 故障排除:如果细胞的CFSE染色后死亡，滴定CFSE的量随着浓度取决于细胞类型; 0.25微米很适合初级骨髓单个核细胞。 </li><li>加65微升撕裂溶液放入48孔组织培养处理板的每孔中。分散液均匀地覆盖井的整个表面上并孵育&gt; 30分钟，在室温。 </li><li>制备细胞悬浮液以0.5×10 6个细胞的10微升的48孔板的1×PBS中，每孔的密度。在1.5ml微量离心管中混合的细胞悬浮液以每孔100μlRBM矩阵。混合细胞和基质RBM轻轻吹打向上和向下，避免引入气泡。置于冰上细胞/基质混合物，以避免基质凝胶。 注:成果管理系统是完全可扩展，建立了系统在非48孔格式的基础上，用板的表面面积调整所有试剂（基质和介质）的款项。比例进行相应的镀细胞的数量。 </li><li>吸出剩余的撕裂，并加入细胞/基质混合物成的阱的中心。很快，散布细胞/基质混合物以覆盖井的整个表面上均匀。放置板30〜60分钟在37℃，5％CO 2的组织培养箱中，使基质固化。请勿摇晃或断开孵化期间TURB板。 故障排除:为了防止细胞分布不均的RBM，拌 ​​匀之前，电镀。混合后，每5 个孔接种，以避免细胞沉降到管底。一次镀，细胞不下沉到由于表面张力在基质中的井的底部。 故障排除:为了防止细胞分布不均的RBM，拌 ​​匀之前，电镀。混合后，每5 个孔接种，以避免细胞沉降到管底。一次镀，细胞不下沉到由于表面张力在基质中的井的底部。 </li><li> Prewarm BMGM在水浴设定为37℃。 </li><li> 30分钟后，请检查矩阵已正确设置，它会形成一个柔软的凝胶状层时，板倾斜不动。用1毫升温BMGM的覆盖细胞/基质层。为了避免矩阵吸管介质撕裂轻轻地使用（点胶滴逐滴）血清学移液管。将组装RBM文化成37℃，5％CO2细胞培养箱和培养所需的时间段。 注:细胞活力可维持至少30天不改变介质;如果需要换液每次只改变介质的50％。 关键的一步:它是至关重要的介质不冷，冷的温度可能扰乱已经设置的矩阵。小心不要喷在介质上的矩阵的顶部就可能会出现软基质层。 故障排除:如果在成果管理系统细胞活力低，细胞密度可能有实验用的细胞比骨髓抽吸初级单核细胞等工作，则必须确定。当用细胞系的工作，降低了细胞数目的10倍，作为永生化细胞系增殖比原代细胞更快。 </li></ol> 4。 "在矩阵&#39;成像<ol><li><strong&gt;光学显微镜<ol><li>图像的培养细胞直接在RBM使用明场，微分干涉对比（DIC），共聚焦或任何其他成像技术，使远距离成像的矩阵是大约1毫米厚。使用上的许多显微镜可用的Z堆叠功能，图像的整个培养从上到下。 </li></ol></li><li> 免疫荧光染色 提示:对于下面的染色协议避免吸染色/洗涤解决方案，而不是倾斜的板，用吸管取出的解决方案。 <ol><li>取出BMGM用移液管，洗2〜3倍，用RT的1X PBS 100μL/洗/孔。确保足够的PBS用于覆盖所述孔的整个表面上。 </li><li>通过加入100微升10％NBF /孔（对于48孔板）15分钟，在室温下固定细胞在基质中。删除NBF并与RT的1X PBS 100μL/洗/孔洗涤细胞两次。 注:避免使用冷NBF，因为它可能液化矩阵。 </li><li>除去PBS中，过夜，以阻止抗体的非特异性结合加1％BSA的1小时在室温或4℃。 </li><li>制备在1％BSA的第一抗体溶液在所需的稀释。稀释会各有不同，每个抗体，请与制造商或滴定，以确定最佳的抗体浓度。加的第一抗体溶液100μl/孔，并根据制造商的说明孵化。 关键步骤:所有涉及缀合的荧光团或荧光染料的抗体孵育应在黑暗中进行。 </li><li>除去第一抗体溶液，并用1×PBS洗涤3次5分钟。 </li><li>用于间接免疫荧光染色，培养与缀合至荧光探针1小时在室温下的二次抗体。稀释的抗体在1％BSA的浓度由制造商建议的。 </li><li>删除辅助一ntibody溶液并用1×PBS洗涤3次5分钟。 故障排除:为了避免高背景成像过程中，滴定荧光标记的二抗和使用提供足够的信号的最低浓度。如果高的背景仍然是一个问题，增加洗涤步骤的数量和持续时间，或使用直接结合的一抗，并跳过步骤4.2.6。 </li><li>染色的细胞核中添加4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI）的核染色溶液中，在由制造商所建议的稀释，并孵育在室温下7-10分钟。 </li><li>除去核染色溶液中，用1×PBS中洗2×5分钟。 </li><li>从最后一次洗涤除去PBS并加入一滴安装介质（常规设置），在样品上保留的荧光。图像上使用荧光或共聚焦显微镜适当的激发/发射设置。 注:成像应在2天内染色完成为了避免矩阵和RBM完整性和荧光信号的损失损耗下降。如果不立即成像，彩色样本应保持在4℃，避光，直到成像。 </li></ol></li></ol> 5。从RBM细胞的分离<ol><li>轻轻取出介质，而不会干扰基质层。不要吸。 </li><li>洗涤细胞2倍，用无菌1X PBS。不要吸。 </li><li>加入0.5毫升冰冷的CRS到每个孔中，并伴随着细胞移除整个基质层，通过大力上下吹打。收集细胞，基质，和CRS在1.5ml微量离心管中。放置于冰上。 </li><li>添加一个额外的0.5毫升CRS在同一井和收集所有剩余的材料。转移到同一个离心管中。 </li><li>涡流细胞，基质，和对照品，将混合物在冰上孵育1小时。涡流细胞每隔15分钟，以促进从基质释放。 </li><li> 1小时，无肉眼可见团块后检查混合物矩阵应该存在。 故障排除:如果是矩阵的团块仍然可见1小时后，转移细胞/矩阵/ CRS混合到一个更大的管中，加入额外2-5毫升的CRS，孵育一个额外的30-60分钟。 </li><li>离心细胞，在CRS在4℃下1,000 rpm离心5-10分钟去除上清，重悬细胞沉淀在1ml冷的1X PBS的。 </li><li>离心机在1,000 rpm下5-10分钟，去除上清。重复的PBS洗一次。 </li><li>第二PBS洗涤完成细胞从基质和分离的细胞中回收，可用于任何下游应用，如DNA / RNA /蛋白质分离，流式细胞术， 在体内移植等 。 </li></ol>

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Physiol. 76, 77-84 (1970).</li><li>Bug, 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=Rho+family+small+GTPases+control+migration+of+hematopoietic+progenitor+cells+into+multicellular+spheroids+of+bone+marrow+stroma+cells.">Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells.</a> J. Leukocyte Biol. 72, 837-845 (2002).</li><li>Provenzano, P. P., 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=Collagen+reorganization+at+the+tumor-stromal+interface+facilitates+local+invasion.">Collagen reorganization at the tumor-stromal interface facilitates local invasion.</a> BMC Med. 4, (2006).</li><li>Tancred, T. M., Belch, A. R., Reiman, T., Pilarski, L. M., Kirshner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Expression+of+Fibronectin+and+Collagens+I+and+IV+in+Multiple+Myeloma+and+Monoclonal+Gammopathy+of+Undetermined+Significance.">Altered Expression of Fibronectin and Collagens I and IV in Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance.</a> J. Histochem. Cytochem. 57, 239-247 (2009).</li></ol>

作者宣称没有利益冲突。

成果管理模型提供其中的细胞在BM的组合物保持离体长达30天的综合系统。骨髓细胞按其功能密切模仿体内发现的BM架构RBM自分层种植。此外，这是第一个系统，它允许对多发性骨髓瘤克隆8的扩张。为确保BM细胞的生长健壮和培养物的整体成功的最关键的步骤是:1），得到的BM细胞与&gt; 70％生存力和较自由的红血细胞的健康人群，2），以确保基质组分充分混合，使细胞均匀地?...

组织培养物的开发是为了研究在受控环境中的细胞行为进行比较在完整有机体的各种处理时，以尽量减少系统的可变性。最初成立于1900年初1,2这种方法，指的是一种技术，其中组织外植体离体培养中玻璃盘。在20世纪中期的系统适应于分散生长的细胞，而不是完整的组织碎片，和术语"组织文化"和"细胞培养"成了代名词3。在这种传统的细胞培养系统中的细胞生长的组织培养塑料覆盖以补充有各种生长因子的生长培养基的表面上。贴壁细胞培养物，其中细胞附着和铺展在组织培养塑料和非贴壁培养物，其中细胞增殖的悬浮液:两种培养物都基于不同细胞的粘附能力出现了。由于细胞的初期文化，多永生细胞系已经建立，如海拉4，第一个人类癌症细胞系。这些细胞系有细胞培养增殖下去的能力，也是最不需要任何特殊处理，以保持活力。 的原始细胞的培养方法的还原论方法的目的是要简化系统尽可能地由仅包括维持细胞生存力和增殖所需要的最低限度的部件。然而，简化的细胞培养方法无法支持体外最主要的人体细胞具有有限寿命的类型。因此，新的培养系统被设计为近似的组织微环境尽可能接近，从而使细胞外植体在生理条件下生长。在对比的常规/还原论的细胞培养方法中，3维（3-D）培养系统正在成为一种优选的方法，以有效地培养各人类细胞系和原代细胞随后学习与之相关的健康和疾病的机制，在支持微环境的上下文。这样的3-D系统通常被设置成使用补充有生长因子的重构的矩阵和/或介质，以概括的组织微环境，研究目的细胞类型。第一这些3维（3-D）培养模型的开发是为了研究乳腺发育。提供细胞的天然条件的乳腺上皮细胞包埋在基质胶中，IV型胶原和细胞外基质（ECM）的富含层粘连蛋白来源，并覆盖有生长培养基中。在这样的条件下，乳腺上皮细胞形成簇相似的乳腺腺泡，并且在刺激催乳激素，这些腺泡分泌的酪蛋白，和其它乳蛋白，成腺泡状结构的中空旭光。在标准的文化，甚至除了催乳素5后酪蛋白的分泌没有观察到，进一步强调了微环境中保持细胞的形态学和表型特征的作用。当细胞被取出，它们的生理微环境的上下文中的正常细胞功能的丧失的另一个示范是一个示范，如果不支持的微环境，角化细胞不能形成分层表皮6。已经建立了一些其他的3-D模型，以允许主单元7,8 的体外繁殖。 微环境中的细胞行为的关键作用是优雅的表现在哪里乳腺上皮细胞形成了"由内而外"腺泡胶原蛋白的培养，当我基质相比，形成于基底膜的正确极化腺泡的研究。当层粘连蛋白产生肌上皮细胞被添加到I型胶原培养9这个损失的适当的形态恢复。此外，正确的微环境所需的精确基因型表现。生长在培养皿中，转染的细胞 - 细胞粘附分子CEACAM1 MCF7乳腺癌细胞行为完全一样的未转染CEACAM阴性细胞。然而，当在基质胶中培养的，在ECM中，野生型形式的MCF7肿瘤样结构接触而MCF7细胞转染CEACAM1恢复到正常的表型和腺泡形态与中空旭光，因为已经建立了与非恶性乳腺上皮细胞10。类似地，阻断β1-整合素在乳腺癌细胞中不改变他们的行为的标准培养条件下，但以3-D培养物进行同样的实验表明，阻断β1-整合恢复恶性细胞为正常表型11。因此，组织的微环境，不仅需要维持细胞生存力，也能保持正常的细胞功能。 除了提供其中细胞行为可以在生理条件下被研究的系统秒，3维文化作为强大和可靠的介质的新型疗法8,12,13临床前试验。在3-D培养细胞可研究化合物的筛选环境介导的耐药性14，在细胞-细胞和细胞-细胞外基质粘附的贡献可以评估的条件下进行。此外，新化合物的脱靶毒性可通过将各种组织的多种细胞区室来确定。这样的屏幕可以更快速地进行，且更具成本效益比在体内 8的可比性研究。 这里，我们提出的重构的骨髓（RBM），其中正常和恶性骨髓（BM）细胞增殖的体外系统中的密切模仿人类骨髓微环境的一个3-D模型的设置。先前尝试在二维培养，液体或BM间质中各种成分粘附共培养成长初级人类骨髓细胞，或3-D，半固体琼脂培养获得了有限的成功，因为它们无法提供细胞组织微环境15-18的组成部分。在这些系统中主要的骨髓细胞有活力差，不能增殖体外 。其中人骨髓祖细胞生长在旋转椭圆体的共培养与骨髓基质细胞的另一个系统是其中造血干细胞的活力和增殖的持续时间至少为96小时，19的三维系统。然而，尽管向造血祖生物学的理解非常有益的，这种系统并不忠实地概括了BM的由于微环境是缺乏细胞外基质成分的，因此，限制了其有用性。这里所描述的RBM模型提出了一个全面的系统，人类骨髓细胞和细胞外车厢被重建体外 。我们证明了RBM文化能够支持正常的人骨髓细胞的体外生长，因为我们LL作为细胞治疗各种血液病隔离。

<table border="0" cellpadding="0" cellspacing="0" width="877">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">CellTrace CFSE proliferation kit-for flow cytometry</td>
			<td style="width:239px;">Invitrogen/Life technologies</td>
			<td style="width:108px;">C34554</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Fibronectin from human plasma</td>
			<td>Millipore</td>
			<td>NG1884448</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Ficoll-Paque PLUS</td>
			<td>GE Lifesciences</td>
			<td>17-1440-02</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Matrigel basement membrane matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:328px;">Rat tail collagen type I</td>
			<td>BD Biosciences</td>
			<td>354249</td>
			<td style="width:203px;">Collagen I from Millipore does not polimerize properly using this protocol</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">Vectashield mounting medium for fluorescence (regular set)</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">SigmaFast Red TR/Napthol AS-MX tablets</td>
			<td>Sigma-Aldrich</td>
			<td>F4648</td>
			<td style="width:203px;">For TRAP staining</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">SigmaFast BCIP/NBT tablets</td>
			<td>Sigma-Aldrich</td>
			<td>B5655</td>
			<td style="width:203px;">For alkaline phosphatase staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Zeiss confocal LSM 510 microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">Zeiss 510 software</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td style="width:203px;">Image analysis software for confocal analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">Zeiss Axiovert 200M inverted microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">MetaMorph software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td style="width:203px;">Image analysis software for Axiovert 200M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:328px;">FACSort flow cytometer&#160;</td>
			<td>BD biosciences</td>
			<td></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:328px;">CellQuest Pro software&#160;</td>
			<td>BD biosciences</td>
			<td></td>
			<td style="width:203px;">Software for the analysis of flow cytometry data</td>
		</tr>
	</tbody>
</table>

a three-dimensional tissue culture model to study primary human bone marrow and its malignancies

组织文化是一个非常宝贵的工具来研究细胞功能的许多方面，从正常发育疾病。常规细胞培养方法依赖于细胞的任一附加到组织培养皿的固体基质或在悬浮液中生长在液体培养基中的能力。多个永生细胞系已经建立和使用这样的方法生长的，然而，当原电池需要被生长的体外这些方法常常失败。这种故障已被归因于缺少从那里组织培养塑料被用作用于细胞生长的表面的标准系统的组织微环境的适当的细胞外基质成分。细胞外基质是组织微环境的一个组成部分，它的存在是对生理功能，如细胞分化，存活和增殖的维持是至关重要的。在这里，我们提出了一个3维的组织培养方法，其中主骨生髓CELLS是生长在细胞外基质配方，概括了人骨（RBM系统）的微环境。嵌在细胞外基质，细胞通过补充了人血浆中的介质供给营养物质，从而提供了一个完整的系统，其中细胞的存活和增殖可以持续长达30天，同时保持了原代组织的细胞组成。使用成果管理系统，我们已经成功地生长正常捐助者和患者的淀粉样变，以及各种血液系统恶性肿瘤原发性骨髓细胞。成果管理系统允许的新型疗法的临床前药效的细胞行为和评价直接在矩阵的实时可视化。此外，细胞可以从RBM中分离，并随后用于体内移植，细胞分选，流式细胞术，以及核酸和蛋白质的分析。综合来看，成果管理方法提供了可靠的系统，用于原发性骨MARRO增长在生理条件下瓦特细胞。

由于原发性骨髓细胞不能标准组织培养条件下增殖，成果管理系统的建立是为了紧密地模仿骨微环境，提供一个系统的文化和扩大骨髓细胞体外 。在BM细胞外基质，纤维连接蛋白，Ⅰ型胶原，Ⅳ型胶原，层粘连蛋白和21的主要组成部分，已被纳入RBM矩阵，以提供结构骨架，以这种3-D的文化。骨内膜，骨及骨髓，富含胶原蛋白I和纤连蛋白之间的接口，被重建通过涂覆组织培养皿与这?...

Watch this Scientific Journal Video about A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies at JoVE.com

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

在标准培养方法细胞被取出来的生理环境，生长在菜的塑料表面。研究原代人骨髓细胞的行为，我们创建了其中细胞条件扼要组织的天然微环境下生长的三维培养系统。

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

a-three-dimensional-tissue-culture-model-to-study-primary-human-bone

Research

JoVE Journal

Medicine

16.6K 次观看.  Purdue University. 在标准培养方法细胞被取出来的生理环境，生长在菜的塑料表面。研究原代人骨髓细胞的行为，我们创建了其中细胞条件扼要组织的天然微环境下生长的三维培养系统。 

视频: A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all 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 ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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 ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

用膝关节关节仪评价大鼠膝关节屈曲收缩的特定组织贡献

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...