Name: robotic production of cancer cell spheroids with an aqueous two-phase system for drug testing
Uploaded: 2015-04-23T15:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing at JoVE.com

The authors acknowledge funding from the National Institutes of Health R21CA182333.

1.准备聚合物双水相体系（ATPS） <ol><li>权衡0.5克聚乙二醇（PEG）（分子量:35000），并把它添加到9.5 ml完全生长培养基在无菌的15毫升的锥形以制备5％10 ml（重量/体积）水的PEG相。 注意:添加一半的培养基，以第一，随后通过添加聚合物和锥形然后介质的剩余量最小化的聚合物与圆锥形壁的密合性，并有助于该聚合物溶解得更快。 </li><li>权衡0.128克的葡聚糖（DEX）（分子量:500,000），并将其添加到0.872毫升完全生长培养基中的无菌的1.5 ml离心管中，制备12.8％（重量/体积）水的DEX阶段1毫升</li><li>约1分钟涡旋两种溶液，以溶解在介质中的聚合物。 </li><li>保持两种溶液在水浴中于37℃1小时，以确保溶解和均匀的混合物。保持水位以上的上限，以避免溶液的污染从浴的可能性水。 </li><li>加载的PEG相溶液进入无菌塑料注射器，并通过0.2微米的孔径的注射器过滤器将它传递以除去杂质。 注意:用PEG溶液填充注射器，将其放置在一个锥形管的顶部，并使用武力滤波器的插入，慢慢推过过滤器的溶液中。这是很正常的抵抗注射器活塞的运动。聚合物溶液的轻微损失发生是由于一些溶液将保留在过滤器，但该聚合物的浓度也不会改变。 </li><li>吸管将10μl的DEX相溶液和稀释于10μl培养基在一个单独的管，以产生6.4％（重量/体积）DEX相溶液。抽吸100微升PEG溶液相的分装到培养皿。 <ol><li>分配0.5微升的6.4％（重量/体积）的DEX相溶液进入的PEG相溶液。目视确认成功ATPS形成通过观察在显微镜下一个DEX下降。下降的边界应保持可见，以表示两个稳定，独立的水相的存在下进行。 </li></ol></li><li>存储库存含水PEG和DEX相溶液在4℃下直到使用。 注意:建议聚合物溶液制备的各试验之前，新鲜和内贮存24小时使用。 </li></ol> 2.准备癌症印刷的细胞球体<ol><li>成长的利益（ 例如，MDA-MB-157乳腺癌细胞），以90％-100％汇合单层癌细胞。对于MDA-MB-157细胞中，使用一个含有10％FBS，1％谷氨酰胺和1％抗生素的DMEM组成介质。 <ol><li>使用细胞解离缓冲液（根据制造商的协议）并装载悬挂入15ml锥形收获细胞。它离心5分钟XG 173.3，除去上清，悬浮细胞1毫升完整的生长培养基。 </li></ol></li><li>称重传感器上血细胞计数和计数它们来计算所需数量的细胞的期望球体细胞密度。在T75瓶中生长的MDA-MB-157细胞的融合单层通常给出〜7×10 6个细胞。 注:1.5×10 4或MDA-MB-157细胞中更大的所需细胞密度为液滴体积，以产生一个单一的球体将取决于细胞类型22例如，密度为每0.3微升DEX相压降，建议确保形成一个球体。 </li><li>离心细胞，在173.3 XG第二时间为5分钟，重悬它们在生长培养基中的适当体积的悬浮液浓缩到所需细胞密度。 注意:例如，如果7×10 6个肿瘤细胞收获，以形成在一个0.3微升DEX相一滴1.5×10 4个细胞密度的球体所需要的细胞悬浮液的总体积将是140微升。然而，由于稀释在下一步对DEX相溶液，只使用70微升细胞培养基重悬细胞。 </li><li&gt;添加到所得的细胞悬浮液的12.8％的相等体积（w / v）含水的DEX相溶液制备1.2。为1.5×10 4个细胞的密度球体的例子中，70微升的DEX相溶液加入到70微升的细胞悬浮液。 </li><li>彻底混合细胞悬浮液，以确保细胞和DEX溶液混合的均匀分布。移液上下应做轻轻以防止气泡形成。 </li></ol> 3.准备联合培养球体印刷<ol><li>生长的癌细胞（ 例如，MDA-MB-157人乳腺癌细胞），并支持细胞（ 例如，人成纤维细胞），以90％-100％汇合单层。收获用细胞解离缓冲液（根据制造商的协议）不同的细胞类型。 <ol><li>加载每个悬挂入15ml锥形，离心它们进行5分钟，在173.3 XG，并且吸出上清液从每个圆锥形。每个圆锥的在1ml完全克悬浮细胞rowth媒介。 注意:荧光染料，如钙黄绿素AM（活细胞染色）和核染料（Hoechst公司）可以用来区分两种类型的细胞。 </li></ol></li><li>分别装入不同的细胞类型上血球计数和它们来计算每种细胞类型的所需数量的癌细胞的所需比率，以在共培养球形体支持细胞。 MDA-MB-157细胞的汇合单层的成纤维细胞和在T75烧瓶培养通常给〜7×10 6和〜6×10 6个细胞。 </li><li>从支持细胞对癌细胞悬浮液，得到两种细胞类型的数目的所希望的比例的悬浮液中添加了正确的量。例如，使用50癌细胞的比率为1成纤维细胞。 </li><li>离心机中含有适当量的混合细胞悬浮液5分钟，在173.3 XG和重悬细胞的锥形导致最终密度包括生长培养基等体积和12.8％的（重量/体积）水溶液的DEX相溶液（在1.2中制备）。 </li><li>轻轻吸取得到的细胞悬浮液，上下，以确保悬浮液的均一性。 </li></ol> 4.印刷肿瘤球体的成96孔板<ol><li>前一天的实验，涂覆未处理的圆底96孔板，用1％（重量/体积）的Pluronic在37℃下24小时。这种涂层可以防止细胞附着在培养周期。 </li><li>免除50微升的过滤5％（重量/体积）水的PEG相到96孔板（目标板）的各孔中。 </li><li>使用移液管，混合的细胞悬浮液（在步骤2或单 - 和共培养，分别步骤3的方法制备），并从一个384-孔板的一列（源板）加入20微升的悬浮液，以每其它公。 </li><li>打开液体处理器和"家"的移液头注册坐标。然后编译（低于4.6）先前定义的协议，以保证实验器具和参数的定义正确的LY。这种"归巢"的步骤可以是用于不同的液体处理程序不同。 注意:该协议的流程，示出一步一步低于4.6，将类似于不管使用的机器人的液体处理程序;然而，编程接口可能看起来不同，由于使用不同的软件。 </li><li>放置源板，目的地板，和枪头拖曳在液体处理程序的工作站上定义的位置， 如图1。 </li><li>执行包括下列步骤的协议: <ol><li>负荷桶从液体处理程序的移液头与来自工作站（ 图1）的混合移液管头（8提示）一列。 </li><li>混合的细胞悬浮液中的源板（ 图1）的孔中。选择一个混合容积比在井，以避免气泡形成细胞悬浮液体积要小。 </li><li>喷射尖端到空废物提示框（ 图1）。 </li&gt; <li>负载从移液头桶一列与10微升分配移液管头（ 图1）。 </li><li>抽吸0.3微升从源盘的细胞悬浮液（ 图1）到每个枪头。 </li><li>分配所述细胞悬液到目标盘的一列（ 图1）的孔中。使用0.5mm的分配高度和小于1微升/秒的分配流量。 </li><li>重复步骤4.6.1至4.6.6直到柱逐列印刷到整个目标板是完整的。 </li></ol></li><li>小心从工作站表面去除目标板，并放置在潮湿培养箱中在37℃和5％的CO 2。携带它，当进入孵化器，以避免含有细胞DEX阶段滴妨害保持板水平。 </li><li>孵育板24小时，以允许细胞的聚集成DEX相压降中的球体。 </li></ol> &lt;P类="j​​ove_title"&gt; 5。肿瘤细胞球体药物治疗需要注意的是下面的协议是一个4天的药物治疗，并且包括用新鲜药物续期2.一天后它可以被修改为其它的治疗期。 <ol><li>视觉上确认球体形成在96-孔板24小时后。如果需要的话，像井球体的大小，测量通过平均每个球体的最小和最大直径。 </li><li>通过在以预定的浓度溶解由制造商推荐的溶剂中溶解它制备所需药物的储备溶液。例如，溶解氯氨铂在水中的2毫克/毫升。 注意:保护药物的储备溶液从光如果药物是光敏感。 </li><li>使用系列稀释技术，制备稀释的药物浓度与在先前步骤中制备的储液细胞培养基。每个稀释应两次所需的浓度。稀ution比率将取决于起始原料浓度变化，并且所希望的工作浓度。 </li><li>加入50微升在先前步骤向每个孔制备药物溶液。这可以利用机器人或通过手动移液来完成。 注意:每个药物浓度可以为一半在每个稀释至所需浓度的50微升的水的PEG相已经很好。 <ol><li>从96孔板（16），对于每个浓度的两列使用球状体。 </li><li>在对照孔中，只添加50微升新鲜培养基到现有50μl的水性聚乙二醇相。 </li></ol></li><li>孵育球体与药物48小时，在37℃和5％的CO 2，避光。 </li><li> 48小时后培养，制备药物的新鲜稀释液在所需的浓度，并通过添加50微升新鲜药液向每个孔续订药物。 注意:井已经包含从第一药物吨所需的药物浓度reatment。因此，在此重建阶段，每个浓度制备成所需浓度，因为不会发生稀释。 </li><li>孵育球体与药物为另一48小时，在37℃和5％的CO 2，避光。 </li></ol> 6.细胞活力的球体分析<ol><li>之后48小时培养以新的药物（ 即 ，治疗球体用药物的总时间为4天），以及通过手动移液测量介质的一个总体积。 注:在一个典型的好体积〜140微升（小减少的发生是由于蒸发）。 </li><li>计算细胞活力试剂（ 例如，PrestoBlue），为总孔体积的10％浓度的体积。细胞活力的试剂这个体积添加至每孔。 注意:使用在140微升在井的现有介质体积，15.6微升的体积，需要得到的总的浓度为10％好。这是通过计算除现有的介质卷9（140微升/ 9 = 15.6微升）。 </li><li>孵育板与细胞活力试剂在37℃下加至每个孔6小时，避光。 </li><li>将板在微板读数器，并在分别为560nm和590nm的激发和发射波长，读取荧光信号。 </li><li>通过从相同的处理条件平均的值后的荧光信号从处理过的孔中，以对照孔中的标准化计算细胞的球状体的百分存活率。 </li></ol>

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Chem. 86, 4052-4057 (2014).</li><li>Petrak, D., Atefi, E., Yin, L., Chilian, W., Tavana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+spatio-temporally+controlled+cell+microprinting+with+polymeric+aqueous+biphasic+system.">Automated spatio-temporally controlled cell microprinting with polymeric aqueous biphasic system.</a> Biotech. Bioeng. 11, 404-412 (2014).</li><li>Tavana, 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=Nanolitre+liquid+patterning+in+aqueous+environments+for+spatially+defined+reagent+delivery+to+mammalian+cells.">Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells.</a> Nat. Mater. 8, 736-741 (2009).</li><li>Tavana, 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=Polymeric+aqueous+biphasic+system+rehydration+facilitates+high+throughput+cell+exclusion+patterning+for+cell+migration+studies.">Polymeric aqueous biphasic system rehydration facilitates high throughput cell exclusion patterning for cell migration studies.</a> Adv. Func. Mater. 21, 2920-2926 (2011).</li><li>Tavana, H., Mosadegh, B., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+aqueous+biphasic+systems+for+non-contact+cell+printing+on+cells:+engineering+heterocellular+embryonic+stem+cell+niches.">Polymeric aqueous biphasic systems for non-contact cell printing on cells: engineering heterocellular embryonic stem cell niches.</a> Adv. Mater. 22, 2628-2631 (2010).</li><li>Tavana, H., Mosadegh, B., Zamankhan, P., Grotberg, J. B., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microprinted+feeder+cells+guide+embryonic+stem+cell+fate.">Microprinted feeder cells guide embryonic stem cell fate.</a> Biotechnol. Bioeng. 108, 2509-2516 (2011).</li><li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Rev Cancer. 6, 392-401 (2006).</li><li>Olsen, C. J., Moreira, J., Lukanidin, E. M., Ambartsumian, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+fibroblasts+stimulate+invasion+of+breast+cancer+cells+in+a+three-dimensional+culture+and+increase+stroma+development+in+mouse+xenografts.">Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts.</a> BMC Cancer. 10, 444 (2010).</li><li>Ozdemir, B. 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=Depletion+of+carcinoma-associated+fibroblasts+and+fibrosis+induces+immunosuppression+and+accelerates+pancreas+cancer+with+reduced+survival.">Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.</a> Cancer Cell. 25, 719-734 (2014).</li><li>Hsiao, A. 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=Microfluidic+system+for+formation+of+PC-3+prostate+cancer+co-culture+spheroids.">Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids.</a> Biomaterials. 30, 3020-3027 (2009).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-based+drug+screen:+considerations+and+practical+approach.">Spheroid-based drug screen: considerations and practical approach.</a> Nat. Protoc. 4, 309-324 (2009).</li><li>Weigelt, B., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+microenvironmental+influences+on+the+normal+mammary+gland+and+breast+cancer.">Unraveling the microenvironmental influences on the normal mammary gland and breast cancer.</a> Semin. Cancer Biol. 18, 311-321 (2008).</li><li>Mueller, M. M., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friends+or+foes+-+bipolar+effects+of+the+tumour+stroma+in+cancer.">Friends or foes - bipolar effects of the tumour stroma in cancer.</a> Nature Reviews Cance. 4, 839-849 (2004).</li><li>Bissell, M. J., Radisky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putting+tumours+in+context.">Putting tumours in context.</a> Nat. Rev. Cancer. 1, 46-54 (2001).</li></ol>

作者什么都没有透露。

球体呈现一个现实的模型，以更好地理解肿瘤生理学和药效，并为抗癌药物开发的有用工具。这样的应用将极大地从简单的球体生成和维护技术，只需要标准的实验室器具，液体处理工具和筛分设备中受益。使用的含水两相体系中滴相中自发地聚集癌细胞允许高效生产和维护球体与机器人液体处理程序，并在原位药物治疗和商业试剂和工具的细胞反应终点分析。因此，该技术的一大优势在?...

基于细胞的测定提供用于开发和发现新的抗癌药物的重要工具。1,2-历史上，癌细胞的单层培养物已被用于研究的候选化合物对特定类型的癌细胞的功效。维护在标准培养板单层培养的易用性，标准板与用于加入试剂的商业机器人工具的兼容性，并用筛分设备对细胞反应的化学化合物下游分析是渲染2D培养一个有吸引力的工具的主要好处用于药物测试。3不幸的是，单层细胞测定往往不能预测在体内化合物的功效，使得药物开发和发现极其昂贵的过程。4,5-尽管显著投资和努力由制药公司和学术单位，只有约1％的抗癌药物在临床试验中被批准由FDA在过去的二十年。2D培养和肿瘤细胞在体内的复杂的三维环境之间差距6是单层培养系统的一个主要缺点。7因此，候选化合物对肿瘤细胞中的一个设置的筛选即更类似于三维肿瘤环境可能会加快新型化疗药物的发展。8 癌细胞球体呈现一个相关的三维肿瘤模型体外 9,10球体是通过癌细胞上非粘附表面或在悬浮液中使用诸如旋转烧瓶，液体覆盖，微加工技术的微自发或诱导的组件，其形成致密簇。井阵列，微流体，及悬滴11-16球体模仿实体肿瘤，包括几何和氧，营养物，和药物化合物有限传输到中央区的关键特征;因此，它们更紧密地再生药物responSE相比单层培养实体肿瘤的17-19尽管这样明显的好处，球体是不经常使用的抗肿瘤细胞的化学化合物的筛选。在一个标准的高吞吐量的设置，与市售的机器人和筛选/成像工具兼容产生均匀大小的球体的困难阻碍球体文化纳入药品开发过程。尽管定制的材料和板最近已成为市售于满足这一需求，成本考虑阻止它们的广泛使用。 两个主要的技术以生产高通量一致尺寸的球状体用一个新的悬滴平台和微制造微井的能力。13,16,20然而，这两种方法都需要特殊的板，并且是昂贵的制造和不方便端点用户设备核心研究中心和医药等行业，其中最为主要的EF堡垒为新的抗癌药物的发现进行的。尽管在含细胞滴与最近悬滴板的设计的稳定性的一些改进，仍培养过程中使用的板的仅每隔一个孔，以避免滴扩频/合并。16此显著降低实验吞吐量。药物添加和更新很难与手工或机器人移液和球状体需要被转移到一个标准的板，用于生化分析，因为该板配置不与常规的筛选设备容易地兼容诸如板的读者。21微井使用软光刻还制作可以控制大小的球体的生产。13,20然而，这个平台与标准的移液工具的不兼容性防止治疗与不同的药物化合物/浓度个体球体，揭露所有的球体，以单一的治疗条件。因此，这种方法不适合于高通量化合物筛选，需要多个化合物/浓度的同时进行测试。 为了克服这些障碍，已经开发了高通量生产的标准96孔板一致尺寸的癌细胞球体的新技术。22,23的方法是基于一种聚合物的水性两相体系（ATPS）与聚乙二醇（上PEG）和葡聚糖（DEX），为相形成聚合物。24 ATPSs最近已应用在各种新的细胞生物学应用，使细胞图案化和局部递送生物试剂对细胞的高含水介质。25-32为了形成球体，癌细胞与含水DEX相和所得悬浮液的一个子微升滴混合的吸移到孔含有所述浸渍水的PEG相溶液。落仍然从浸没相和局限的细胞不混溶的，以便形成一个球状体。恶魔ortantly，高含水浸渍阶段提供营养物质的球状体的细胞和减少的介质蒸发的众所周知的问题通用的一些其它测定法，导致在介质渗透压变化和药物浓度的波动。这项技术使球体的生产和仅使用市售的试剂和移液工具在标准的96孔板的药物治疗。重要的是，在同一个板块使用标准生化分析和板块读者进行的球体细胞反应的分析。与ATPS的方法来机器人液体处理的适应性和加工的便利性，使高通量产生两个单培养和共培养球形体一个简单的实验室技术。这种新方法将是向前迈进一大步走向融合的肿瘤细胞球体与提高测试吞吐量和成本效益（增加测试的化合物和热度数量药物开发和发现过程CED试剂消耗）和效率（降低手工操作时间）。 机器人的生产癌细胞球体在96孔板使用ATPS方法的一个详细的协议如下所述。此外，所得的球状体和使用商业生物化学测定细胞应答下游分析的药物治疗的信息呈现。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents and Consumables&lt;/strong&gt;</td></tr><tr><td>Polyethylene glycol, MW: 35,000</td><td>Sigma-Aldrich</td><td>94646</td><td></td></tr><tr><td>Dextran, MW: 500,000</td><td>Pharmacosmos</td><td>5510 0500 9007</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>12306C</td><td></td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Antibiotic</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>Clacein AM</td><td>Life Technologies</td><td>C3100MP</td><td></td></tr><tr><td>Hoechst</td><td>Life Technologies</td><td>33342</td><td></td></tr><tr><td>Cisplatin</td><td>Spectrum Chemicals</td><td>15663-27-1</td><td></td></tr><tr><td>PrestoBlue</td><td>Life Technologies</td><td>A-13261</td><td></td></tr><tr><td>Pluronic F-108</td><td>Sigma Aldrich</td><td>542342</td><td></td></tr><tr><td>Disposable Tips (10 &amp;micro;l)</td><td>Fluotics</td><td>C-P10V11.ST</td><td></td></tr><tr><td>Disposable Tips (70 &amp;micro;l)</td><td>Fluotics</td><td>C-P70V11.ST</td><td></td></tr><tr><td>Round-bottom 96-well plates</td><td>Nunc</td><td>268200</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Liquid Handler</td><td>Agilent Technologies</td><td>SRT Bravo</td><td></td></tr><tr><td>Microplate Reader</td><td>Biotek Instruments</td><td>Synergy H1M</td><td></td></tr></tbody></table>

robotic production of cancer cell spheroids with an aqueous two-phase system for drug testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

机器人液体处理程序的工作站如图1的移液头，并且在第4.6节球体的机器人印刷中使用的所有站都进行标记。该图像示出了使用两种不同的站前端箱（一组用于混合的提示和用于抽吸细胞悬浮液水相的DEX混合物/分配第二组）。整个设置容纳一个标准的生物安全柜，以保持无菌内。 图2示出了"打印"的过程的示意性与水的两相体系。装有细胞悬浮在水相的DEX（蓝色，图案化相...

Watch this Scientific Journal Video about Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing at JoVE.com

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

癌症细胞球体机器人生产与药物测试的双水相系统

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

robotic-production-cancer-cell-spheroids-with-an-aqueous-two-phase

Research

JoVE Journal

Bioengineering

9.0K 次观看.  The University of Akron. A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

视频: Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

科研

癌症研究

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

生产尺寸控制肿瘤细胞球体采用微孔板大数

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

生物工程

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

生成用于药物评估研究的 3D 肿瘤微球

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

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 ...

生物化学

用于基于 FLIM 的代谢和缺氧分析的多细胞球状体形成

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

作者聚焦:使用荧光寿命成像显微镜标准化用于代谢和氧合分析的球状体形成方法

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

癌症细胞球体机器人生产与药物测试的双水相系统

摘要

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此视频中的章节

高通量三维肿瘤球体药物筛选的生成

用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析

一种用汽车 t 细胞进行的切杀检测

生产尺寸控制肿瘤细胞球体采用微孔板大数

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

生成用于药物评估研究的 3D 肿瘤微球

功能性 3D 球状体细胞培养物的半自动表型分析

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

癌症细胞球体机器人生产与药物测试的双水相系统

摘要

探索更多视频

此视频中的章节

高通量三维肿瘤球体药物筛选的生成

用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析

一种用汽车 t 细胞进行的切杀检测

生产尺寸控制肿瘤细胞球体采用微孔板大数

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

生成用于药物评估研究的 3D 肿瘤微球

功能性 3D 球状体细胞培养物的半自动表型分析

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）