Name: gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation
Uploaded: 2015-07-31T13:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

我们深深体会到池谷健博士和技术人员东洋合成，东京，日本提供热敏微图案培养板以及科学建议。我们也感谢小仓里美小姐，SAE铃木小姐，明日香三好女士和Katsue森井女士与动物实验的技术援助。这项工作是财务部分由日本学术振兴会KAKENHI格兰特 - 在急救科学研究的支持，创新（COI）计划的中心，并从日本科学技术振兴机构（JST）的S-创新项目，以及JSPS核心 - 到核心方案，A.高级研究网络。

所有的动物进行了研究，东京大学的日本动物护理和使用委员会，东京的批准。 1.细胞的制备<ol><li>对于原代肝细胞，通过改良的两步胶原酶消化过程13,14按照协议，从大鼠肝细胞分离。 <ol><li>麻醉只SD（SD）大鼠（雄性，5周龄）吸入麻醉下异氟醚。放置老鼠在连接到麻醉机，以提供异氟醚为所述腔室的腔室。取出鼠入睡后，并使用掩模通风设置在手术台上。控制异氟烷流大约为0.4 - 通过检查大鼠的条件0.7升/分。灌注只SD（SD）大鼠（雄性，5周龄）的从肝门静脉肝脏用特殊溶液8克/升的氯化钠组成（氯化钠），400毫克/升的氯化钾（KCl）中，78毫克/ L磷酸二氢钠二水合物（和NaH 2 PO 4·2H 2 O），151毫克/升磷酸氢二钠十二水合物（的Na 2 HPO 4·12H 2 O），2.38克/升的2- [4-（2-羟乙基）-1-哌嗪基]乙磺酸（HEPES），190毫克/升乙二醇四乙酸（EGTA），350毫克/升碳酸氢钠（ 碳酸氢钠 ），和900毫克/升葡萄糖。 </li><li>循环通过肝脏胶原酶溶液。 注意:溶液由500毫克/升的胶原酶，9.8克/升Hank氏缓冲盐，2.38克/毫升HEPES，556毫克/毫升氯化钙水合物（氯化钙2·H 2 O），350毫克/升碳酸氢钠 ，和50毫克/升胰蛋白酶抑制剂，以将pH调节至7.2。 </li><li>小心地取出肝，并轻轻剁碎它用手术刀刀片一个菜，添加的Dulbecco氏改良的Eagle氏培养基（DMEM），补充有10％胎牛血清（FBS），并通过100微米过滤细胞悬浮液尼龙网。以除去额外碎片，离心细胞悬浮液，在20×g下1分钟。在此步骤中，肝细胞在上清液中。 </li><li>重复离心步骤两次（总共三个离心）。最后，离心肝细胞，在50×g下3分钟，以恢复它们以颗粒的形式。 </li><li>重悬的肝细胞，以4×10 5个细胞/ ml在DMEM构成的特殊培养基中的浓度补充有10％FBS，1％青霉素-链霉素-戊二醛（PSQ），1％二甲基亚砜（DMSO），0.1微摩尔/ L的地塞米松，0.5微克/毫升的胰岛素，10毫摩尔/升烟酰胺，0.2毫摩尔/升磷酸抗坏血酸（ASC-2P），和10ng / ml人表皮生长因子（hEGF的）15。这个特殊的介质是强制性的在体外条件下保存肝功能。 </li></ol></li><li>为了获得大鼠MSCs，安乐死只SD（SD）大鼠（雄性，5周龄）由isoflur过度管理烷。切除股骨和胫骨，并通过插入一个22克针进入骨的轴冲洗出来，用10ml的DMEM补充有10％FBS的收集骨髓。通过100微米的尼龙筛收集细胞，通过过滤。 </li><li>种子细胞上使用含有10％FBS和1％青霉素/链霉素的DMEM10厘米培养皿。对于球体的实验中，使用干细胞在5代。 </li></ol> 2.准备三维细胞球体<ol><li>市售获得微图案培养板，在其上的细胞粘附区域规则地排列在直径100微米的二维方式，通过涂布了的PEG基质的非粘合区所包围。 注:对于细胞移植，与热敏性聚合物PIPAAm一个附加的涂层是必要的，让细胞分离，通过冷却板（参见步骤5.1）。温度波动可以在该聚合物的化学诱导的变化8-10。在37℃，PIPAAm稍疏水的，使得在正常条件下被培养的细胞。的降低在低于32℃的结果在聚合物的快速水合的温度，从而导致细胞的自发脱离。 </li><li>种子上的12孔微图案板的肝细胞或干细胞，在4×10 5细胞/孔andincubate它们在37℃下在含有5％的CO 2的潮湿气氛中的密度。细胞将积聚在微图案粘附区域逐步形成圆球状体在2天。 注意:对于在单层培养制备对照细胞，用一般的12孔板并如上所述种子细胞以相同的密度，按照类似的程序。 </li></ol> 3.准备Polyplex公司纳米胶束的<ol><li>合成的嵌段共聚物，PEG-的PAsp（DET）（聚[N&#39; - [N - （2-氨基乙基）-2-氨基乙基]天冬酰胺]）（聚乙二醇的Mw = 12，000，聚合度的PAsp（DET）链段的（DP）= 59），以及仅由该阳离子段[的PAsp（DET）]（DP = 55）的，按照先前由作者11中描述的方法均聚物， 16，17。 </li><li>通过调节聚合物浓度制备的PEG-的PAsp（DET）嵌段共聚物中的残留的氨基在10mM HEPES缓冲液（pH值7.3）等摩尔比的混合溶液和的PAsp（DET）均聚物33.3微克/毫升和19.1微克/毫升。 注意:上述的聚合物浓度可用于制备纳米胶束与残余摩尔总氨基的两种聚合物中的磷酸基团中的pDNA（N / P比）的10的N / P比比率可以变化的代表值根据不同的细胞类型和用途（详见讨论）。这两种聚合物的结合使用可同时实现有效的PEG屏蔽的PAsp（DET）的运作，以加强内体逃脱（详见讨论）18。 </li><li>通过克隆表达各自基因导入pCAG-GS质粒段（http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html制备的质粒DNA编码Gaussia萤光素酶，GL4萤光素酶，或促红细胞生成素）下使用根据制造商的协议市售试剂盒的CAG启动子/增强子得到表达。扩增质粒DNA在主管大肠杆菌菌株，并用无内毒素的质粒DNA纯化系统纯化它。确定质粒DNA的浓度在260纳米的吸光度，得到150微克/毫升溶液在10mM HEPES缓冲液（pH7.3）中。 </li><li>为了制备多聚物纳米胶束，彻底混合的pDNA溶液（在10mM HEPES缓冲液中150微克/ ml），将两种聚合物以2:1的比例预混合的溶液:1（体积比）。 </li></ol> 4.基因转染到球体<ol><li>孵育细胞（肝细胞或MSCS）播种到微图案板允许球体成熟，形成后72小时。对于基因转染，用1ml新鲜培养基更换培养基后添加100微升的polyplex纳米胶束溶液（含有10μg质粒DNA）到每个孔中。继续24小时的纳米胶束解决方案的孵化。 </li><li>对于使用基于脂质的转染试剂对照，将混合的pDNA溶液与试剂在3重量比试剂/ pDNA的调整的pDNA的最终剂量为等于两个所述基于脂质的试剂和纳米胶束的方法。 </li></ol> 5.恢复细胞球体和移植<ol><li>替换用200μl冷冻的PBS的培养基中，然后将板在冰上。 注意:通常，球状体可以在约15分钟分离，并且可以在悬浮液中进行移植的形式被回收。 </li><li>轻轻吸出细胞在200微升悬挂使用注射器用23 G或27G的针头在体内的注射。 </li></ol> 6.评估转基因表达<ol><li>正是24小时用新鲜培养基替换后100微升培养基的-为Gaussia萤光素酶分泌到培养基中的表达的体外评价，收集50个。估计使用商业海肾萤光素酶测定系统，并根据制造商的协议光度计的萤光素酶表达。 注:Gaussia荧光素酶保持稳定在培养基中超过一个星期。因此，为了评价转基因表达的实时效率，事先更换新鲜的培养基中收集的样品介质。介质变化的时间可以灵活。 </li><li>为在宿主动物中的转基因表达以下细胞移植的体内评价，麻醉的BALB / c裸鼠（雌性;7周旧）吸入麻醉下异氟醚。 <ol><li>放置一个鼠标连接到麻醉机，以提供异氟醚为所述腔室的腔室。取出小鼠入睡后，并使用掩模通风设置在手术台上。控制异氟烷流大约为0.2 - 通过检查小鼠的条件0.5升/分。 </li><li>注入200μl的含有球状体转染GL4荧光素酶表达质粒DNA（如在4.1中描述）到腹部区域的皮下组织的细胞悬浮液。 </li><li> （150毫克/公斤;静脉内途径）立即注射D-萤光素后，根据制造商的协议使用IVIS成像系统测量萤光素酶表达。 </li></ol></li><li>为了评价细胞移植的治疗效果，注入200μl的含有球状体转染的促红细胞生成素编码质粒DNA进入细胞悬液腹部区域的皮下组织。 <ol><li>采集血样 ​​通过出血颌下获得约200微升血19。测量使用的血液试样分析仪的血红蛋白和血细胞比容。 注意:用于移植的细胞数量是由接种于板的数量调节。不幸的是，它是难以确定，因为内部的球体的数量不能测量准确的细胞数。 </li></ol></li><li>实验之后，放置在加热垫上有温度控制器相连的小鼠，直到从麻醉中苏醒。 </li></ol>

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The authors declare that they have no competing financial interests.

在这个协议中，关键是要在基因导入和球体恢复的步骤保持球状体的三维结构。它是为了保持有利的微环境必需的细胞，以避免细胞死亡或细胞活性的损失。例如，白蛋白分泌，肝细胞的具有代表性的固有功能，保存完好，在肝细胞的球状体，而在常规的单层培养的肝细胞播种12后迅速失去其分泌能力几天。对于干细胞，所述球状体显著提高分化的效率为脂肪细胞或成骨细胞，与参与脂肪...

细胞移植疗法已经引起了广泛的关注用于治疗各种顽固性疾病。是由移植的细胞分泌的活性和生物活性因子半衰期是用于细胞移植系统的改进的治疗效果是必不可少的。移植前细胞的遗传修饰是一个有利的技术规范和操纵细胞功能，包括的生物活性因子的分泌。同样重要的是保持一个有利的微环境的细胞，以避免细胞死亡或细胞活性的损失。三维（3D）球体细胞培养物，其中细胞 - 细胞相互作用被保存完好，是有希望的用于此目的，例如，用于改善从原代肝细胞的白蛋白分泌和促进从间质干细胞的多谱系分化（间充质干）1-7。 spheroi在这项研究中，一种新的组合系统ð培养和基因转染被用来作为遗传修饰的细胞移植的平台。用于创建球体细胞，在微图案化培养板一个球体培养系统被使用。在这些平板上，直径100微米的细胞粘附区域被规则地排列在一个二维的方式，并通过涂覆的PEG基质3的非粘合区所包围。由种子细胞的足够数量，100微米3D球体直径阵列形成对应于该微图案培养床。 所述球状体被回收而不破坏其三维结构通过使用热敏细胞培养板，涂覆有热敏聚合物，聚（异丙基丙烯酰胺），该（PIPAAm）8-10。该微图案架构上的热敏板构造（定制）。通过简单地降低所述板的温度，所述球状体从培养床分离并分散d。在磷酸盐缓冲盐水（PBS）中。因此，可以以可注射悬浮液的形式得到了大量的球体为100微米的大小均匀的。 <img alt="图1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> 图1中的球体培养系统上的微图案化板示意图。遗传修饰通过使用原始非病毒基因载体，多聚物纳米胶束基因转染来实现。它是由质粒DNA（质粒DNA）和聚乙二醇（PEG）-polycation嵌段共聚物11。这些有特点的核壳结构包括一个PEG外壳和浓缩质粒DNA的内芯，使安全有效的基因导入细胞用于治疗目的11。 <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">请点击此处查看THI的放大版本。</a>的身影。 <img alt="图2" src="/files/ftp_upload/52384/52384fig2.jpg" /> 图由核酸和PEG嵌段聚阳离子嵌段共聚物形成的复合物的多聚物纳米胶束的2.结构。在这项研究中，该技术的主要优点在于，球体结构不是由纳米胶束基因转染期间破坏。后大鼠原代肝细胞的球状体的纳米胶束介导的转染，可以得到延长的转基因表达对于一个多月与来自肝细胞连续白蛋白分泌的水平比得上未转染球状体12。从球状体的转基因表达和分泌白蛋白的热敏板恢复之后也保持不变。显而易见的是，纳米胶束可以安全地促进基因导入不损害熟知的先天功能atocytes。因此，球体细胞在微图案热敏板的使用纳米胶束基因导入是一种很有前途的平台，为转基因细胞移植体外培养的组合。 <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Pen-Strep-Glut</td><td>GIBCO</td><td></td><td></td></tr><tr><td>Dexamethasone</td><td>Wako Pure Chemical Industries</td><td>041-18861</td><td></td></tr><tr><td>Nicotinamide</td><td>Wako Pure Chemical Industries</td><td>141-01202</td><td></td></tr><tr><td>Hank&amp;rsquo;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td><td>Sigma-Aldrich</td><td>A8960</td><td></td></tr><tr><td>Human epidermal growth factor (hEGF)</td><td>Toyobo</td><td>PT10015</td><td></td></tr><tr><td>Cell-able multi-well plates</td><td>Toyo Gosei</td><td>PP-12</td><td></td></tr><tr><td>Thermosensitive cell culture plates (Upcell&lt;sup&gt;)&lt;/sup&gt;</td><td>CellSeed Inc</td><td></td><td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td></tr><tr><td>Lipid-based transfection reagent (FuGENE HD)</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>Renilla Luciferase Assay System</td><td>Promega</td><td>E2810</td><td></td></tr><tr><td>pGL4 Luciferase Reporter Vector</td><td>Promega</td><td>E6651</td><td></td></tr><tr><td>pDNA expressing Gaussia luciferase</td><td>New England BioLabs</td><td>N8082S</td><td></td></tr><tr><td>Mouse erhthropoietin-expressing vector</td><td>Origene</td><td>MC208445</td><td></td></tr><tr><td>pCAG-GS</td><td></td><td></td><td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; DH5&amp;alpha; competent cells</td><td>Takara</td><td>9057</td><td></td></tr><tr><td>Endotoxin-free plasmid DNA purification system</td><td>Nippon Genetics</td><td>NucleoBond Xtra EF</td><td></td></tr><tr><td>Collagenase</td><td>Wako Pure Chemical Industries</td><td>639-00951</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>GIBCO</td><td>R-007-100</td><td></td></tr><tr><td>Luminometer</td><td>Promega</td><td>GloMax&amp;trade; 96 Microplate Luminometer</td><td></td></tr><tr><td>IVIS Imaging System</td><td>Xenogen Corp.</td><td>Xenogen IVIS Spectrum in vivo imaging system</td><td></td></tr><tr><td>Blood sample analyzer</td><td>Sysmex</td><td>pocH-100i Automated Hematology Analyzer</td><td></td></tr></tbody></table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

改善细胞移植的治疗功效，一个移植系统的转基因，可注射的球状体被开发。将细胞球粒上涂敷有热敏聚合物微图案板的培养体系制备。形成在板的一些球状体中，对应于直径100微米的细胞粘附区域被以二维方式规则地排列，由非粘合区所包围是由一个聚乙二醇（PEG）基体涂覆。所述球状体可容易地回收作为液体悬浮液通过降低板的温度，并且它们的结构是由具有足够大口径（超过27 G）将它们通过注射针良好的维护。遗传修饰是通过基因转染使用原始非病毒基因载体，多聚物纳米胶束，其能够将基因导入细胞，而不破坏球体结构的实现。对于PRIM转染了荧光素酶表达基因进制肝细胞的球状体，在萤光素酶是可持续在移植动物中获得，以及保存肝细胞功能，通过白蛋白的表达所指示的。该系统可应用于多种细胞类型，包括间质干细胞。

在通过使用多聚物纳米胶束或控制基于脂质的转染试剂12中的肝细胞或干细胞形成的球状体进行的Gaussia荧光素酶表达质粒DNA的基因转染。所述纳米胶束诱导球体结构几乎没有变化与非转染的球体上的微图案化板相比，而对照试剂显著转染（ 图3）后一天破坏的结构。使用纳米胶束，一致白蛋白分泌和转基因（Gaussia荧光素酶）的表达转染后进行很好的维护了将近一个月。然而，...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K 次观看.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

视频: Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

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

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

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

生产尺寸控制肿瘤细胞球体采用微孔板大数

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

生物工程

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

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

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

生成用于药物评估研究的 3D 肿瘤微球

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

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

发育生物学

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 ...

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

使用新型 Stamp 装置从二维细胞培养物中生成三维球状体/类器官

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver ...

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

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

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.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

摘要

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此视频中的章节

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

生产尺寸控制肿瘤细胞球体采用微孔板大数

生成用于药物评估研究的 3D 肿瘤微球

建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性

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

使用新型 Stamp 装置从二维细胞培养物中生成三维球状体/类器官

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

三维球体制作一个高效，灵活细胞聚集方法