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>

<ol>
	<li>Landry, J., Bernier, D., Ouellet, C., Goyette, R., Marceau, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroidal+aggregate+culture+of+rat+liver+cells:+histotypic+reorganization,+biomatrix+deposition,+and+maintenance+of+functional+activities.">Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.</a> J Cell Biol. 101 (3), 914-923 (1985).</li><li>Yuasa, C., Tomita, Y., Shono, M., Ishimura, K., Ichihara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+cell+aggregation+for+expression+of+liver+functions+and+regeneration+demonstrated+with+primary+cultured+hepatocytes.">Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.</a> J Cell Physiol. 156 (3), 522-530 (1993).</li><li>Otsuka, 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=Two-dimensional+multiarray+formation+of+hepatocyte+spheroids+on+a+microfabricated+PEG-brush+surface.">Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.</a> Chembiochem. 5 (6), 850-855 (2004).</li><li>Wang, W., 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=3D+spheroid+culture+system+on+micropatterned+substrates+for+improved+differentiation+efficiency+of+multipotent+mesenchymal+stem+cells.">3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells.</a> Biomaterials. 30 (14), 2705-2715 (2009).</li><li>Bartosh, T. J., 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=Aggregation+of+human+mesenchymal+stromal+cells+(MSCs)+into+3D+spheroids+enhances+their+antiinflammatory+properties.">Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties.</a> Proc Natl Acad Sci U S A. 107 (31), 13724-13729 (2010).</li><li>Frith, J. E., Thomson, B., Genever, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+three-dimensional+culture+methods+enhance+mesenchymal+stem+cell+properties+and+increase+therapeutic+potential.">Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential.</a> Tissue Eng Part C Methods. 16 (4), 735-749 (2010).</li><li>Nakasone, Y., Yamamoto, M., Tateishi, T., Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+spheroids+underlayered+with+nonparenchymal+cells+for+biomedical+applications.">Hepatocyte spheroids underlayered with nonparenchymal cells for biomedical applications.</a> IEICE Transactions on Electronics. E94, 176-180 (2011).</li><li>Nishida, K., 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=Corneal+reconstruction+with+tissue-engineered+cell+sheets+composed+of+autologous+oral+mucosal+epithelium.">Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.</a> N Engl J Med. 351 (12), 1187-1196 (2004).</li><li>Ohashi, K., 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=Engineering+functional+two-+and+three-dimensional+liver+systems+in+vivo+using+hepatic+tissue+sheets.">Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets.</a> Nat Med. 13 (7), 880-885 (2007).</li><li>Sekine, 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=Cardiac+cell+sheet+transplantation+improves+damaged+heart+function+via+superior+cell+survival+in+comparison+with+dissociated+cell+injection.">Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection.</a> Tissue Eng Part A. 17 (23-24), 2973-2980 (2011).</li><li>Itaka, K., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+prospects+of+polyplex+nanomicelles+for+plasmid+DNA+delivery.">Progress and prospects of polyplex nanomicelles for plasmid DNA delivery.</a> Curr Gene Ther. 11 (6), 457-465 (2011).</li><li>Endo, T., Itaka, K., Shioyama, M., Uchida, S., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfection+to+spheroid+culture+system+on+micropatterned+culture+plate+by+polyplex+nanomicelle:+a+novel+platform+of+genetically-modified+cell+transplantation.">Gene transfection to spheroid culture system on micropatterned culture plate by polyplex nanomicelle: a novel platform of genetically-modified cell transplantation.</a> Drug Deliv and Transl Res. 2 (5), 398-405 (2012).</li><li>Howard, R. B., Christensen, A. K., Gibbs, F. A., Pesch, 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=The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver.">The enzymatic preparation of isolated intact parenchymal cells from rat liver.</a> J Cell Biol. 35 (3), 675-684 (1967).</li><li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J Cell Biol. 43 (3), 506-520 (1969).</li><li>Tateno, C., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cultivation+of+adult+rat+hepatocytes+that+undergo+multiple+cell+divisions+and+express+normal+parenchymal+phenotypes.">Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes.</a> Am J Pathol. 148 (2), 383-392 (1996).</li><li>Kanayama, N., 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=A+PEG-based+biocompatible+block+catiomer+with+high+buffering+capacity+for+the+construction+of+polyplex+micelles+showing+efficient+gene+transfer+toward+primary+cells.">A PEG-based biocompatible block catiomer with high buffering capacity for the construction of polyplex micelles showing efficient gene transfer toward primary cells.</a> ChemMedChem. 1 (4), 439-444 (2006).</li><li>Itaka, K., Ishii, T., Hasegawa, Y., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polyamino+acid-based+polycations+as+safe+and+effective+gene+carrier+minimizing+cumulative+toxicity.">Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity.</a> Biomaterials. 31 (13), 3707-3714 (2010).</li><li>Uchida, S., 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=PEGylated+Polyplex+With+Optimized+PEG+Shielding+Enhances+Gene+Introduction+in+Lungs+by+Minimizing+Inflammatory+Responses.">PEGylated Polyplex With Optimized PEG Shielding Enhances Gene Introduction in Lungs by Minimizing Inflammatory Responses.</a> Mol Ther. 20 (6), 1196-1203 (2012).</li><li>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+simple,+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet.">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Anim (NY). 34, 39-43 (2005).</li><li>Uchida, S., 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=An+injectable+spheroid+system+with+genetic+modification+for+cell+transplantation+therapy.">An injectable spheroid system with genetic modification for cell transplantation therapy.</a> Biomaterials. 35 (8), 2499-2506 (2014).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<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&#8217;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)</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>Escherichia coli DH5&#945; 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&#8482; 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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

改进的可视化和定量分析，药物使用Micropatterned细胞的影响

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

在机械生物学研究的轨道摇床上的 6 井板中分段内皮细胞的生长

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...