Name: propagation of dental and respiratory cells and organs in microgravity
Uploaded: 2021-05-25T15:00:00
Description: Watch this Scientific Journal Video about Propagation of Dental and Respiratory Cells and Organs in Microgravity at JoVE.com

研究得到了国家牙科和颅面研究所（UG3-DE028869和R01-DE027930）的慷慨资助。

获得了所有必要的机构批准，以确保该研究符合TAMU机构动物护理指南。
1. 生物反应器组装和灭菌
<ol><li>按照制造商的建议，在塑料仪器循环的高压釜中对生物反应器的四个高纵横比容器（HARV）进行灭菌20分钟。</li>
	<li>灭菌后，通过拧紧生物反应器随附的螺钉将容器组装在细胞培养罩中，然后容器即可使用。</li>
</ol>2. 用于生物反应器共培养实验的支架
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<ol>
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在微重力下细胞生长方案的关键步骤包括生物反应器，支架，用于3D培养的细胞以及作为诱导细胞分化手段的支架涂层。我们研究中使用的生物反应器类型包括RCCS-4生物反应器，这是NASA开发的原始旋转细胞培养系统（RCCS）旋转圆柱形组织培养装置的最新修改，用于在模拟微重力下生长细胞。这种RCCS-4环境提供了极低的剪切应力，从而增强了传质并改善了培养性能。这个版本的生物反应器允许同时...

重力影响地球上生命的方方面面，包括单个细胞的生物学及其在生物体内的功能。细胞通过机械感受器感知重力，并通过重新配置细胞骨架结构和改变细胞分裂来响应重力的变化1，2，3。微重力的其他影响包括流体填充囊泡中的静水压力，细胞器的沉降以及浮力驱动的流动和热量对流4。关于重力损失对人体细胞和器官影响的研究最初是为了模拟太空飞行任务期间宇航员的失重环境5。然而，近年来，这些最初由NASA开发的用于模拟微重力的3D生物反应器技术正变得越来越重要，作为细胞群培养的新方法，否则不适合2D培养系统。
3D生物反应器通过在悬浮液中培养细胞来模拟微重力，从而产生持续的“自由落体”效果。旋转生物反应器的其他优点包括器官培养系统中没有空气暴露，剪切应力和湍流减少，以及持续暴露于不断变化的营养供应中。旋转细胞培养系统（RCCS）生物反应器提供的这些动态条件有利于空间共定位和单细胞聚集体的三维组装6，7。
以前的研究已经证明了旋转生物反应器在骨再生8，牙胚培养9和人类牙囊细胞培养10方面的优势。还有一份报告表明，RCCS增强EOE细胞增殖和分化为釉母细胞11。然而，分化的细胞被认为是基于釉质母细胞蛋白免疫荧光和/或仅釉质生成素表达的釉质母细胞11 ，而不考虑其细长的形态或极化细胞形状。
除了NASA开发的旋转壁容器（RWV）生物反应器外，从细胞生成3D聚集体的其他技术包括磁悬浮，随机定位机（RPM）和clinostat12。为了实现磁悬浮，用磁性纳米颗粒标记的细胞使用外部磁力悬浮，从而形成无支架的3D结构，这些结构已用于脂肪细胞结构的生物制造13，14，15。模拟微重力的另一种方法是通过控制围绕两个轴的同时旋转来产生多向 G 力，从而消除称为 clinostat16 的设备中心的累积重力矢量。当骨髓干细胞在抑溪恒温器中培养时，通过抑制成骨细胞分化来抑制新的骨形成，说明了微重力16的去分化作用之一。
促进釉质母细胞忠实培养的体外系统将为牙釉质组织工程迈出重要一步17。不幸的是，到目前为止，鳞翅目鳞翅目到目前为止，已经报道了五种不同的釉质母细胞样细胞系，包括小鼠釉质母细胞系细胞系（ALC），大鼠牙齿上皮细胞系（HAT-7），小鼠LS8细胞系20，猪PABSo-E细胞系21和大鼠SF2-24细胞系22。然而，这些细胞中的大多数在2D培养中失去了其独特的极化细胞形状。
在本研究中，我们转向旋转细胞培养生物反应器系统（RCCS），以促进与间充质祖细胞共培养的宫颈环上皮的釉母细胞样细胞的生长，并克服2D培养系统的挑战，包括营养物质流动减少和重力引起的细胞骨架变化。此外，RCCS为研究与牙周组织工程相关的细胞/支架相互作用以及检查炎症介质对体 外肺泡组织的影响提供了新的途径。总之，这些研究的结果强调了基于微重力的旋转培养系统在分化上皮的繁殖和评估环境对 体外生长细胞的影响方面的益处，包括细胞/支架相互作用和组织对炎症状况的反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>ThermoFisher Scientfic</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma Aldrich</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>BGJb Fitton-Jackson Modification media</td>
			<td>ThermoFisher Scientfic</td>
			<td>12591</td>
			<td></td>
		</tr>
		<tr>
			<td>BIOST PGA scaffold</td>
			<td>Synthecon</td>
			<td>Custom</td>
			<td>Available from the company through a custom order</td>
		</tr>
		<tr>
			<td>BMP-2</td>
			<td>R&#38;D Systems</td>
			<td>355-BM</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Sigma Aldrich</td>
			<td>D6429-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher Scientfic</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibricol</td>
			<td>Advanced Biomatrix</td>
			<td>5133-20mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Galanin</td>
			<td>Sigma Aldrich</td>
			<td>G-0278</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin disc</td>
			<td>Advanced Biomatrix</td>
			<td>CytoForm 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphene sheets</td>
			<td>Advanced Biomatrix</td>
			<td>CytoForm 300</td>
			<td></td>
		</tr>
		<tr>
			<td>hEGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>hFGF</td>
			<td>ThermoFisher Scientfic</td>
			<td>AA1-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyapatite disc</td>
			<td>Advanced Biomatrix</td>
			<td>CytoForm 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Il-6 protein</td>
			<td>PeproTech</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte SFM media (1X)</td>
			<td>ThermoFisher Scientfic</td>
			<td>17005042</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Corning</td>
			<td>354259</td>
			<td></td>
		</tr>
		<tr>
			<td>LRAP peptide</td>
			<td>Peptide 2.0</td>
			<td>Custom made sequence: MPLPPHPGSPGYINLSYEVLT 
			PLKWYQSMIRQPPLSPILPEL 
			PLEAWPATDKTKREEVD</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore Nitrocellulose membrane</td>
			<td>Merck Millipore</td>
			<td>AABP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>RCCS Bioreactor</td>
			<td>Synthecon</td>
			<td>RCCS 4HD</td>
			<td></td>
		</tr>
		<tr>
			<td>SpongeCol</td>
			<td>Advanced Biomatrix</td>
			<td>5135-25EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Syring valve one way stopcock w/swivel male luer lock</td>
			<td>Smiths Medical</td>
			<td>MX5-61L</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes with needle 3cc</td>
			<td>McKESSON</td>
			<td>16-SN3C211</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA (0.25%)</td>
			<td>ThermoFisher Scientfic</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

propagation of dental and respiratory cells and organs in microgravity

重力是人体细胞功能、增殖、细胞骨架结构和取向的关键决定因素之一。旋转生物反应器系统（RCCS）模拟在太空中发生的重力损失，而是通过连续旋转培养的细胞或组织来提供微重力环境。这些RCCS确保营养，生长和转录因子以及氧气的不间断供应，并解决了静止的2D（二维）细胞或器官培养皿中重力的一些缺点。在本研究中，我们使用RCCS共培养宫颈环细胞和牙髓细胞成为釉质母细胞，表征牙周祖细胞/支架相互作用，并确定炎症对肺泡的影响。RCCS环境促进了釉母细胞样细胞的生长，促进了牙周祖细胞增殖以响应支架涂层，并允许评估炎症变化对培养的肺泡的影响。这份手稿总结了沿途的环境条件、材料和步骤，并突出了关键方面和实验细节。总之，RCCS是掌握 体外 细胞培养和3D（三维）生长的创新工具，并允许研究不适合经典2D培养环境的细胞系统或相互作用。

生物反应器的内室为细胞增殖和分化、附着在支架上或聚集形成类似组件的组织提供了环境。每个 HARV 容器可容纳多达 10 mL 的培养基，并促进营养物质的持续循环，使每个细胞都有极好的存活机会。图1A说明了注射器端口与连接无菌一站式阀的容器前板上的连接。这些阀门充当培养室的守门人。介质更换需要打开截止旋塞阀，以方便连接含有新鲜培养基的无菌注射器。生物?...

Watch this Scientific Journal Video about Propagation of Dental and Respiratory Cells and Organs in Microgravity at JoVE.com

Propagation of Dental and Respiratory Cells and Organs in Microgravity

该协议提出了一种在微重力下培养和3D生长釉质母细胞样细胞的方法，以保持其细长和极化形状以及牙釉质特异性蛋白质表达。还描述了微重力下牙周工程结构和肺器官培养的培养条件。

牙齿和呼吸细胞及器官在微重力下的繁殖

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

在培养物中拉长和极化分泌釉质生成素的釉质母细胞。这也是第一个在微重力下生长釉质母细胞的方案。釉质母细胞在培养中的生长一直是牙釉质领域的主要挑战之一。对我们来说，它使用了这种生物反应器模型，这是我们第一次在牙釉质研究中实现这一标志。该模型可用于了解成釉细胞的生物学。该模型将由Mirali Pandya博士演示。首先将出生后六天小鼠收集的组织放在解剖显微镜下并解剖颈环。通过将无菌阀连接到注射器端口来设置生物反应器。将两个细胞，宫颈环和牙髓以及涂层支架添加到生物反应器中，并用补充有生长因子和细胞外基质蛋白的10毫升角质形成细胞SFM培养基填充生物反应器容器。关闭并拧紧生物反应器容器的填充口盖，然后打开无菌阀。使用两个无菌的三毫升注射器进行培养基更换。为此，用新鲜培养基填充一个注射器，并将另一个注射器留空。打开两个注射器阀门，轻轻地将气泡移向空注射器端口。一旦将来自整个注射器的新鲜培养基小心地注入容器中，通过空注射器端口清除所有气泡。用盖子关闭注射器端口。将每个容器连接到旋转器底座。打开电源并将速度设置为每分钟 10.1 转。调整速度以确保脚手架处于悬挂状态并且不接触船壁。对于每24小时更换一次培养基，将容器直立放置以确保细胞沉降在底部。打开注射器端口，将无菌注射器连接到端口。如前所述，吸出75%的营养耗尽培养基，并通过另一个端口注入新鲜培养基。石墨烯支架和胶原蛋白支架的宏观图如图所示。石墨烯支架具有平行的表面浮雕阵列，而胶原蛋白支架则表现出多孔结构。解剖骨骼化的小鼠下颌骨，露出下部小鼠门牙的最远端。切除的颈袢的精确位置在出生后六天的小鼠门牙中划定，而成年骨骼化小鼠下颌骨中的类似区域则作为参考。半颌骨由三颗下颌臼齿和一个不断生长的门牙组成，而颈袢细胞壁龛包含牙釉质形成所需的各种细胞群。宫颈环细胞在定制的微环境中成功分化导致釉母细胞的形成，其典型特征是极化细胞，一端是细胞核，另一端是长细胞过程。在没有生长因子和支架涂层的培养基中单独培养细胞导致宫颈环细胞分泌关键牙釉质蛋白，但没有伸长或极化。与含有未包衣支架的对照组相比，甘丙素包衣支架组表现出显着更高的增殖率。从E15小鼠收获的肺组织片段在生物反应器的3D微重力环境中成功培养10天。向培养基中添加白细胞介素-6导致与炎症相关的肺泡形态变化，类似于体内观察到的变化。在介绍中，我告诉过你牙釉质团队提出一个釉质母细胞培养模型是多么困难。现在，我们已经通过使用高度创新的程序解决了这一挑战，该程序侧重于两个方面：海绵胶原蛋白支架以及使用基质来分化釉母细胞。这两项创新共同导致了3D培养中釉母细胞样细胞的极化和生长。这些3D培养的细胞是了解成釉细胞生物学的绝佳模型。为了理解这种生物学，我们可以使用各种技术，包括电子显微镜、蛋白质组学和基因组学。成淀粉细胞是奇妙的细胞。是的，它们有能力分泌棱柱形牙釉质。现在，这个生物反应器模型使我们能够了解釉质母细胞如何分泌基质并分泌磷灰石矿物质来生长棱柱状牙釉质。

Research

JoVE Journal

Bioengineering

Use of Human Perivascular Stem Cells for Bone Regeneration

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

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 ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

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 ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...