Name: construction of a multilayered mesenchymal stem cell sheet with a 3d dynamic culture system
Uploaded: 2018-10-20T15:00:00
Description: Watch this Scientific Journal Video about Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System at JoVE.com

这项工作得到了中国国家自然科学基金 (31771064 号赠款) 的支持;广东省科技规划项目 (赠款编号2013B010404030、2014A010105029、2016A020214012);广州市科技规划项目 (授权号 201607010063);本科创新与创业培训项目 (授权编号 201610559028);中国青年科学家国家科学基金会 (赠款号 31800819)。

所有干细胞和动物实验程序均按照《国家实验室动物护理指南》的道德准则进行, 并经济南大学动物护理与使用委员会 (中国广州) 批准。
1. 用 PLA2去细胞方法制备民进党脚手架14
注: 有关 PLA2去细胞方法的示意图, 请参见图 1A 。
<ol><li>准备100毫升 200 U/毫升 PLA2溶液。在198毫升碳酸盐缓冲液?...

<ol>
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F., 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=Composite+scaffold+provides+a+cell+delivery+platform+for+cardiovascular+repair.">Composite scaffold provides a cell delivery platform for cardiovascular repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (19), 7974-7979 (2011).</li><li>Liu, Y., 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=Electrospun+nanofibrous+sheets+of+collagen/elastin/polycaprolactone+improve+cardiac+repair+after+myocardial+infarction.">Electrospun nanofibrous sheets of collagen/elastin/polycaprolactone improve cardiac repair after myocardial infarction.</a> American Journal of Translational Research. 8 (4), 1678-1694 (2016).</li><li>Arana, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+delivery+of+collagen+patches+with+adipose-derived+stem+cells+in+rat+and+minipig+models+of+chronic+myocardial+infarction.">Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction.</a> Biomaterials. 35 (1), 143-151 (2014).</li><li>Wang, Y., 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=Preparation+of+high+bioactivity+multilayered+bone-marrow+mesenchymal+stem+cell+sheets+for+myocardial+infarction+using+a+3D-dynamic+system.">Preparation of high bioactivity multilayered bone-marrow mesenchymal stem cell sheets for myocardial infarction using a 3D-dynamic system.</a> Acta Biomaterialia. 72, 182-195 (2018).</li><li>Wu, Z., 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=The+use+of+phospholipase+A(2)+to+prepare+acellular+porcine+corneal+stroma+as+a+tissue+engineering+scaffold.">The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold.</a> Biomaterials. 30 (21), 3513-3522 (2009).</li><li>Degano, I. R., 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=The+effect+of+self-assembling+peptide+nanofiber+scaffolds+on+mouse+embryonic+fibroblast+implantation+and+proliferation.">The effect of self-assembling peptide nanofiber scaffolds on mouse embryonic fibroblast implantation and proliferation.</a> Biomaterials. 30 (6), 1156-1165 (2009).</li><li>Lampe, K. J., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+stem+cell+niches+from+the+molecule+up+through+engineered+peptide+materials.">Building stem cell niches from the molecule up through engineered peptide materials.</a> Neuroscience Letters. 519 (2), 138-146 (2012).</li><li>Cui, X. 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本议定书报告了构建多层 MSC 表的有效方法。该细胞表具有最佳的机械强度、高细胞播种密度和良好的干细胞生物活性。以骨髓基质细胞为例, 用 RAD16-I 多肽水凝胶快速构建3D 单元结构。在动态灌注系统中培养后, 成功地获得了多层 BMSC 片, 骨髓基质细胞保持了干细胞标记的高表达。
构建临时多层单元结构是协议的关键步骤。RAD16-I 是一种商业水凝胶肽, 由1% 氨基酸和99% 水组成?...

干细胞治疗已被报道为许多疾病的有效治疗;然而, 在传统的干细胞注射之后, 低细胞保留率和目标区内存活的不良仍然是关键问题。为了解决这个问题, 组织工程科学家开发了细胞表技术。采用温度-反应培养皿1制备了 monolayered 细胞表, 并对其后续研究报告了在梗死中干细胞保留和存活的显著改善。面积2,3。在这些方法中, 建立多层细胞表是提高细胞存活和细胞表治疗效果3、4的有效策略。从那时起, 科学家们一直在开发不同的细胞表结构方法, 以增加细胞数量, 干细胞属性和细胞表的力学性能。目前, 在心肌梗死5、软骨损伤6、皮肤创面7的治疗中, 已经建立和研究了某些类型的细胞表。
干细胞在移植前的生物活性表现出对损伤组织再生的影响, 不同的细胞表构建策略对干细胞有不同的影响。一方面, 汇合细胞表仅由高密度干细胞组成, 而天然细胞外基质可通过堆叠 monolayered 细胞表8或使用磁性组织工程技术9获得。另一方面, 研究人员开发了不同的支架, 以提供足够的机械强度和支持细胞生长10,11,12, 使低干细胞播种密度, 以确保营养供应。然而, 尽管有这些方法, 多层细胞板结构中的低效营养供应仍然是体外构建过程中的一个重要问题。因此, 迫切需要一种高效、可行的电池板施工系统。
本协议描述了准备 multilayeredmesenchymal 干细胞 (MSC) 细胞表的步骤。在这个施工系统中, 单元板的机械强度由 DPP 提供。基于该脚手架, 可利用 RAD16-I 肽水凝胶快速构建3D 细胞结构, 并采用动态灌注系统培养多层细胞表, 以稳定3D 细胞表结构, 提供足够的营养电池的供应。采用该系统, 成功制备了多层 BMSC 片, 对大鼠心肌梗死模型13进行了最佳的治疗效果。

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			<td height="25" style="height:25px;width:216px;">Phospholipase A2</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:167px;">P6534</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium deoxycholate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:167px;">D6750-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate buffer</td>
			<td style="width:108px;">Gibco BRL</td>
			<td style="width:167px;">89033</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin streptomycin / amphotericin&#160;</td>
			<td style="width:108px;">Gibco BRL</td>
			<td style="width:167px;">15640055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buffer bicarbonate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:167px;">C3041</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Table concentrator</td>
			<td style="width:108px;">Changzhou Aohua Instrument Co.</td>
			<td style="width:167px;">KT20183</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium(DMEM)</td>
			<td style="width:108px;">Corning Cellgro</td>
			<td style="width:167px;">10-014-CVR</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">South American fetal bovine serum&#160;</td>
			<td style="width:108px;">Gibco BRL</td>
			<td style="width:167px;">10270-106/P30-3302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutamine</td>
			<td style="width:108px;">Corning Cellgro</td>
			<td style="width:167px;">25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin/2.21 mM EDTA</td>
			<td style="width:108px;">Corning Cellgro</td>
			<td style="width:167px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biosafety cabinet</td>
			<td style="width:108px;">Esco,Singapore</td>
			<td style="width:167px;">AC2-2S1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Constant temperature incubator&#160;</td>
			<td style="width:108px;">Esco,Singapore</td>
			<td style="width:167px;">CLS-170B-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge tube&#160;</td>
			<td style="width:108px;">Corning</td>
			<td style="width:167px;">430790</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EP tube</td>
			<td style="width:108px;">Axygen</td>
			<td style="width:167px;">31617934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifugal machine</td>
			<td style="width:108px;">TOMOS</td>
			<td>1-16R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:167px;">S9378-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pura Matrix&#160;</td>
			<td style="width:108px;">BD</td>
			<td style="width:167px;">354250</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:216px;">Dynamic perfusion culture system</td>
			<td style="width:108px;">Minucells and Minutissue</td>
			<td style="width:167px;">D-93077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:108px;">Ismatec</td>
			<td style="width:167px;">IPC N8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pump tubing</td>
			<td style="width:108px;">Ismatec</td>
			<td style="width:167px;">Nr.1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MINUSHEET 1300&#160;</td>
			<td style="width:108px;">Regensburg</td>
			<td style="width:167px;"></td>
			<td>tissue carrier components&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MINUSHEET</td>
			<td style="width:108px;">Regensburg</td>
			<td style="width:167px;"></td>
			<td>dynamic perfusion system&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MINUSHEET 0006</td>
			<td style="width:108px;">Regensburg</td>
			<td style="width:167px;"></td>
			<td>gas exchange equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MINUSHEET 0002</td>
			<td style="width:108px;">Regensburg</td>
			<td style="width:167px;"></td>
			<td>500 mL glass bottle&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MINUSHEET 1301</td>
			<td style="width:108px;"></td>
			<td style="width:167px;"></td>
			<td>perfusion culture container&#160;</td>
		</tr>
	</tbody>
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construction of a multilayered mesenchymal stem cell sheet with a 3d dynamic culture system

干细胞治疗在再生损伤器官和组织中具有广阔的发展前景, 细胞表技术的发展, 提高了靶区内细胞的低滞留率和存活率。然而, 在体外构建过程中, 迫切需要一种维持干细胞生物活性和增加细胞表中细胞数量的解决方案。本协议提出了一种具有良好的干细胞生物活性和最佳可操作性的多层细胞表的构建方法。脱细胞猪心包 (DPP) 由磷脂酶 A2 (PLA2) 去细胞法制备, 作为细胞表支架, 大鼠骨髓间充质干细胞 (骨髓基质细胞) 被孤立并扩展为种子细胞。采用 RAD16-I 肽水凝胶构建了临时多层细胞表结构。最后, 用动态灌注系统培养细胞表以稳定三维 (3D) 结构, 并在体外48 小时培养后获得细胞表。该方案为多层干细胞的构建提供了一种有效可行的方法, 今后可作为一种有利的干细胞治疗产品开发出细胞表。

多层干细胞片结构示意图如图 1所示。采用 PLA2去细胞法制备细胞表支架是第一步。以脚手架为基础, 通过将干细胞与 RAD16-1 肽水凝胶混合, 构建一个临时的3D 细胞结构。为了获得具有良好的干细胞生物活性和最佳机械强度的多层细胞表, 在动态灌注系统中培养细胞表。在动态营养供应的条件下, 干细胞可以在多层细胞片中增殖和建立细胞接触, ...

Watch this Scientific Journal Video about Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System at JoVE.com

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

3D 动态培养系统构建多层间充质干细胞片材

本文为构建具有良好干细胞性质的多层干细胞片提供了一种高效可行的方法。

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

干细胞疗法被广泛报道为许多疾病的有希望的治疗方法。细胞表技术已经开发，以提高细胞的保留和在目标内的生存，太。在细胞表构建过程中，营养供给不足仍然是干细胞功能丧失的一个关键问题，干细胞性能不尽人意，最终将影响体内的治疗效果。我们的目的是准备可用的多层干细胞片产品，我们的团队开发了细胞化极化、百化和细胞片支架。基于脚手架的多层细胞启动器将快速构建与非肽氢和动态灌注系统，以确保干细胞的营养供应在体外。拿出一个干燥的DBP脚手架，把它放在黑色底座的中心。在 DBP 脚手架上放一个白色张力环，并贴在组织载体上。确保 DBP 支架完全固定在组织托架中。在 DBP 基架上添加 100 种微升培养基。将脚手架放入37摄氏度的培养箱中，浸泡15分钟。将细胞从培养箱中取出，从培养皿中取出培养剂，用两毫升温暖的PBS轻轻清洗细胞。从培养皿中去除所有 PBS，并确保没有液体保留。在菜中加入两毫升至1.25%的三辛酸菜溶液，并孵育细胞三分钟。通过添加两毫升培养介质来停止尝试素效果。轻轻地从盘子中清洗细胞。将细胞悬浮液转移到新的15毫升离心管中。在重力220倍的离心5分钟，获得细胞沉积物。取出上流液，清除细胞沉积物。用三毫升10%蔗糖溶液重新悬浮细胞。吸气10微升细胞悬浮液，内容细胞数读取血细胞计，提取300万个细胞，并在新的1.5毫升离心管中转移。将细胞在220倍平方根的细胞中心5分钟，完全去除超生体，获得细胞沉淀物。准备 20 微升 10%蔗糖溶液。轻轻重新悬浮细胞并获得均匀的悬浮。在悬浮液顶部加入20微升肽水凝胶，轻轻混合肽水凝胶，将细胞悬浮液与T混合，拿出DBP支架，离开组织载体，然后用T.吸气混合物轻轻吸气，将混合物均匀地添加到DBP脚手架中。在组织载体底部加入一毫升钙化。在培养皿中轻轻加入四毫升的培养，然后出现细胞片。将细胞放入培养箱两小时，让它培养。动态灌注系统包括灌注培养容器、气体交换容器和玻璃瓶。装配是动态灌注系统，如图所示。在玻璃瓶中添加两个水力毫升培养中位数。将细胞片插入培养容器的腔室，在容器中加入三毫升培养中位数，将动态灌注系统放入培养箱中，然后开始泵送。在双假设卧式中打开区域性容器。从培养容器中拿出细胞表，放入培养皿中。使用一个钳子固定组织载体，并使用另外两个钳子将白色张力环与黑色底座分离。最后，获取多层单元格板。DBP 基架是透明的。SEM 转售显示所捕获的学院和组件完全保留。细胞片可以通过钳子获得和保存，以进行短期保存。这些细胞片可以转移到1.5毫升的环状体管。例如，作为一个模型，从BMFC对干细胞标记CD 9O和CD 29的细胞显示高度阳性开始。在细胞结构后，BMFC的离开细胞板保持CD 90和CD 29的高膨胀水平。构建多层细胞结构是协议的关键步骤。当PH值从SA变化为中性时，平移时间需要四个水凝胶，因此，用蔗糖溶液清洗细胞以去除表面离子非常重要。此外，细胞悬浮液和泛氢凝胶的体积比不是构建临时多层结构的好朋友。3D干细胞结构形成后，动态灌注系统对稳定多层结构、保持干细胞活力十分重要。培养中位的动态渗透可以促进干细胞在多层结构中扩散和推断细胞背景，动态培养温度计也应当根据不同的干细胞类型进行调整。

Research

JoVE Journal

Bioengineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Optimizing Attachment of Human Mesenchymal Stem Cells on PolyÃƒÅ½Ã‚Âµ-caprolactone Electrospun Yarns

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Micropatterning and Assembly of 3D Microvessels

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...