This work was funded by the University of Calgary, under a new investigator start-up grant to Dr. Ungrin.

注：微孔板中可用不同的格式，这取决于所期望的结果。规格以及对细胞应与每种格式中使用的最小和最大数目的近似的准则示于表1中 。较小的微孔逐渐变细至一尖点，并且因此不存在下的大小限制，虽然球状体之间的变异性变得在较小的尺寸更显著。常规生产的球状物由平均每少至20个细胞的是直接的8。较大微孔尺寸没有完全锥形的，因此试图以形成由小数量的细胞球状体可导致在各微孔多个较小的球状体。由于表面性质略有不同，使用AggreWell 400EX板时，准备用表面活性剂溶液（步骤1.2）是不可选的。 该协议是基于使用AggreWell 400板 - 为其他格式的利弊ULT 表1。细胞在每孔加载的数量乘以它包含由细胞被群集的每个球体的数目微孔的数目来确定。例如，为了产生从1000个细胞每个球状体，每孔必须装入（1200倍球体1,000个细胞每=）1.2×10 6个细胞。细胞密度必须计算给定的细胞将在0.4毫升的体积被加载。因此，对于从各1000个细胞形成的球状体的例子中，1.2×10 6细胞在0.4毫升转化为每毫升3×10 6个细胞的密度。 1。准备微孔以接收细胞<ol><li>微孔板是作为提供无菌的，并且应该只从它们的包装中，在无菌的环境中，例如一个层流生物安全柜中移除。不育应保持整个协议。应无菌受到损害，该井是使用70％的乙醇在水中使用重新消毒​​的以下可选步骤。 <ol><li>加入0.5毫升70％乙醇中，每个未使用的井。一些微孔将捕获的气泡，尽管这可以通过增加液体的一侧上，并允许它在表面上扩散，而不是垂直地落下它在表面上被降低。更换盖子。 </li><li>虽然乙醇蒸气应迅速渗透任何气泡，如果有大量的，可能需要将其删除。离心2分钟，2,000×g的。请注意，重要的是确保转子是否正确以这样的速度平衡是很重要的。 </li><li>使用低倍率倒置显微镜，确认气泡已经从微孔中删除。 </li><li>孵育5分钟，用盖子上在室温下进行。 </li><li>吸乙醇。板现在可以用无菌水或PBS中，可直接使用，或风干进行存储。 </li></ol></li><li>为了确保电池不会与微孔表面相互作用，可以使用表面活性剂来制备。一般来说它是可取的初始执行该步骤，并随后比较使用和不使用表面活性剂包覆的微孔所产生的球粒。 <ol><li>加入0.5毫升的冲洗液到每个孔中。一些微孔将捕获的气泡，尽管这可以通过增加液体的一侧上，并允许它在表面上扩散，而不是垂直地落下它在表面上被降低。更换盖子。 </li><li>离心2分钟，2,000×g离心以去除气泡。请注意，重要的是确保转子是否正确以这样的速度平衡是很重要的。 </li><li>使用低倍率倒置显微镜，确认气泡已经从微孔中删除。 </li><li>孵育至少30分钟，盖子上，在室温下过夜或在4℃下板可以被存储在4度的孔中的表面活性剂溶液中，直至需要的，如果适当地密封，以防止干燥。 </li><li>用无菌水或PBS在使用前立即冲洗板。不要让板涂布后干燥。 </li></ol></li></ol> 2。细胞的准备注：细胞制备的细节将有所变化与所研究的特定的细胞类型。然而，我们观察到这些条件是有效的与广泛的细胞，包括这里所讨论的线路，以及人类和鼠胚胎干细胞（ESC），人的诱导多能干细胞（IPSC），成纤维细胞，假定原始内胚层的，和基质细胞。其他团体已采用此系统成功用于广泛的应用范围包括从间充质干细胞的软骨9，标准化的一代从多能干细胞10的神经前体，在肝细胞11和在山椒12脊髓再生的评估毒理分析。与HT29细胞工作的具体协议如下所示。 <ol><li>从HT29细胞的T75烧瓶，培养在DMEM+10％FBS </li><li>从瓶中吸出培养基，并加3毫升的胰蛋白酶。 </li><li>孵育细胞5分钟，在37℃下</li><li>加入3ml生长培养基的停止胰蛋白酶消化。取一等份用于细胞计数，其余转移到15ml离心管中。 </li><li>离心5分钟，在200×g下。 </li><li>而在离心分离过程中，计数细胞。 </li><li>吸胰蛋白酶/中混合，然后换上由上述计算所需的细胞密度测定新鲜生长培养基，体积。 </li><li> （可选）通过一个过滤器传递细胞悬液，以消除任何团块。注：电池的收割条件不同而有所变化线之间。如果离解的协议，例如传代感兴趣的细胞是可用的，应该作为起点。敏感细胞系使用胰蛋白酶，可能会导致过度的细胞死亡。在这种情况下，我们使用的TrypLE作为替代dissociati观察到很好的效果对试剂8。如果离解过程中的细胞成为结块和失去活力，除了DNA酶的酶溶液可以解决这个问题。减少分离时间，采用研磨的步骤，打破了细胞团块也可以提高生存能力。 </li></ol> 3。球体的形成注：球体​​可以在各种介质中的制剂产生，但是初步试验应进行使用，其中所述细胞进行培养，以区分从改变介质组合物的后果过渡到三维培养系统的后果的介质。 <ol><li>加0.4毫升生长培养基到每个孔中。 </li><li>离心2分钟，2,000×g离心以去除气泡。请注意，重要的是确保转子是否正确以这样的速度离心之前平衡是很重要的。 </li><li>使用低倍率倒置显微镜，确认气泡已经从微孔中删除。 </li><li>添加0.4毫升细胞悬液（以上计算）所需的密度。移液器向上和向下轻轻混匀，而不引入气泡进入微孔。以确保将细胞均匀分布在整个井，以便获得一致的球状体是很重要的。 </li><li>离心5分钟，在200×g下。如果板没有离心分离期间自我水平，对流可能产生的结果在细胞穿过微孔分布不均匀。因此，板应在内部平衡以及对其他板，从而使重量分布均匀的片托架的两侧。 </li></ol>注：如果单元格不均匀分布的证据可见，它可能需要将少量的润滑，以板架在枢轴点 - 与离心机制造商的说明进行咨询。 注意：一旦细胞已被离心分离成微孔，它们是合理的电阻tant要洗出从板的轻微运动。应避免导致介质的晃荡突然动作，但该板从离心机到显微镜或培养箱转移不应导致显著细胞位移。 <ol start="6"><li>使用倒置显微镜，确认细胞已在微孔内聚集，并且它们均匀地分布在井。 </li><li>孵育过夜，在37°C。 </li><li>使用倒置显微镜，观察球体的形成过程。某些细胞系将形成致密的球体在24小时内，其他人可能需要更长的时间。从集群中的HT29细胞聚集的级数示于图2。 </li><li>从微孔中恢复球体，并轻轻地喷射出来用移液器转移到悬浮培养。另外，保持在球体与原位更换培养基8 -持续时间取决于其初始大小和增长速度，这DEFINE的时间，直到他们长大了在它们形成的微孔。 <ol><li>要更换培养基中不失球体，吸出培养基在井的边缘使用巴斯德吸管。慢慢把尖，直到它开始从半月板画液，并按照半月板下来，因为它下降。然后加入新鲜培养基对井壁在同一个地方。一些球粒可能会丢失，但是，如果介质总是抽吸和在相同的位置取代，这个数目将维持小相比，大量的好。 </li></ol></li></ol>

<ol>
	<li>Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., 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=Multicellular+tumor+spheroids:+An+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: An underestimated tool is catching up again.</a> Journal of Biotechnology. 148 (1), 3-15 (2010).</li><li>Sherar, M. D., Noss, M. B., Foster, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+backscatter+microscopy+images+the+internal+structure+of+living+tumour+spheroids.">Ultrasound backscatter microscopy images the internal structure of living tumour spheroids.</a> Nature. 330 (6147), 493-495 (1987).</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), 2720 (2011).</li><li>Naber, H. P. H., Wiercinska, E., Ten Dijke, P., Van Laar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+Assay+to+Measure+TGF-&#946;-induced+Invasion.">Spheroid Assay to Measure TGF-&#946;-induced Invasion.</a> J. Vis. Exp. (57), e3337 (2011).</li><li>Ungrin, M. D., Joshi, C., Nica, A., Bauwens, C., Reproducible Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ultra+high-throughput+formation+of+multicellular+organization+from+single+cell+suspension-derived+human+embryonic+stem+cell+aggregates.">ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.</a> PLoS ONE. 3 (2), e1565 (2008).</li><li>Bauwens, C. L., Song, 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=Geometric+control+of+cardiomyogenic+induction+in+human+pluripotent+stem+cells.">Geometric control of cardiomyogenic induction in human pluripotent stem cells.</a> Tissue Engineering. Part A. 17 (15-16), 1901-1909 (2011).</li><li>Bratt-Leal, A. M., Carpenedo, R. L., Ungrin, M. D., Zandstra, P. W., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+biomaterials+in+multicellular+aggregates+modulates+pluripotent+stem+cell+differentiation.">Incorporation of biomaterials in multicellular aggregates modulates pluripotent stem cell differentiation.</a> Biomaterials. 32 (1), 48-56 (2011).</li><li>Ungrin, M. D., Clarke, 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=Rational+bioprocess+design+for+human+pluripotent+stem+cell+expansion+and+endoderm+differentiation+based+on+cellular+dynamics.">Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics.</a> Biotechnology and Bioengineering. 109 (4), 853-866 (2012).</li><li>Markway, B. D., Tan, G. K., Brooke, G., Hudson, J. E., Cooper-White, J. J., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+chondrogenic+differentiation+of+human+bone+marrow-derived+mesenchymal+stem+cells+in+low+oxygen+environment+micropellet+cultures.">Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures.</a> Cell Transplantation. 19 (1), 29-42 (2010).</li><li>Kozhich, O., Hamilton, R., Mallon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+Generation+and+Differentiation+of+Neural+Precursor+Cells+from+Human+Pluripotent+Stem+Cells.">Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells.</a> Stem Cell Reviews and Reports. , 1-6 (2012).</li><li>Fey, S. J., Wrzesinski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Drug+Toxicity+Using+3D+Spheroids+Constructed+from+an+Immortal+Human+Hepatocyte+Cell+Line.">Determination of Drug Toxicity Using 3D Spheroids Constructed from an Immortal Human Hepatocyte Cell Line.</a> Toxicological Sciences. , (2012).</li><li>Mchedlishvili, L., Mazurov, V., 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=Reconstitution+of+the+central+and+peripheral+nervous+system+during+salamander+tail+regeneration.">Reconstitution of the central and peripheral nervous system during salamander tail regeneration.</a> Proceedings of the National Academy of Sciences. , (2012).</li><li>Butler, C. M., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Aggregate+Cohesion+by+Tissue+Surface+Tensiometry.">Measurement of Aggregate Cohesion by Tissue Surface Tensiometry.</a> J. Vis. Exp. (50), e2739 (2011).</li><li>Xu, K., Buchsbaum, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mammary+Epithelial+Cells+from+Three-dimensional+Mixed-cell+Spheroid+Co-culture.">Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture.</a> J. Vis. Exp. (62), e3760 (2012).</li><li>Baraniak, P. R., Cooke, M. T., Saeed, R., Kinney, M. A., Fridley, K. M., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffening+of+human+mesenchymal+stem+cell+spheroid+microenvironments+induced+by+incorporation+of+gelatin+microparticles.">Stiffening of human mesenchymal stem cell spheroid microenvironments induced by incorporation of gelatin microparticles.</a> Journal of the Mechanical Behavior of Biomedical Materials. 11, 63-71 (2012).</li><li>Purpura, K. A., Bratt-Leal, A. M., Hammersmith, K. A., McDevitt, T. C., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+engineering+of+3D+pluripotent+stem+cell+niches+to+guide+blood+development.">Systematic engineering of 3D pluripotent stem cell niches to guide blood development.</a> Biomaterials. , (2012).</li></ol>

Ungrin博士拥有财务利益的AggreWell技术作为一个发明家。

We have established a system whereby large numbers of uniform spheroids may be generated from multiple cell lines from different sources. We have yet to encounter an adherent cell line that does not form spheroids under these conditions. We have previously observed cell loss in populations prone to anoikis5,8, however to date this issue has not arisen with tumor lines. The system is arbitrarily scalable with surface area, with behavior consistent across microwells in 24-well and 6-well format, as well as prototype bioreactors containing 50,000 microwells each presently under development.
Should spheroid asymmetry be a concern, the incubation time in step 4.8 may be increased to two or three days. Spheroids may also be incubated for a period after extraction from the microwells to increase symmetry, however in this case care must be taken to keep culture densities sufficiently low, as spheroids in contact with one another will often fuse into larger structures. For this reason, we have previously maintained cultures within the microwells in which the aggregates were formed, with the primary limitation being the size of the spheroid. This in turn is a function of both growth rate and initial size8, and if extended culture within the microwell plate is planned, this should be considered in advance. Options to prevent overgrowth, should it occur, include either starting with smaller spheroids, or employing the larger microwells of the AggreWell 800 plate.
Should spheroids fail to increase in coherence over time, one potential cause may be cell death. Particularly in larger aggregates of highly metabolically active cells, mass transport limitations on the delivery of oxygen and essential nutrients can result in a necrotic core2 - thus a non-cohesive spheroid may simply be a consequence of excessive cell death. Alternatively, measurements of the mechanical cohesion of spheroids have been used to assess intercellular binding forces, in relationship to the metastatic potential of a given cell line13. It would be interesting to investigate the relationship between spheroid shape and metastatic potential, perhaps using morphometric parameters such as roundness and perimeter to area ratio.
In addition to investigating the mechanical and morphometric properties of spheroids, assembly in the microwell system permits large-scale production of mixed-composition spheroids, consisting of combinations of multiple cell types and / or biomaterials6,7. The interactions of tumor cells with other cell types are important to more closely model the behavior of tumors in vivo14, thus it may be of interest to generate spheroids from tumor cells in combination with fibroblasts and endothelial cells, for example. Microparticles of various biomaterials may also be incorporated, and can affect spheroid properties both directly through their interactions with cells7,15, and also as reservoirs for the controlled release of growth factors and cytokines into the interior of the spheroid16.
If there is any concern about sterility, for example when working with an microwell plate in which some wells have previously been used, that may have spent some time in an incubator as part of a previous experiment, the wells may be resterilized with 70% ethanol in water.
Once spheroids have been formed, they may also be separated from residual unincorporated cells by passing the suspension over a cell strainer. Individual cells will pass through, while the spheroids will be retained. If the spheroid is suspected of shedding potentially metastatic cells over the course of culture, it may be desirable to perform this procedure multiple times - initially to remove unincorporated cells, and subsequently to isolate purified populations of the cells given off by the spheroids.

有越来越多的证据体，肿瘤细胞有不同的行为在三维培养物比它们在塑料培养时以及确定在常规组织培养平台的治疗剂时，转变到多种生理上相关的系统1可能会失去功效。因此，需要研究癌症细胞的行为在这些条件下，既要深入了解其潜在的生物，并且还增加过渡的新的治疗剂的筛选设施到诊所的成功率。一个有用的模型系统，具有悠久的历史采用被称为肿瘤球体1,2癌细胞的三维集群。理想情况下，技术球体的形成将允许生产大量均匀的球体，其大小和组成由实验者所控制的。而悬滴和孔板接近3,4能够满足其中的一些重quirements，吞吐量一般是有限的，并且产生大量的球状物变得劳动密集的任务。 我们最近开发了一个系统，以解决在再生医学领域5类似的挑战。在一系列密集微米级孔的用人被迫聚集（ 见图1），该方法允许球状体的生成从任意数量的细胞，包括多种细胞类型6的混合物，以及各种生物材料7的掺入。球状体形成大量-几千到几十万或更多-而可以立即提取或保持在其上，形成具有至少一个8到2（未发表的观察）周培养基交换的微孔。因此，该系统非常适合于大量的均匀和可再现的肿瘤球状体的产生为EFFE的评估ctiveness新型抗肿瘤药物或生物的基本调查。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>AggreWell 400 plate</td>
			<td>StemCell Technologies Inc.</td>
			<td>27845/27945</td>
			<td></td>
		</tr>
		<tr>
			<td>Rinsing Solution</td>
			<td>StemCell Technologies Inc.</td>
			<td>07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (37 <span id="docs-internal-guid-263ae0a0-aa3d-bddd-2a36-2ae1901f1604" style="font-size:15px;font-family:Arial;color:#000000;background-color:#ffffff;font-weight:normal;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;">&#181;m)</td>
			<td>StemCell Technologies Inc.</td>
			<td>27215</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>VWR / LONZA</td>
			<td>CA12001-676</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene (EDTA)</td>
			<td>VWR / LONZA</td>
			<td>CA12001-660</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>VWR / LONZA</td>
			<td>CA12002-212</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>VWR / LONZA</td>
			<td>CA-95042-112</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Invitrogen / Life Technologies</td>
			<td>12605010</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>VWR / Motic</td>
			<td>CA19000-610</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X15R centrifuge with carriers for standard well plates</td>
			<td>VWR / Beckman</td>
			<td>CABKA99465, CABK369704, CABK392806</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow biosafety cabinet</td>
			<td>ESBE / Baker</td>
			<td>BKR-SG603AHEUVSP</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of large numbers of size-controlled tumor spheroids using microwell plates

肿瘤球状体正日益被视为一个重要的体外模型中对肿瘤细胞在三维空间中的行为。比传统的贴张文化更加生理有关，他们更准确地概括了复杂性和目前的实际肿瘤的相互作用。为了利用这一模型，以更好地评估肿瘤生物学，或新的治疗剂的功效，这是必要的，以便能够重复地产生球状体，以受控的方式并在显著号码。 该AggreWell系统由金字塔形微孔的高密度阵列，成单细胞悬浮液进行离心分离的。细胞的数量聚类在各微孔的底部，并涉及不同的细胞类型的数量和比率仅依靠由实验者引入悬浮液的性质。因此，我们能够生成任意大小和组成的肿瘤与球体出需要修改底层平台技术。反过来数百每培养面积的平方厘米的微孔确保极高的生产水平可以通过一个简单的，非人工密集的过程来实现。因此，我们预计，该协议将是广泛有益的研究人员在肿瘤球体领域。

可以容易地产生和提取到的悬浮培养从别共解剖起源多种肿瘤细胞系的球状体， 图3示出了一致的尺寸控制得到通过这种方法，用各条件下高度均匀的球体种群。在行为上清除行间的差异也可见，与HT29结肠癌细胞和TE6食管癌细胞形成密集的球体与轮廓分明的界限，而前列腺癌细胞LNCaP引起了那么一致球体不规则边界（见讨论可能的原因）。更强的内力在更连贯的聚集也导致崩溃到一个更对称的形式甚至仍然在其形成的微孔，而LNCaP细胞聚集，特别是在较大的尺寸，明显保留了方形锥体几何微孔。输入的细胞数和所述phy之间的关系<html>所得到的球状体的iCal的大小将依赖于许多变量。根据每个细胞的体积和细胞的雇员的数目的乘积的理论量可以通过细​​胞的损失，这反过来又可以包括细胞内的单细胞悬液阶段损失，以及从过小的聚集体造成的损失降低数字邻居的不足，并从由于运输中的核心局限性坏死和过大的聚集体。大小也将通过在聚合过程中可能出现的任何细胞增殖的影响。因为这些参数会有所不同细胞系的细胞系，如特定物理尺寸的球粒所需细胞，介绍了正确数量的必须通过实验确定。作为第一近似值，预计球体直径以增加与掺入的细胞数的立方根- 例如在细胞的数目的8倍的增加应该导致球体直径增加一倍。 <html> _content“FO：保持together.within页=”总是“&gt; <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50665/50665fig1highres.jpg" src="/files/ftp_upload/50665/50665fig1.jpg" /> 图1。球体制作示意图。的微孔系统由紧凑的四方锥微孔阵列（625每平方厘米为<span id="docs-internal-guid-263ae0a0-aa35-f9c7-69a1-8c5aa197db53" style="font-size:15px;font-family:Arial;color:#000000;background-color:#ffffff;font-weight:normal;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;">400μM号），为使细胞可以被离心的。将细胞聚集了由倾斜的侧壁，以及所得到的旋转椭圆体的大小是通过改变所用的细胞悬浮液的密度来控制。 50665fig2highres.jpg“SRC =”/ files/ftp_upload/50665/50665fig2.jpg“/&gt; 图2。细胞聚集成球状体的组装。球体从每个七百HT29细胞形成。立即离心后（A）中，细胞被松散地聚集在各微孔的底部。注意，该簇被朝右下方在这种情况下，均匀地偏移。这是可以接受的簇大小保持在阵列中是一致的。如果显著簇大小的差异是从阵列的一侧看到对方，参见附注在步骤3.5。培养24小时后（B），细胞间粘附力所造成的簇聚集并形成连贯的球状体，然后可将其提取的（C）。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50665/50665fig3highres.jpg" src="/files/ftp_upload/50665/50665fig3.jpg" /><b<html>转/&gt; 图3。在微孔板产生的球体。从球状体形成七百（A，C，E）或一千五百（B，D，F）的 HT29结肠癌细胞（A，B），前列腺癌细胞LNCaP（C，D）或TE6食管癌细胞（E，F）。注意球状体的稠度的制剂中，并且还细胞系之间的变化 - 尤其是宽松的LNCaP聚集体相比，更密集的HT29和TE6细胞。标尺为200微米。 <table border="1"><tbody><tr><td></td><td colspan="3"> 微孔格式 </td></tr><tr><td></td><td> AggreWell 400 </td><td> AggreWell 40EX </td><td> AggreWell 800 </td></tr><tr><td>微孔宽度（al;color:#000000;background-color:#ffffff;font-weight:normal;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;&quot;&gt;µm ) </td><td> 400 </td><td> 400 </td><td> 800 </td></tr><tr><td>每平方厘米的微孔</td><td> 625 </td><td> 625 </td><td> 156.25 </td></tr><tr><td>每孔微孔</td><td> 1,200 </td><td> 4,700 </td><td> 300 </td></tr><tr><td>最小的细胞每微孔* </td><td> N / A </td><td> N / A </td><td> 2000 </td></tr><tr><td>每个微孔的最大细胞* </td><td> 2,000 </td><td> 2,000 </td><td> 10,000 </td></tr><tr><td>最小的细胞，每孔* </td><td> N / A </td><td> N / A </td><td> 6×10 5个</td></tr><tr><td>每孔最大细胞* </td><td> 2.4×10 6个</td><td> 9.4×10 6个</td><td> 3×10 6个</td></tr><tr><td> 1.1.1 - 70％乙醇的体积（ml） </td><td> 0.5 </td><td> 2.0 </td><td> 0。5 </td></tr><tr><td> 1.2.1 - 冲洗液体积（ml） </td><td> 0.5 </td><td> 2.0 </td><td> 0.5 </td></tr><tr><td> 3.2 - 中预压量（ml） </td><td> 0.4 </td><td> 1.6 </td><td> 0.4 </td></tr><tr><td> 3.5 - 电池装载量（ml） </td><td> 0.4 </td><td> 1.6 </td><td> 0.4 </td></tr><tr><td colspan="4"> *每微孔细胞的数量只是一个近似值作为该值将与所使用的特定细胞的大小而变化，并且应该根据经验确定</td></tr></tbody></table> 表1中。 AggreWell选择和协议的修改。

Watch this Scientific Journal Video about Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates at JoVE.com

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

我们引进的粒度均匀，并组成控制肿瘤球体的协议大数代（几千到几十万），使用市售微孔板。

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 ...

production-large-numbers-size-controlled-tumor-spheroids-using

Research

JoVE Journal

Bioengineering

17.3K 次观看.  University of Calgary. 我们引进的粒度均匀，并组成控制肿瘤球体的协议大数代（几千到几十万），使用市售微孔板。 

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

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

科研

生物工程

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

在球体哺乳动物细胞大规模扩张营养调控连续通

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

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.

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 ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

使用3D生物印刷创建心脏组织展示球体的机械整合

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

化学品生产<em&gt;肺炎克雷伯菌</em使用竹水合物作为原料

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

利用自动 Microbioreactor 技术优化异种蛋白质生产的通用协议

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

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 ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

用于组织工程应用的尺寸控制和无乳化壳聚糖 -染料木酚微凝胶的制备

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...