Name: gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation
Uploaded: 2015-07-31T13:00:00
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?...

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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>

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. ...

Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

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

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

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

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

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

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

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

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

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

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, ...