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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents and Consumables</td>
			<td></td>
			<td></td>
			<td></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&#39;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 &#181;l)</td>
			<td>Fluotics</td>
			<td>C-P10V11.ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Tips (70 &#181;l)</td>
			<td>Fluotics</td>
			<td>C-P70V11.ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom 96-well plates</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></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

8.9K 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

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

科研

生物工程

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

机器人镜治疗系统偏瘫武器功能恢复

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

使用 3D 声学组装装置可扩展生成均匀的单元球体

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

作者聚焦:用于高质量 3D 细胞球状体培养的无支架声学组装方法的开发 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

用于细胞球体批量生产的三维声学装配装置

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...