Name: sample preparation strategies for mass spectrometry imaging of 3d cell culture models
Uploaded: 2014-12-05T13:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models at JoVE.com

This work was supported by the Walther Cancer Foundation under the Advancing Basic Cancer Research program to the Harper Cancer Research Institute of the University of Notre Dame (DARW) and the 2011 Starter Grant from the Society for Analytical Chemists of Pittsburgh (ABH). Thanks also to the staff of the Mass Spectrometry and Proteomics Facility for useful discussions.

1. 3D细胞培养和准备成像<ol><li>培养一合适的细胞系， 即，HCT116结肠癌细胞系，在二维，单层培养。 </li><li>成长的HCT 116细胞中的T-25烧瓶中的McCoy的5A介质，在5％的CO 2培养箱直至50-70％汇合，通常需要几天的时间+ 10％胎牛血清。 </li><li>释放的细胞用0.25％胰蛋白酶-EDTA溶液，并计数使用血球细胞的一部分和台盼蓝染色以检查细胞密度和生存力。 </li><li>计数后，稀释细胞用完全培养基以达到6000细胞的一个合适的接种密度/孔，或30000个细胞/ ml。 </li></ol> 2.准备琼脂糖涂层的96孔板<ol><li>添加0.19克琼脂糖至10ml标准培养基的50ml锥形管中（在介质的1.5％琼脂糖溶液）10。确保纳入轻轻搅拌。高压灭菌琼脂糖在水浴20分钟。 </li><li>使用多道移液器，吸50微升琼脂糖+媒体混合到平底96孔板培养板的60家中央水井。作为孔在板的外周边缘具有稍高的蒸发率，添加200μl的1×磷酸盐缓冲盐水（PBS）代替琼脂糖这些边缘。 </li><li>一旦琼脂糖混合物已被添加到所述内孔中，除了设置在板冷却到37〜 °C中的加入细胞前。 </li></ol> 3.种子，往往在三维培养注:更多或更少的细胞可以取决于所涉及的文化的要求进行接种，但已经确定，这些条件后10-14天培养物导致在大约1毫米直径的三维细胞培养物的结构（数据未显示） 。 <ol><li>加入200μl适当的细胞溶液（稀释含有〜6000细胞）的琼脂糖涂层96孔板中。覆盖板并在潮湿的培养箱，用5％的CO 2设定在37℃下细胞生长。 </li><li>改变细胞的媒体至少每48小时，或者出现媒体时，会改变颜色。 <ol><li>通过仔细吸出旧媒体从周围的小三维细胞培养物（可见的肉眼由第2天），在琼脂糖的底部改变介质。加入200微升新鲜培养基轻轻在三维细胞培养物。 </li><li> （可选步骤），在这些媒体的变化，增加化疗药物或其他药物进行测试。稀的首选药物，以提供完整的媒体的终浓度并添加必要的200μl到每个孔中。 </li><li>监控三维细胞培养物的生长通过光学显微镜，直到三维细胞培养物大约1毫米的直径。 </li></ol></li></ol> 4.收获的3D细胞培养<ol><li>用2毫升血清吸管从琼脂糖床上轻轻地取下3D细胞培养。 </li> &lt;LI&gt;轻轻吹打成约1毫升PBS在等待24孔板洗净3D细胞培养与PBS。 </li><li>传输通过血清​​吸管收获3D细胞培养的离心管中的蛋白质或代谢物的提取，或将它们嵌入在切割中，以协助对切片成像应用。 </li></ol>在明胶5.嵌入的3D细胞培养<ol><li>准备〜175毫克/毫升明胶的高纯度水（18MΩ首选），22,23混合明胶大力。观察溶液变得粘稠，难以操作。将含有明胶的锥形管在水浴中在约60℃，温暖，直到溶液变得清晰和容易抽吸。 注:温暖明胶溶液变硬迅速，因为它的冷却，所以冷冻前快速地执行所有以下步骤。 </li><li>之后将其加热，立即采取明胶到细胞培养罩。保持管明胶在BEAKER用预热的水在约60 °C至避免明胶溶液硬化。 </li><li>使用2ml的血清吸液管，沉积0.6毫升暖明胶溶液成24平底板的孔中。此体积是足以覆盖一薄层的底部。轻轻沉积立即上使用不同的2毫升吸管，以避免破裂的三维结构中的明胶层的顶部的三维细胞培养物。 注意:必须注意在此阶段不放置的PBS放入明胶。如果发生这种情况，但是，使用的是200微升吸管从明胶的顶部取出PBS中。 </li><li>后的三维细胞培养物放置在明胶层的表面上，小心地吸取另一个0.6暖明胶混合物在三维细胞培养物的毫升，以便不扰乱培养物的位置。之后的第二层放置，冻结明胶包埋三维细胞培养物在-80℃下。 </li></ol> 6切片和解冻日山Ë明胶嵌入式3D细胞培养质谱成像<ol><li>除去24孔板含有从冷冻的三维细胞培养物。暖板与热的底部，从你的手，直到镊子或者手术刀会轻轻滑下井的一侧。 </li><li>顺畅滑动钳或手术刀，释放明胶没有解冻的中心。取一滴的水，低温恒温器的支持，并以此来坚持明胶盘的支持。 <ol><li>嵌入在明胶三维细胞培养物是最适合于切片在-30℃。清洁低温恒温器叶片和玻璃在使用前用70％的乙醇。使用刀片或每个三维细胞培养物，明胶盘，以防止变钝的刀片的一个新的刀片的不同部分。 </li></ol></li><li>使用低温恒温器，轻轻面临关闭多余的明胶，直到3D细胞培养变得可见。 </li><li>此时慢慢切片3D细胞培养在12-16微米的部分一个平稳的运动。 注:关键因素切片包括玻璃到叶片的距离，刀片角度，低温恒温器的温度，并在盘上的支撑件的角度，因此，所有的这些都必须进行检查，以确保结果的一致性。 </li><li>小心解冻片和它们安装于氧化铟钛（ITO）涂覆的玻璃载片适当标记，并将其存储在干燥器中。使用切片尽快为最高质量。 </li></ol>对于MALDI成像7.矩阵的应用和样品制备<ol><li>此前MALDI成像，准备切片用适当的矩阵。 <ol><li>对于小分子分析，一般不需要洗涤步骤。为完整蛋白质的检测，用冷100％丙酮的切片，卡诺液，或70％/ 95％的异丙醇，以除去小分子，例如脂类，可能掩盖信号24 - 26 </li><li>轻轻洗脸不超过30秒的时间。别惊动任何片。 </li></ol></li><li>制备基质中有机溶剂和水的溶液，用0.1％的TFA。对于小分子诸如α氰基-4-羟基肉桂酸（CHCA），使用60％的ACN:40％的H 2 O:0.1％TFA（10毫克/毫升）。用于蛋白质检测，例如芥子酸（30毫克/毫升），使用相同的溶剂溶液。用于芥子酸的最佳溶解性，在乙腈首先溶解加入TFA中之前:H 2 O溶液。其它基质可以适当地使用。 注:基质可以与几种不同的方法被应用，但应当注意的一些矩阵已经发现共crystalize与明胶，如阿魏酸，使该样品不能使用。较小的晶体产生更一致的质谱，允许更多的分子种类进行检测，并归因于生理差异，而不是基体效应。 </li><li>通过handspotting方法应用矩阵。 <ol><li>用细尖的注射器适用0.5微升的MATR的三维细胞培养物切片上IX溶液，并允许矩阵crystalize。 </li></ol></li><li>或者，使用喷枪方法。填补了商业级喷枪与基体的解决方案。 <ol><li>持喷雾器8-12英寸远离幻灯片，瞄准精细喷雾在片。在道次之间，允许基质干燥1分钟，以允许最佳晶体形成。可达10-20通行证的喷枪的要求产生矩阵22的一个最佳的层。 </li></ol></li><li>或者，使用升华法在其他地方详细说明。25,27 </li><li>干片在干燥器中至少30分钟前MALDI-MSI，不管矩阵应用的方法的。25 </li></ol> 8. MALDI-MSI使用MALDI-TOF系统分析（ 即 AUTOFLEX III） 注:本部分包含的说明和建议的设置参数的MALDI-TOF成像实验。取决于插件trument制造商，所使用的软件，该方法可能会发生变化。 <ol><li>适合于制成以牢固地保持75毫米×25毫米幻灯片将图像附加到ITO涂覆的载玻片的三维细胞培养物的切片的适配器的幻灯片。加载幻灯片适配器插入仪器。 </li><li>选择的质量范围问题的适当校准标准准备仪器MALDI-MSI。对于小分子的询问，对应于所使用的基质，α-CHCA检测到的信号，是校准物的一个共同的基团。对于蛋白质分析，已知蛋白质标准（ 即，泛素，胰蛋白酶）的质量范围的兴趣被选择和斑点邻近于三维细胞培养物作为校准物。在进行方法开发之前校准仪器。 </li><li>在成像之前，开发的数据采集方法的选择，用由仪器制造商提供的软件的质量范围。参见图1和2为小分子和蛋白质成像。为小分子成像，使用反射模式的最高质量分辨率。 <ol><li>调整质量范围为小分子的数据采集与100-1,000 米/ z和对之间8,000-25000米/ Z蛋白质。调整质量范围以涵盖所选择的区域，同时提供与仪器的最高质量分辨率成为可能。 </li><li>调整激光功率，频率，激光射击和光斑尺寸使用校准溶液和附近的"牺牲"三维细胞培养物切片的数量。观察主要仪器控制软件这些设置。使用以下设置为推荐的出发点:60％的功率，400激光枪，超小尺寸光斑。这些设置将整个仪器和方法各不相同，因此优化仪器参数，从而为每个特定仪器的最高质量的光谱。可以调整其它参数包括检测器的增益，采样率，和电子增益。 SAVE这种方法在成像软件的使用（ 即 ，自动执行方法FlexImaging将借鉴）。 注意:下面的质量范围的兴趣抑制离子（在仪器控制再次发现），以便不干扰检测。 </li><li>开发蛋白质数据采集方法选择的质量范围。按照上面概述的相同的步骤，但对于中到高的质量范围，高于约3 kDa的，采用线性模式。 注:设置将不同于那些用于小分子的方法。 </li></ol></li><li>使用由仪器制造商，如FlexImaging对于布鲁克MALDI-TOF系统所提供的成像软件设置特定MS仪器参数。 <ol><li>创建一个新的影像文件和文件夹，使用前制定并保存方法。选择仪器参数，如光栅光斑尺寸（从默认的变化约200微米至50微米），设置仪器控制方法（设置步骤8.3.2或8.3.3），和POSI灰其中扫描整个三维细胞培养物。 </li><li>在样本选择三个合适的教点，激光移动到每个反过来。获得使用"打印屏幕"的MALDI-TOF激光控制软件的光学照片。 注:许多成像软件程序所需要的样本为&#39;教&#39;，可以用扫描仪或常仪器相机来获得软件的激光的位置，的光学照片。 </li></ol></li><li>然后开始从成像软件（未仪器控制）在成像过程并从每个切片获得质谱。观察最群众横跨三维细胞培养物和其他本地化到特定的区域。 </li></ol> 9.可选数据分析方法<ol><li>进行有针对性的分析，通过单击平均频谱群众进行光谱的人工分析，或允许成像软件自动选择以上特定阈值群众（ 如 ，AB峰奥雅纳前20％的阈值）。对于质量控制，检验的基质峰分布或平均频谱。 </li><li>执行统计分析，在数学软件提取的数据集，例如R或Matlab。 <ol><li>以ASCII格式，可读的文本文件，加载到所选择的软件出口单光谱。 </li><li>各个光谱转换为ASCII的mzML格式，mzXML，对于由几个不同的软件程序读取，28 - 31或数据导出为分析（.IMG）格式的文件中，用于其它成像应用例如MRI共同格式32分析两个开源选项MSiReader和BioMap 32,33。 </li><li>一旦文件被导出到一个可读的格式，通过软件，通过在各自的软件指令加载数据集。装载之后，执行采集后处理，诸如基线去除和正常化。 </li><li>有了这个数据集，执行为staticstical治疗，如使用任何自定义脚本或内置算法软件如Matlab 18聚类的主成分分析。 </li></ol></li></ol>

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B., Bohn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+Mass+Spectrometry+Imaging+and+Confocal+Raman+Microscopy+for+Studies+of+Three-Dimensional+Cell+Culture+Sections.">Correlated Mass Spectrometry Imaging and Confocal Raman Microscopy for Studies of Three-Dimensional Cell Culture Sections.</a> Analyst. 139 (18), 4578-4585 (2014).</li><li>Derda, R., Tang, S. K. Y., 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=Multizone+paper+platform+for+3D+cell+cultures.">Multizone paper platform for 3D cell cultures.</a> PloS one. 6 (5), 18940 (2011).</li><li>Imbeault, S., Valenzuela, N., Fai, S., Bennett, S. a. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+protein+in+3D+neural+stem+cell+culture:+a+hybrid+visualization+methodology.">Localizing protein in 3D neural stem cell culture: a hybrid visualization methodology.</a> J Vis Exp. 2 (46), 2-7 (2010).</li><li>He, W., Kuang, Y., 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=Proteomic+Comparison+of+3D+and+2D+Glioma+Models+Reveals+Increased+HLA-E+Expression+in+3D+Models+is+Associated+with+Resistance+to+NK+Cell-Mediated+Cytotoxicity.">Proteomic Comparison of 3D and 2D Glioma Models Reveals Increased HLA-E Expression in 3D Models is Associated with Resistance to NK Cell-Mediated Cytotoxicity.</a> J Proteome Res. 13 (5), 2272-2281 (2014).</li><li>Chen, R., Cape, S. S., Sturm, R. M., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometric+Imaging+of+Neuropeptides+in+Decapod+Crustacean+Neuronal+Tissues.">Mass Spectrometric Imaging of Neuropeptides in Decapod Crustacean Neuronal Tissues.</a> Methods Mol Biol. 656, 451-463 (2010).</li><li>DeKeyser, S. S., Kutz-Naber, K. K., Schmidt, J. J., Barrett-Wilt, G. A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+of+Neuropeptides+in+Decapod+Crustacean+Neuronal+Tissues.">Imaging Mass Spectrometry of Neuropeptides in Decapod Crustacean Neuronal Tissues.</a> J Proteome Res. 6 (5), 1782-1791 (2007).</li><li>Van Hove, E. R. A., Smith, D. F., Fornai, L., Glunde, K., Heeren, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+paper+based+tissue+washing+method+for+mass+spectrometry+imaging:+localized+washing+and+fragile+tissue+analysis.">An alternative paper based tissue washing method for mass spectrometry imaging: localized washing and fragile tissue analysis.</a> J Am Soc Mass Spectrom. 22 (10), 1885-1890 (2011).</li><li>Yang, J., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Sublimation/Recrystalization+for+Imaging+Proteins+by+Mass+Spectrometry+at+High+Spatial+Resolution.">Matrix Sublimation/Recrystalization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution.</a> Anal Chem. 83 (14), 5728-5734 (2012).</li><li>Seeley, E. H., Oppenheimer, S. R., Mi, D., Chaurand, P., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+protein+sensitivity+for+MALDI+imaging+mass+spectrometry+after+chemical+treatment+of+tissue+sections.">Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections.</a> J Am Soc Mass Spectrom. 19 (8), 1069-1077 (2008).</li><li>Gemperline, E., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-mass+spectrometric+imaging+for+the+investigation+of+metabolites+in+Medicago+truncatula+root+nodules.">MALDI-mass spectrometric imaging for the investigation of metabolites in Medicago truncatula root nodules.</a> J Vis Exp. 1 (85), 1-11 (2014).</li><li>Deutsch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mzML:+a+single,+unifying+data+format+for+mass+spectrometer+output).">mzML: a single, unifying data format for mass spectrometer output).</a> Proteomics. 8 (14), 2776-2777 (2008).</li><li>Pedrioli, P. G. a., Eng, J. K., 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+common+open+representation+of+mass+spectrometry+data+and+its+application+to+proteomics+research.">A common open representation of mass spectrometry data and its application to proteomics research.</a> Nat Biotechnol. 22 (11), 1459-1466 (2004).</li><li>Lin, S., Zhu, L., Winter, A., Sasinowski, M., Kibbe, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+mzXML+good+for.">What is mzXML good for.</a> Expert Rev Proteomics. 2 (6), 839-845 (2005).</li><li>Orchard, S., Hoogland, C., Bairoch, A., Eisenacher, M., Kraus, H. -. J., Binz, P. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+the+data+explosion.+A+report+on+the+HUPO-PSI+Workshop.+August+2008,+Amsterdam,+The+Netherlands.">Managing the data explosion. A report on the HUPO-PSI Workshop. August 2008, Amsterdam, The Netherlands.</a> Proteomics. 9 (3), 499-501 (2009).</li><li>Robichaud, G., Garrard, K. P., Barry, J. a., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSiReader:+an+open-source+interface+to+view+and+analyze+high+resolving+power+MS+imaging+files+on+Matlab+platform.">MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform.</a> J Am Soc Mass Spectrom. 24 (5), 718-721 (2013).</li><li>Andersson, M., Groseclose, M. R., Deutch, A. Y., Caprioli, R. 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This article is part of a special issue on Multimodal Pre-Clinical Imaging, sponsored by Bruker Biospin. The MALDI-TOF system used to obtain the representative results is a Bruker AutoFlex III.

使用以上概述的方法中，三维细胞培养物可以生长并用MALDI-MSI进行分析。许多永生化细胞系将形成三维结构的培养适当9,10应注意培养，但没有通过次数过多，即，具有低传代数的细胞时。在一般情况下，细胞10和下部的通路数是最佳生长的三维细胞培养物。越来越多的3D细胞培养在传统的琼脂糖支持提供了具有成本效益的方式为有志于3D细胞培养来尝试这个制度的研究小组。此方法利用少数部件尚未在大多数哺乳动物细胞培养实验室。仔细必须注意的琼脂糖板成长的准备，这样的三维细胞培养物的大小和形状相一致;一些方面中，需要小心注意包括检查没有气泡被截留到该混合物中，并且形成在每个孔的底部瓦特适当弯月ELL。如果弯液面不形成正常，细胞可生长在非球体形状或小团块。此外，必须注意不要将PBS与球状体的明胶。如果发生这种情况，使用200微升移液器明胶的顶部取出PBS。如果成本不是一个因素，超低结合圆底板是可商购的，并提供对这些步骤的简化。而其它组织包埋方法也可能是昂贵的和非质谱兼容，明胶包埋已被证明是既便宜和通用性。上述制备的策略已被优化为三维细胞培养物，以获得最高质量的数据。而明胶包埋已被证明是与质谱分析兼容，该方法确实缩小由于共结晶的可用矩阵。一旦冻结，所述三维细胞培养物可稳定几个月，只要它们不冻结 - 解冻。冻结当明胶变得不透明，并且3D细胞文化是一个类似的颜色，所以它是冻结记录三维细胞培养安置切片，以帮助前为宜。 当制备载玻片，基体可以用几种不同的方法被应用，但应注意的是，一些矩阵已经发现共crystalize与明胶，如阿魏酸，使该样品不能使用。较小的矩阵晶体产生更一致的质谱。而handspotting是最简单的矩阵应用的方法，该大的溶剂体积可以导致较大的晶体尺寸和显著分析物的迁移。 在质谱仪，先进的MALDI-MSI技术已经降低了所需的开始分析的专门知识。数据采集​​和分析是直接在大多数系统上，让即使是新手，以获得ITO涂层幻灯片高质量的信息。仔细选择仪器参数，优化对相邻的非图像值得三维细胞文化性ES，是为了获得高品质的数据的关键。例如，增加激光功率可能会增加峰值强度，但降低激光功率可提高分辨率并给予更对称的峰形。 3D细胞培养应迅速切片，以确保最佳的样品质量后使用。蛋白信号的退化可能在不到一周的时间没有适当的存储（干燥器/ -80℃冷冻器）中可以看出，特别是在高质量区域（高于20 kDa的）。由于不断增长的需求，许多不同的可视化的软件程序已经开发并可以自由地研究公开。大多数成像质谱仪已经安装了必要的可视化单一群众对每台仪器使用的专有格式的软件。这些软件程序可以用于获得单质的图像和导出数据。大多数软件还允许两个或多个群众利益的叠加。此外，许多不同的观众MSI的数据都可以免费下载，如MSiReader的ðBioMap。32,33获得，也可以处理收购后的轻松，带来了更多有用的信息，以最前沿的质谱数据。 从制药到脂质与蛋白质，上面概述的系统允许快速采集的数据，以评估感兴趣分子的整个三维细胞培养结构的分布。连续切片可以通过从三维细胞培养物的一个片段的每个2D图像构建被带到图像整个3D结构。在协议的技术和注意事项将使用以希望开始三维细胞培养为单层培养和动物模型之间的桥梁的研究人员。一旦掌握，这些技术可以应用到多种类型的三维细胞培养物，组织和细胞培养物，使研究人员能够像它们选择任何样品。

Mass spectrometry imaging (MSI) is a powerful technique to examine the distribution of molecular species across samples of interest. Researchers have imaged biological samples from whole animals down to single cells with this technique.1&#8211;4 Using MSI hundreds of analytes may be imaged at a single time, rather than single-substrate techniques requiring the development of additional fluorophores for traditional confocal microscopy, or staining for histology.5&#8211;7 Additionally, MSI can be used in either a targeted or a discovery-based fashion, making it an attractive approach to look at the overlap between the distribution of chemical species. Presented within are methods for the growth, preparation, and matrix assisted laser desorption ionization/desorption (MALDI) imaging of small samples (~1 mm in diameter) of three-dimensional multicellular cell cultures.8&#8211;11
3D cell cultures are of particular interest to the biological community as an intermediary between traditional monolayer culture and in vivo tumors. As the name implies, 3D cell cultures are heterogeneous cellular aggregates that contain many of the chemical gradients in the microenvironment observed in small avascular tumor nodules, making them a more realistic model system to the native tumor environment than monolayer culture.10 Many human cell lines can be grown as 3D cultures, including cell lines derived from colon, breast, ovary, prostate, kidney and lung tissues.10 There are inherent challenges in imaging three dimensional cell cultures as they are smaller than many samples commonly used for mass spectrometry imaging and therefore require careful sample preparation.7 However, the advantages of these tractable model systems include lower cost and faster growth and analysis time compared to animal models. As a result, studies involving three dimensional cell cultures can be higher throughput than traditional animal studies.12&#8211;14
All of these advantages have resulted in increasing popularity for three dimensional cell cultures in biological research; there is a corresponding need to develop new analytical strategies to explore both the composition and spatial distribution of molecules in these systems.12,15&#8211;21 The goal of this manuscript is to describe the adaptation of mass spectrometry imaging methods and sample preparation to three dimensional cell cultures.

<table><tbody><tr><td>HCT116 Cell line</td><td>ATCC</td><td>ATCC CCL-247</td><td>Potential hazard, handle with care</td></tr><tr><td>McCoy&#039;s 5A media</td><td>Gibco</td><td>16600-108</td><td></td></tr><tr><td>Fetal Bovine serum</td><td>HyClone</td><td>SH30071.03</td><td></td></tr><tr><td>0.25% Trypsin-EDTA solution</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Gelatin, unflavored</td><td>Knox</td><td></td><td>Available at most grocery stores in single-packets</td></tr><tr><td>ITO-coated glass slides</td><td>Delta Technologies, Limited</td><td>CG-90IN-S115</td><td></td></tr><tr><td>&amp;alpha;-Cyano-4-hydroxycinnamic acid (CHCA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C8982</td><td></td></tr><tr><td>Sinapic Acid</td><td>Sigma-Aldrich</td><td>85429</td><td></td></tr><tr><td>0.3 mm Gravity Feed Dual-Action Airbrush Paint Spray Gun Kit Set</td><td>RoyalMax Airbrush</td><td></td><td>Many options available on sites such as Amazon.com</td></tr></tbody></table>

sample preparation strategies for mass spectrometry imaging of 3d cell culture models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

微星成像有揭示在三维细胞培养物中的许多不同的分子的分布的可能性。使用上文概述的方法，由小分子（ 图1）到大的蛋白质（ 图2）物种可能跨越文化进行跟踪。这些结果显示与游离工具MSiReader 32结果可用于单离子与可视化软件在整个三维细胞培养物的兴趣或者在感兴趣或一个区域中的整个扫描区域进行分析的单个离子的可视化。另外，大多数软件会自动可视高于某个阈值由用户，这可能有助于发现努力设置离子。一旦单个离子的地图获得，大多数软件包括叠加图像，查看离子共存的能力。无论是在发现为基础的方法或者以靶向方式，质谱成像是一种强大的工具，来分析三维细胞培养物。 图3D细胞培养中生长和切片。左）一个完整的大肠癌HCT-116 3D细胞培养结构为前收获观看第14天光学图像1.代表性的结果 。右图 ​​）大肠3D细胞培养解冻大约赤道片的光学影像安装在涂有ITO的幻灯片图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52313/52313fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52313/52313fig2highres.jpg" /> 图中的小分子的MALDI-MSI 2.代表性结果的三维细胞培养。顶）平均质谱中的低质量（小分子）的范围实现了与α-CHCA 的赤道部分 。底部）代表图像单质:327.8米/ Z主要定位于所述三维细胞培养物和内部429.7米/ Z主要定位于所述三维细胞培养物的边缘。虚线表示球体的近似边缘。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52313/52313fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52313/52313fig3highres.jpg" /> 图蛋白成像的代表性的结果的三维细胞培养物。顶）平均质谱实现了与芥子酸在较高（蛋白）的质量范围的赤道部分的通过MALDI-MSI。底部）单质的代表性图像:10871米/ Z主要定位于球体的边缘，并12184米/ Z更朝向旋转椭圆体的内部进行本地化。虚线表示旋转椭圆体的近似边缘。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52313/52313fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models at JoVE.com

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

永生化癌细胞系可以种植三维细胞培养物，生物研究的一个有价值的模型。本协议描述的3D细胞培养，包括改进的样品制备平台质谱成像。该协议的目的是为了指导用户以制备三维细胞培养物用于质谱成像分析。

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

sample-preparation-strategies-for-mass-spectrometry-imaging-3d-cell

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

18.1K 次观看.  University of Notre Dame. 永生化癌细胞系可以种植三维细胞培养物，生物研究的一个有价值的模型。本协议描述的3D细胞培养，包括改进的样品制备平台质谱成像。该协议的目的是为了指导用户以制备三维细胞培养物用于质谱成像分析。 

视频: Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

利用成像质谱技术捕获组织与细胞之间的小分子通信

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

科研

癌症研究

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

生物化学

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

化学

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

生物工程

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

功能性 3D 球状体细胞培养物的半自动表型分析

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

通过三维细胞培养基质的紫外光图案化产生多库细胞微环境

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

单探头的应用:根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

生物学

iPSC 来源的神经元和星形胶质细胞的 3D 生物打印共培养物

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

作者聚焦:推进 3D 细胞建模 – 一种用于神经共培养的高通量方法 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

样品制备策略的3D细胞培养模型质谱成像

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此视频中的章节

利用成像质谱技术捕获组织与细胞之间的小分子通信

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

制备及分析

功能性 3D 球状体细胞培养物的半自动表型分析

通过三维细胞培养基质的紫外光图案化产生多库细胞微环境

单探头的应用:根据环境条件质谱成像和单细胞分析

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用