The authors thank M. Gordon of the Queen's University Biomedical Imaging Centre for assistance. This work was supported by operating grants from the Cancer Research Society of Canada (19439) and the Canadian Institutes for Health Research (MOP-142303) (LMM), and by Ontario Graduate Scholarships and studentships from the Terry Fox Research Institute Training Program in Transdisciplinary Cancer Research (SMM, EYL), and by a Craig Jury Summer Studentship (SMM).

注:所有试剂和耗材均列在材料清单 。 1.球体由生产细胞聚集<ol><li> 甲基纤维素溶液:准备100毫升100毫克/毫升甲基纤维素。 <ol><li>热火50毫升超纯H 2 O操作80℃。加入10克甲基纤维素粉末和搅动，直到颗粒被均匀地分散。 </li><li>带给用冷超纯的 H 2ö终体积，并在4℃下搅拌，直到溶液变得清晰，稻草色，并且不包含可见固体。 </li><li>通过0.45微米的过滤器通过除去未溶解的固体。制备的溶液可以被等分并储存于4℃多达12个月。 注:甲基纤维素用作无细胞毒性的，惰性的，悬浮剂以增强细胞 - 细胞粘附和阻止粘附细胞单层的形成。甲基纤维素是可溶于水，并且可以被洗去以下spherOID生成。 </li></ol></li><li> 球状体形成中 注意:使用前，立即准备球体形成网上平台。不要储存。 <ol><li>稀甲基纤维素溶液至1-5毫克/毫升在适当的培养基中。 </li><li>通过0.22微米的过滤器传送到消毒和去除未溶解的固体。 </li></ol></li><li> Dulbecco磷酸盐缓冲盐水（PBS） <ol><li>稀释至1x和使用之前，将通过一个0.45微米的过滤器。制备的溶液可以无限期地在室温下储存。 </li></ol></li><li> 细胞球体的生产（图1） 注:事先准备好所有试剂。由灰尘，纤维或其他不溶性微粒，以避免溶液的污染是重要的。这些污染物的存在会球体形成产生不利影响。培养基和其他解决方案应通过一个0.45微米的过滤器被传递到当POS除去这些颗粒sible。传输解决方案时，这些会带来额外的纤维避免使用棉过滤移液器。 <ol><li>培养细胞单层在适当的培养70％汇合培养基补充有10％血清。抽吸培养基并用1×PBS冲洗以除去碎片。添加3mL的胰蛋白酶-EDTA（0.05％胰蛋白酶）解离缓冲液，以10cm的细胞培养皿中，在37℃下孵育细胞和5％ 的 CO 2。 </li><li>检查显微镜下的细胞在10倍的放大倍率，以确认支队。中和分离缓冲液与7毫升血清补充培养基。 </li><li>在500×g离心10分钟，轻轻地沉淀细胞离心细胞悬浮液。 </li><li>除去上清液。使用具有过滤嘴一个P1000吸管在1mL球体形成培养基悬浮细胞沉淀。 注:细胞悬液应无团块。 <ol><li>以磨碎的细胞沉淀，压在管的底部枪头以微小的角度，而移液剪切细胞沉淀。避免捣碎超过3 - 5次，否则会损坏细胞。吸管慢慢地，也不排除枪头的全部内容，以避免产生气泡。 </li></ol></li><li>算使用血球细胞和稀释细胞悬浮液，以1×10 4个细胞/ mL。 注:细胞数可以被优化，以产生不同大小的球体。受损细胞的锥虫蓝染色可以用于确认悬浮细胞的生存力。 </li><li>转移细胞悬液至无菌的，无尘的多道移液器贮存和使用过滤嘴分配100微升到96孔U型底细胞斥板的各孔中。 <ol><li>用过滤超纯H 2Ø冲洗水库去除灰尘和纤维。 </li><li>混合周期性而接种，以防止细胞从沉降到储存器的底部的细胞悬浮液。为了避免产生气泡，使用反向移液技术，同时混合和dispensinG。 </li></ol></li><li>转移板到组织培养箱（37℃和5％的CO 2），并每日检查为球体的形成。细胞应解决的每一个的底部内6小时以及和通常聚合成球状体24-48小时（ 图2）内。 </li><li>对于成功的球体形成的确认，使用P10尖，轻轻吸取媒体在球体，而在放大10倍的显微镜下观察。适当地形成球体将从板和轧辊松开，确认其三维结构。 注:甲基纤维素和血清浓度应通过滴定来确定对于每个细胞系，以产生形成一个单一的球状体的每孔。以这种方式对选定的细胞系的优化条件示于表1，并形成在图2B的球状体的例子。球状体可生长时间超过指示但是，当他们变得更大他们的成长越来越困难处理无碎片。如果细胞形成一个单层，卫星球状体，或不沉降到井的底部，甲基纤维素和血清的浓度可能需要为最佳的球体形成（ 图3B）来进一步调整。不要使用含有污染物的球体，如纤维或灰尘（ 图3A）。预形成-球状体可以与如生长因子，活细胞的污渍，或用于分析的抑制剂的感兴趣的化合物进行治疗。 </li></ol></li></ol> 2.球体侵袭试验（图4） <ol><li> 胶原蛋白中和 <ol><li>酷所有的液体到4°C和胶原蛋白的合作以防止不必要的聚合反应时，应举行冰上。 </li><li>通过0.22微米的过滤器在超纯H 2 O.通过所有的解决方案，准备NaOH和新鲜的0.1M的新鲜的0.1 M和0.01M的解决方案。 </li><li>混合型牛胶原（3.1毫克/毫升的库存），0.01M NaOH和10倍的PBS（8:1:1的比例以体积计），并调整到使用冷0.1M NaOH中和HCl pH为7.4。胶原的最终浓度为2.5毫克/毫升。 </li><li>准备足够的中和胶原填充成像容器的2-3毫米的深度。 100微升的最小需要在材料清单中列出的8孔组织培养室滑动的每个孔中。 </li></ol></li><li> 嵌入在胶原球体（图4） <ol><li>涂层具有最小的50微升中和胶原的8孔组织培养室滑动的各孔的底部。 <ol><li>使用反向移液技术，以避免在胶原产生气泡。用枪头过孔表面均匀地涂抹胶原蛋白。 注:此胶原底层将持有悬挂的球体，通常是1-2毫米厚。基底层最小化的形成上层胶原层上的弯月面的。如果基底层太薄，球体可能使接触与腔室滑动件的底部和形成细胞单层。 </li></ol></li><li>放置玻片，并充满超纯H 2Ø35毫米的组织培养皿，在10cm的组织培养皿。在37℃下孵育直至胶原聚合，通常为1小时。请将剩余的中和冰上胶原蛋白。 注:充满水的35mm培养皿将提供湿度和防止干燥。该室幻灯片应保持在整个试验这种湿度试验箱。胶原基底层可以留聚合过夜。较长的孵育通常导致更硬的凝胶。 </li><li>使用P1,000滤嘴，收集来自96孔U型底电池斥板于1.5mL单元顶部管预形成球状体（步骤1.4.7）。吸管慢慢地，避免反复或剧烈的移液，以尽量减少球体的流体剪切。多个球状体可用于转移一个单管被汇集到8孔组织培养室滑动的一个孔中。 </li><li>离心收集球体在TABL在2000 XG 2-5秒和ETOP离心使用P200移液器上清。避免离心比需要更长的，因为这可能会导致球体掰开或聚集在一起。 注:吹打除去​​大部分上清液后，该管可以被仔细地在一个干净的纸巾吸走多余的液体反转。不要让球体干燥。 </li><li>使用大口径P200尖，轻轻悬浮在50微升中和胶原蛋白球体。任何球状体转移到室幻灯片之前做此收集球体每管。保持一直悬浮在胶原蛋白中和冰上直至准备转移到玻片的球体。 </li><li>从孵化器中取出室幻灯片，认真吸取胶原基础层顶部的球体胶原混合。在37℃和5％的CO 2，直至胶原聚合。 <ol><li>不要免除吸管的全部内容，以避免产生气泡。 Dropw伊势移液减少干扰胶原基层的机会。 </li></ol></li><li>慢慢加入至少100微升温热含有期望的趋化因子，抑制剂，或其他治疗的腔室滑动的每孔培养基中。如果液体吸到太大力球体含胶原层撕裂。培养基可缓慢分配向下一个井的侧面，以避免扰乱胶原。 </li><li>孵育室滑动，在37℃和5％的CO 2，以允许细胞侵袭。细胞侵袭到周围的胶原可通过明场或荧光成像观察和通过各种使用落射荧光，共焦或者光片显微镜方法定量。 </li></ol></li></ol> 3.入侵的量化:明场显微镜<ol><li>在设定的时间间隔，图像在10X放大率用明场显微镜（步骤2.2.8）胶原嵌入球体。 注意:长期活细胞的实验中，加热的显微镜载物台和CO 2调节的室可以用于保持在37℃ 的 CO 2而成像球状体和5％。 </li><li>使用软件如ImageJ的测量细胞侵入周围的胶原蛋白和在每个时间点入侵的平均距离的数量量化侵袭。 </li></ol> 4.定量入侵:荧光显微镜用活细胞染色<ol><li>以下步骤2.2.8，补充用荧光染色，例如DAPI，钙黄绿素AM，或适当的细胞系按照制造商的说明其他细胞跟踪化合物在玻片的介质。 注:对于侵袭的定量，在整个球体均匀染色不是必需的。对于需要染色更深的渗透，较长的孵育应用（&gt; 3小时）可能是必要的。 </li><li>清除污渍，用500微升温暖1X PBS代替。在cubate在37℃下进行10分钟，以除去多余的污渍。重复至少两次。 </li><li>使用荧光显微镜，通过侵入球状体获得光学截面。 注:长期暴露于激光重复应该在成像过程中被最小化，以限制光毒性和光漂白。 </li><li>使用软件如ImageJ的生成的Z突起，其可用于量化球体的总面积，侵入细胞，和距离的数字侵入到胶原基质。 </li><li>作为一个例子，以量化侵袭，调整图像阈排除背景，突出仅球体和侵入细胞，并测量它们的面积。原始球体的面积可以从该总减去量化侵袭。 </li></ol> 5.定量入侵:免疫荧光注:所有的解决方案和缓冲区应通过一个0.45微米的过滤器来传递使用前圣雷莫已经碎片，它可以染色产生不利影响。 <ol><li>准备PBS +洗涤液。制备1×PBS中并加入氯化钙和氯化镁至0.1毫米的最终浓度。 氯化钙和氯化镁加入后不要高压缓冲。溶液可以过滤灭菌，并储存在室温下。 </li><li>制备中性缓冲福尔马林（NBF）固定缓冲区。加入5滴的1M的NaOH至0.6g多聚甲醛。带来至20毫升，用PBS +和在60℃下孵育直至低聚甲醛溶解（约1小时）。冷却至室温并调节pH值至7.4，用1M HCl中。 注:NBF固定缓冲区应立即使用前准备。 </li><li>制备牛血清白蛋白（BSA）封闭缓冲液。在3％w / v的溶解的BSA在PBS +。溶液可以过滤灭菌并且储存于4℃几天，但最好是新鲜制备。不要加热。 </li><li>准备通透缓冲区。稀释的Triton X-100以在PBS + 0.2％体积/体积。 注:通透缓冲区应立即使用前准备。 </li><li> （可选）准备安装MOWIOL中。结合2.4克MOWIOL，6克甘油，和6毫升超纯H 2 O混合在在室温下在旋转器3小时。加入12毫升0.2M的三Cl（pH值为8.5）。与在50℃下混合10分钟孵育。 <ol><li>离心机在5000×g离心15分钟以沉淀不溶物质。添加抗漂白剂，如2.5％DABCO。储存在-20°C 500微升等分。 </li></ol></li><li> 球状体的免疫荧光染色（图5） 注:使用吸管慢慢地从室幻灯片中添加或删除液体。避免使用吸气的，因为这可能破坏胶原层。 <ol><li>准备球状体和嵌入在胶原填充室载玻片，如在章节1中记载为2。 注:胶原蛋白的自发荧光可以通过使用薄层或c的小体积最小化ollagen。 </li><li>从每一个腔室滑动除去尽可能多培养基越好。 </li><li>简单地说用500微升PBS +洗涤液冲洗胶原层以去除残留的网上平台。 </li><li>加入500微升新鲜PBS +的洗涤缓冲液到每个孔中，并在37℃下孵育5分钟以洗涤球粒。重复两次。 </li><li>用500微升NBF固定缓冲液更换PBS +洗涤液。在室温下孵育20 - 25分钟。 </li><li>删除NBF固定缓冲区，并用500微升PBS +洗涤液冲洗。洗5分钟，用新鲜的PBS +洗涤缓冲液至少3次。 注:固定球体可以储存在4℃下在新鲜的PBS +的洗涤缓冲液几天。 0.01％叠氮化钠可以加入到洗涤缓冲液贮存期间，以抑制微生物的生长。 </li><li>更换PBS洗涤+缓冲区500μL通透缓冲区。在室温下孵育10-15分钟。 </li><li><stro纳克&gt;替换透化缓冲液500微升BSA封闭缓冲液并在室温下孵育1小时。 <ol><li>也可以删除BSA封闭液。使用手术刀或剪刀，消费球体和胶原层围绕着三个x 3毫米的区域。使用大口径P1000尖，用新鲜的BSA封闭液轻轻漂洗去除胶原蛋白的切除块。胶原蛋白转移块1.5毫升管。 </li><li>离心2000 XG 2分钟，弃上清。该管可以仔细倒置在干净的纸巾吸走多余的液体。 </li></ol></li><li>初级抗体添加到球状体并在室温下孵育1小时。添加第一抗体的足够体积，以使整个胶原层被均匀地淹没。上述可选步骤（5.6.8.1-5.6.8.2）通常允许使用抗体的小卷。 </li><li> 淹没玻片与至少10毫升的PBS +洗BUF的容器FER 10分钟以洗涤。重复至少两次。 <ol><li>可选，如果球体从以前的胶原层（步骤5.6.8.1）切除，增加至少1毫升的PBS +洗缓冲到1.5毫升管，轻轻地搅动。允许浸泡至少10分钟。 </li><li>除去尽可能多PBS +的洗涤缓冲液尽可能并重复洗涤用PBS +洗涤缓冲至少两次。离心机在2000 XG数分钟沉淀胶原块。 注:通过浸没在洗涤缓冲液前，用70％的乙醇擦拭室滑动的清洁外表面。 </li></ol></li><li>重复步骤5.6.9-5.6.10使用适当的二级抗体。 注:如果球状体在成像器直接染色，继续执行步骤5.6.13。 </li><li> 可选的，如果球状体由胶原层以前切出（步骤5.6.8.1），清洁载玻片并用70％的乙醇共焦显微镜兼容盖玻片。<ol><li>取下胶原块，重悬在超纯H 2尽可能多的洗涤液尽可能O.在安装之前，立即执行此步骤。不要把彩色块浸泡在水中。 </li><li>用细尖镊子，把握胶原块，并转移到载玻片的边缘。 </li><li>使用镊子举行的地方胶原蛋白，用干净的棉签胶原块灯芯多余的液体，确保不要直接触摸的胶原蛋白。 注意:如果胶原卡住到棉签它可以使用镊子轻轻除去，或通过在水中浸没。 </li></ol></li><li> 使用反向移液技术，免除100微升MOWIOL安装介质上的胶原蛋白块，地点在盖玻片样品。 <ol><li>使用棉签或纸巾从盖玻片下灯芯过剩MOWIOL封固剂和水了。 </li><li>允许MOWIOL安装介质在4℃过夜硬化盖玻片C.封边用指甲油。 </li></ol></li><li>利用共聚焦或荧光显微镜图像染色球体观察的兴趣，形成侵入结构等 （ 图5B，5C）蛋白质的定位。 注:彩色球体应加以保护，以防止漂白。 </li></ol></li></ol> 6.失巢凋亡分析注:球体形成协议很容易适应量化锚定非依赖性生长并失巢凋亡抗性的各种细胞类型。 <ol><li> 接种细胞失巢凋亡测定（图6） <ol><li>遵循球体生产指令1.4节，但至少使用30毫克/毫升甲基纤维素制备球状体形成网上平台。 注:当回温，30毫克/毫升甲基纤维素应该产生一个厚厚的凝胶保存细胞悬浮液中。 </li><li>孵育几天细胞周和在在用显微镜放大10倍定期SPECT。能抵抗失巢凋亡细胞应增殖并形成菌落。 </li><li>通过测量形成在每个孔集落的数目和大小量化失巢凋亡。 注:一旦菌落形成的，它们可以被洗涤以除去甲基纤维素和用于基于球状体的测定法，如所描述。 </li></ol></li></ol>

<ol>
	<li>Zimmermann, M., Box, C., Eccles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+vs.+three-dimensional+in+vitro+tumor+migration+and+invasion+assays.">Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays.</a> Methods Mol Biol. 986 (986), 227-252 (2013).</li><li>Albini, A., Noonan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'chemoinvasion'+assay,+25+years+and+still+going+strong:+the+use+of+reconstituted+basement+membranes+to+study+cell+invasion+and+angiogenesis.">The 'chemoinvasion' assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis.</a> Curr Opin Cell Biol. 22 (5), 677-689 (2010).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat. Rev. Mol. Cell Biol. 8 (10), 839-845 (2007).</li><li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends Biotechnol. 31 (2), 108-115 (2013).</li><li>Carletti, E., Motta, A., Migliaresi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+for+tissue+engineering+and+3D+cell+culture.">Scaffolds for tissue engineering and 3D cell culture.</a> Methods Mol Biol. 695, 17-39 (2011).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J Vis Exp. (51), (2011).</li><li>Handschel, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+of+micromass+culture+technology+in+tissue+engineering.">Prospects of micromass culture technology in tissue engineering.</a> Head Face Med. 3, 4 (2007).</li><li>Stuckhoff, A. P., DelValle, L., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuronal Cell Culture Methods and Protocols Vol. 1078 Methods in Molecular Biology. 1078, 119-132 (2013).</li><li>Stewart, G. J., Wang, Y., Niewiarowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+protects+the+ability+of+anchorage-dependent+cells+to+adhere+following+isolation+and+holding+in+suspension.">Methylcellulose protects the ability of anchorage-dependent cells to adhere following isolation and holding in suspension.</a> Biotechniques. 19 (4), 598-604 (1995).</li><li>Vinci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+establishment+and+analysis+of+three-dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biol. 10, 29 (2012).</li><li>Nagelkerke, A., Bussink, J., Sweep, F. C. G. J., Span, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+of+breast+cancer+cells:+How+to+go+three-dimensional.">Generation of multicellular tumor spheroids of breast cancer cells: How to go three-dimensional.</a> Anal. Biochem. 437 (1), 17-19 (2013).</li></ol>

The authors have nothing to disclose.

我们提出了生产3D细胞球体使用廉价的和广泛使用的试剂在体内组织的架构模型快速，灵活的方法。我们的协议利用MC 8,9的非细胞毒性和粘合促进性质介导的细胞聚集和降低细胞单层的形成。不像从动物来源分离的基于蛋白质的基质，MC是惰性的，不包含生长因子，并且很容易通过洗涤除去，允许球状体在各种应用中使用的隔离无残余基质的存在?...

的肿瘤细胞的行为生物相关的评估是使用传统的二维（2D）细胞培养的方法，部分是因为这些不充分地反映在体内发现的细胞微环境挑战。结合细胞外基质成分进入文化（ 如 Boyden小室测定）的替代方法是体内组织的环境更生理代表。然而，它们可被限定在单个细胞行为的评估，并且不概括在有助于组织或肿瘤生长1，2，3细胞-基质和细胞-细胞相互作用的体内组合的复合物。 使用多细胞球体是最近的做法更准确地再现体内细胞生长1的结构紧凑，4。球状体可以用于研究正常细胞的细胞-基质相互作用，而且还可以作为肿瘤类似物肿瘤进展的特征，如转移性生长或药物抗性4建模。 球状体可以通过嵌入在基体5，或更快速的单细胞的增殖而形成，通过促进多种细胞的聚集，以形成一单细胞群（ 例如 ，悬滴，离心方法）6,7。现有的细胞聚集技术可能需要昂贵的材料或专门设备。此外，这些球状体具有宽范围的尺寸和形态，并且可以是难以生产大批量，使得生长条件或处理困难之间的比较。最后，通过这些方法产生的球状体可能难以从蛋白质extracel隔离细胞性矩阵，其中它们嵌入在其他应用中使用。 在这里，我们描述了用于快速形成使用市售U型底细胞斥板和惰性粘合促进基质，甲基纤维素一致大小细胞球体的一个强大的和易于修改细胞聚集方法。一旦形成，这些多细胞球状体可容易地分离出一种用于在广泛的应用中使用。该协议也容易适于产生通过细胞增殖，其可用于评估其他细胞过程球状体。这里，我们表明细胞侵袭测定法，免疫荧光染色定量，一个失巢凋亡测定中，由于这两种不同的球体形成的协议的应用实例。

<table><tbody><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Phosphate buffered saline</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9625</td><td></td></tr><tr><td>Calcium Chloride Solution</td><td>Sigma-Aldrich</td><td>21114</td><td>Used for PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer; Do not autoclave PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer upon addition of calcium chloride</td></tr><tr><td>Magnesium Chloride Solution</td><td>Sigma-Aldrich</td><td>M1028</td><td>Used for PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer; Do not autoclave PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer upon addition of magnesium chloride</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Spheroid Formation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well U-bottom Cell-Repellent Plate</td><td>Greiner Bio-One</td><td>650970</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma-Aldrich</td><td>D5546</td><td>For culturing SH-SY5Y, PANC-1, TPC-1 cell lines</td></tr><tr><td>F12K Medium</td><td>Thermo Fisher Scientific</td><td>2112722</td><td>For culturing TT cell line</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F1051</td><td>Filter prior to use to remove particulate contaminants</td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>M7027</td><td>Prepare in water to 100 mg/mL</td></tr><tr><td>Roswell Park Memorial Institute Medium</td><td>Sigma-Aldrich</td><td>R8758</td><td>For culturing HCT-116, BxPC-3 cell lines</td></tr><tr><td>TrypLE Express</td><td>Thermo Fisher Scientific</td><td>12605028</td><td>Dissociation buffer</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Invasion Assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine Type I Collagen</td><td>Corning Incorporated</td><td>354231</td><td>Stock 3.1 mg/mL; Maintain on ice when in use</td></tr><tr><td>DMEM Phenol Red Free Low Glucose&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11054-20</td><td>Less background fluorescence compared to Phenol Red supplemented medium</td></tr><tr><td>Glial Cell Line Derived Neurotrophic Factor</td><td>Peprotech</td><td>450-10</td><td>Chemoattractant</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Immunofluorescence Microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>#1.5 Coverglass</td><td>Electron Microscopy Sciences</td><td>72225-01</td><td>For mounting excised spheroids</td></tr><tr><td>Alexa-Fluor 488 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td>Used to stain actin at 1:200</td></tr><tr><td>Bovine Serum Albumin</td><td>Bioshop Canada Incorporated</td><td>ALB001</td><td>Used in BSA blocking buffer</td></tr><tr><td>Dabco 33-LV</td><td>Sigma-Aldrich</td><td>290734</td><td>Antifade</td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Bioshop Canada Incorporated</td><td>GLY001</td><td>Used in MOWIOL mounting medium</td></tr><tr><td>ImageJ Software</td><td>Freeware, NIH</td><td>-</td><td>Used for image analysis&amp;nbsp;</td></tr><tr><td>Microslides</td><td>VWR International</td><td>48312-024</td><td>For mounting excised spheroids</td></tr><tr><td>MOWIOL 4-88</td><td>EMD-Millipore</td><td>475904</td><td>Used in MOWIOL mounting medium</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>EMD-Millipore</td><td>PX0055-3</td><td>Used in fixation buffer</td></tr><tr><td>Triton X-100</td><td>Bioshop Canada Incorporated</td><td>TRX777</td><td>Used in permeabilization buffer</td></tr></tbody></table>

an efficient and flexible cell aggregation method for 3d spheroid production

单层细胞培养不充分模拟组织的体内行为，这涉及复杂的细胞-细胞和细胞-基质相互作用。三维（3D）细胞培养技术是最近开发用于解决贴壁细胞培养的缺点一个创新。而在体外产生组织类似物几种技术已经开发，这些方法往往是复杂，以建立价格昂贵，需要专门的设备，并且通常由兼容性只有某些细胞类型的限制。在这里，我们描述了用于凝集细胞成一致的大小是与多种肿瘤和正常细胞系的生长兼容的多细胞三维球状体一个快速和灵活的协议。我们利用不同浓度的血清和甲基纤维素（MC），以促进依赖贴壁球体的产生和防止在一个高度可再生的方式的细胞单层的形成。逐张为最佳条件idual细胞系可以通过在球体形成介质调整MC或血清浓度来实现。生成的3D球体可被收集用于在广泛的应用范围，包括细胞信号传导或基因表达研究中，候选药物筛选，或在细胞过程如肿瘤细胞侵袭和迁移的研究中使用。该协议也容易地适用于产生从单个细胞克隆的球状体，并可以适于评估贴壁依赖性生长和失巢凋亡抗性。总的来说，我们的协议提供了用于产生和利用为了概括组织的微环境的3D模型和正常和肿瘤细胞的体内生长三维细胞球状体的容易修改的方法。

我们描述一个灵活，高效的方法来生成利用细胞排斥板和补充MC球体形成媒体离散球体。下MC和血清的适当的条件下，各个细胞沉降并在井的中心粘附在一起以形成具有最小粘附到孔底部的球体。使用该协议，是从多种细胞系中（ 图2B）产生球体。 MC和血清浓度的滴定所需的每个细胞系，以确定在形成仅单个球体即足够强大，以允许操纵未经分段的最佳条件。?...

Watch this Scientific Journal Video about An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production at JoVE.com

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

an-efficient-flexible-cell-aggregation-method-for-3d-spheroid

Research

JoVE Journal

Biology

23.7K 次观看.  Queen's University Cancer Research Institute. Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions. 

视频: An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>通过 3D 打印的邮票状设备生成组织球体

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

通过 3D 打印的邮票状设备生成组织球体

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

生物化学

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 ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

用于生成多细胞三维球体的 CD 实验室平台

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

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 ...

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 ...

三维球体制作一个高效，灵活细胞聚集方法

摘要

探索更多视频

一个简单的悬滴细胞生成三维球体文化协议

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

功能性 3D 球状体细胞培养物的半自动表型分析

用于生成多细胞三维球体的 CD 实验室平台

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

用于细胞球体批量生产的三维声学装配装置

用于细胞球体批量生产的三维声学装配装置

用于细胞球体批量生产的三维声学装配装置

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）

三维球体制作一个高效，灵活细胞聚集方法

摘要

探索更多视频

一个简单的悬滴细胞生成三维球体文化协议

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

功能性 3D 球状体细胞培养物的半自动表型分析

用于生成多细胞三维球体的 CD 实验室平台

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

用于细胞球体批量生产的三维声学装配装置

用于细胞球体批量生产的三维声学装配装置

用于细胞球体批量生产的三维声学装配装置

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）