作者感谢W. Sun和K·詹森的批判性的讨论，并承认支持纳米生物力学实验室，在新加坡国立大学。 NAK承认支持由居里夫人IIF奖学金。

1.细胞收获<ol><li>得到的MD-MBA-231细胞从37℃，5％CO 2的培养箱中培养。分离，以0.5％胰蛋白酶-EDTA溶液从组织培养板上的细胞。使用1个毫升胰蛋白酶-EDTA溶液用于在T25培养瓶中的细胞进行培养。 </li><li>沉淀细胞在15ml锥形管中，离心，在200×g下4分钟，吸出上清液，并悬浮细胞在5ml培养基。 </li><li>计数细胞密度，ρ，使用血球。 注意：为了制备细胞接种内凝胶，将细胞悬液稍后会稀释10×达到最终细胞接种密度。因此，10×浓缩的细胞悬浮液是必需的。 </li><li>计算达到10×细胞浓度所需介质的体积： <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/52735/52735eq1.jpg" width="175" /> 注：最后一个细胞接种密度，C 最终 ，大约215; 10 6个细胞/ ml的被推荐用于MD-MBA-231细胞，并用于在此协议。其他播种密度也可探索其他类型的细胞。 </li><li>沉淀细胞再一次在一个15毫升的锥形试管离心，在200×g下4分钟，吸出上清液。 </li><li>重悬的无血清细胞培养基中的细胞所需要的量（V 培养基在步骤1.3计算的）彻底以最小化细胞聚集。 注意：酚红是自体荧光，并且可以与荧光/反射成像干涉。使用不含酚红的培养基中可以考虑实现最佳的图像质量。 </li></ol> 2.准备胶原蛋白解决方案<ol><li>获取股票胶原溶液，10×PBS缓冲液，的Milli-Q H 2 O，0.1M NaOH和几个离心管。保留所有冰上以防止过早胶原蛋白的聚合，并保持无菌状态。 </li><li>平衡无菌玻璃 -底菜在37℃培养箱预热吧。 注：在本协议中的所有卷进行了优化的12 mm孔玻璃底菜。如果其他菜类型时，相应地调整卷。 </li><li>计算需要准备50微升2.4毫克/毫升的胶原溶液为基于所述胶原原料浓度的内凝胶（溶液Ⅰ）所需的体积。 注意：也可以使用其它的胶原蛋白的浓度为所述内凝胶。 </li><li>在无菌的环境（通常是生物安全罩），慢慢加入5微升10×PBS缓冲液至所需的胶原原液量（在步骤2.3计算的）伴以轻轻旋转。注意避免气泡的形成。 </li><li>调节混合物的7.4使用0.1M NaOH中使用校准的pH计的pH。作为一个粗略的指导，使用约5微升以使pH接近7.4（该量取决于该原料浓度和pH下）。 注意：请注意，参与这一卷步骤太小使用标准pH计。请使用以下招数之一： <ol><li>制备用于多个样品的胶原蛋白溶液。使用标准pH计调整在散装的pH值和整个样本分配胶原溶液。 </li><li>可替代地，调整的胶原溶液的pH值在大的体积（ 即 ，体积，允许使用标准的pH计）。注意，以使pH至最终pH所需的NaOH的量。缩小的容积，并使用氢氧化钠适量实验。确认使用石蕊试纸的pH值。 </li><li>否则，使用微pH电极能够更准确地调节小批量的pH。 </li></ol></li><li>使溶液于45体积微升使用的H 2 O.执行上冰的所有步骤，以防止过早胶原蛋白的聚合。 </li></ol> 3.形成同心凝胶文化<ol><li>就拿预热的玻璃底菜（见步骤2.2）从日Ë孵化器。 </li><li>添加5微升10×浓细胞悬液（步骤1.5准备）来解决一悬浮彻底。注意避免气泡的形成。该混合物现在有50微升的体积，并含有终胶原浓度（2.4毫克/毫升）和细胞密度（C 最终 = 2×10 6个细胞/ ml）。 </li><li>慢慢加入20微升所述含细胞溶液Ⅰ至井的中心，以便它形成一圆顶状的液滴（ 图1A）。注意避免泡沫形成在这一步。如果泡沫形成，但仔细赶快试试要么破裂，或用枪头吸出来。轻轻地放在培养皿放回培养箱让内部凝胶聚合45分钟。 </li><li>准备解决O（用于外，无细胞胶原凝胶）的孵育步骤结束前约15分钟。 注意：外凝胶条件可以在胶原蛋白浓度和聚合pH值而言，以改变获得网络的不同的微观结构10。在这个协议中，着眼于一个1.5 - 聚合在pH 7.4 4.0毫克/毫升的胶原凝胶。 <ol><li>基于该胶原原料浓度，计算需要准备200微升胶原溶液0在最终浓度所需的体积。 </li></ol></li><li>加入20微升10×PBS中缓慢缓冲到所需的胶原原液（在步骤3.4计算的）伴以轻轻旋转量。调节混合物的pH至用0.1M的NaOH与使用校准pH计的最终pH值。见注有关调节pH值至2.5步。 </li><li>使溶液至200的最终体积使用微升的H 2 O.执行上冰的所有步骤，以防止过早胶原蛋白的聚合。 </li><li>采取从孵化器的菜45分钟聚合内部凝胶后（见步骤3.3）。轻轻的内凝胶上添加180微升溶液O，以使溶液完全覆盖内凝胶和填充井（ 图1B）。 <ol><li>小心执行此步骤不搅拌的溶液中，这可能会导致在外部凝胶不均匀纤维取向。注意避免接触内部凝胶用枪头，避免气泡或气泡的形成。如果泡沫形成，但仔细赶快试试要么破裂，或用枪头吸出来。轻轻将菜放回孵化器，让外胶聚合。 </li></ol></li><li>取从培养箱培养皿后聚合45分钟。凝胶应已相当固化，在这一点上，虽然它仍可以从底面脱落如果处理大致。 <ol><li>轻轻倾2毫升温热细胞培养基的菜（ 图1C）。确保该凝胶在培养基中完全浸没。刷新介质每2 - 3天整个培养的持续时间。 </li></ol></li></ol> 4。活细胞成像<ol><li>使用倒置共聚焦显微镜配备了长期活细胞成像能力进行成像。包括一个内置的孵育室的温度（37℃）和CO 2（5％）控制。接通显微镜和开始实验之前预热阶段至少1小时。 注意：使用物镜具有长工作距离，以优化细胞的观测和定位在3D凝胶。 </li><li>孵育在含有5微升荧光细胞追踪染料30分钟介质的凝胶，以允许细胞在3D系统的准确定位。随后，通过洗涤用1×PBS中去除未结合的染料三次。之后，加入细胞培养基的培养皿中。 </li><li>取菜从孵化器，并把它放在显微镜阶段（ 图1D）。 注：活细胞成像能够在原则外凝胶的聚合反应后立即启动。然而，在这点上，小区in中的内部凝胶尚未普及。要检查已侵入外硅胶，活细胞成像可以开始24小时培养开始后（取决于细胞类型）的第一个细胞的迁移。作为粗略导，约12 - 需要14天培养的大多数细胞中的内凝胶进入外凝胶。 </li><li>查看（两用粉饼的）的地区选择卷在内部凝胶周围外胶。后培养24小时，该细胞群体将已经扩散，越过内侧和外侧的凝胶之间的界面，并开始侵入外凝胶。 <ol><li>对于两用粉饼的，包括凝胶区域紧邻凝胶界面，中间区，和区域靠近外侧的凝胶7的郊区。排除的区域的距离小于50微米的底面和侧面，以及从该凝胶的顶部，以避免可能的边缘效应。每个两用粉饼一般测量647×647×100微米3（在X，y和z方向上，分 ​​别地），以在z -stack5μm的间隔。 </li></ol></li><li>确保成像模式，信道/过滤器，曝光时间，和图像分辨率被正确地选择。对于胶原蛋白网络的无标记成像，同时采用激光共聚焦显微镜反射时间推移活细胞成像过程。 </li><li>取样品的图像，画出了强度直方图，并调整增益和偏移来观察足够的信号，并避免饱和通过确保直方图在于零与最大强度之间。不整个实验的持续时间的任何更改变这些设置。 </li><li>取10分钟，8小时（或更长，如果必要的话）的选择的两用粉饼的时间推移的图像，以一时间间隔，ΔT，。 </li></ol> 5.细胞跟踪和数据分析<ol><li>使用appropria 的 Z -stack图像进行定量图像后处理<html>德的图像处理软件。 <ol><li>段的时间推移的图像来自动选择在（X，Y，Z，t）的三维细胞的位置。 </li><li>对于每一帧，手动检查的定位精度和除去由于细胞碎片和细胞突起可能已被误认为细胞误报。除去从分析和分割重叠或连接成细胞的不同对象增殖性细胞。 </li></ol></li><li>生成由按时间顺序连接的每个单元的位置，在前面的步骤中获得的细胞的坐标（X，Y，Z，t）的三维时间推移单元轨道。 </li><li>通过删除曲目大于阈轨道长度（通常为20分钟）较短消除随机和系统噪音。 </li><li>如果必要，通过减去总净位移的轨道校正样品漂移。 </li><li>计算单元的位移，尔斯/ ftp_upload / 52735 / 52735eq2.jpg“WIDTH =”80“/&gt;，和细胞迁移的距离， <img alt="公式3" fo:content-width="1in" src="/files/ftp_upload/52735/52735eq3.jpg" width="100" />从所观察到的细胞的轨迹，其中，是表示一个小区的时间的三维位置的矢量和是时间点的总数。 <ol><li>计算单元速度为S = D /（正•Δt），其中Δt是帧之间的时间间隔。计算使用P =量Δd/ D细胞迁移的方向性（或持久性）。持久性的这种简单措施意味着，对于P = 0的净位移是零和用于P = 1的轨迹是直线方向线。 </li></ol></li></ol>

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In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration13, which has to date been lar...

细胞迁移起着各种生理反应，如胚胎发育，止血，和免疫反应，以及在病理过程，如血管疾病，炎症，癌症和1的关键作用。解剖底层细胞迁移的生物化学和生物物理因素，因此从根本上重要的是不仅要了解的细胞功能的基本原则，又能促进各种生物医学应用，例如在组织工程，抗转移和抗炎药物开发。由于体内观察是技术上的挑战，有很多的努力一直专注于体外再演细胞迁移。 体外方法来研究细胞迁移已经基本上被设计为检测在二维（2D）表面上，最显着的划伤或伤口愈合测定法2。这些试验提供了简单的实验装置，方便在线 -细胞成像，并提供有益的见解底层细胞迁移的各种生化机制。但是，这些测定法不占外基质（ECM）架构和重塑，这是在理解体内迁移关键方面。最近，人们已越来越多地认识到，3D培养模型，常常在基于胶原的基质3中，提供了更好的类似于体内情况的一个平台。事实上，细胞表现出的迁徙动力学是从那些在2D表面明显，特别是由于不同的维数的环境4。此外，基体的生物物理和机械性能灵敏影响细胞迁移5，包括在肿瘤细胞侵袭6的上下文中。 在这里，我们提出研究三维细胞迁移行为的ECM与生物物理特性，可以轻松地用不同的制备条件的方法。细胞是接种在“内凝胶”和被允许逃逸到并侵入最初无细胞“外凝胶”。该方法依赖于细胞的能力来识别的存在，并倾向成，无细胞迁移区域中的外凝胶，这是密切相关的细胞mechanosensing 7。在这项研究中，我们采用的胶原蛋白网络，通过高度侵袭性​​乳腺癌细胞，MD-MBA-231入侵的ECM。机械性能和两者的内部和外部的凝胶的微结构可以被调谐8和9，其特征实现生理学相关的条件。重建单元轨道和分析允许在两个群体水平和个体细胞水平的时空迁移行为的详细的定量检查。重要的是，同心凝胶系统的设置模仿面临迁移细胞，尤其是侵入癌细胞，从而提供了重要的见解的体内组织的拓扑对细胞迁移和转移的物理机制。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell culture incubator</td>
			<td>Fisher Scientific Pte Ltd</td>
			<td>Model: 371, S/No 318854-6055</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon A1R</td>
			<td></td>
			<td>Inverted confocal laser scanning microscope equipped with incubator chamber</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent CellTracker dye CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom dish</td>
			<td>IWAKI Cell Biology</td>
			<td>3931-035</td>
			<td>35 mm diameter dish with 12 mm diameter glass-bottom well</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>iN CYTO</td>
			<td>DHC-N01 (Neubauer Improved)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprocessor pH meter</td>
			<td>Hanna Instruments</td>
			<td>pH 211</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutragen Collagen</td>
			<td>Advanced BioMatrix</td>
			<td>#5010-D</td>
			<td>Acid-solubilized bovine collagen type I (stock pH ~ 2)</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sartorius</td>
			<td>S/No 29153352</td>
			<td>Basic pH Meter PB-11</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
	</tbody>
</table>

concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration

细胞迁移的能力是在各种各样的细胞功能，在整个从胚胎发育寿命和伤口愈合肿瘤和肿瘤转移的关键。尽管强烈的研究努力，细胞迁移的基本生物化学和生物物理原理仍然没有完全了解，特别是在生理相关的三维（3D）的微环境。在这里，我们描述了在体外试验设计允许的三维细胞迁移行为定量检查。该方法利用了细胞的mechanosensing能力和倾向迁移到先前未被占据的细胞外基质（ECM）。我们使用高度侵袭性​​乳腺癌细胞的侵袭，MDA-MB-231，在胶原凝胶作为模型系统。细胞群的传播和单个细胞的迁移动力学数周的文化可以用活细胞成像进行监测和分析，以提取spatiotemporally分辨数据。此外中，该方法很容易适应于不同的细胞外基质，从而提供了一种简单而功能强大的方法来研究的生物物理因素对细胞迁移的微环境中的作用。

使用高度侵袭性乳腺癌细胞进行这里提出的同心凝胶测定中，MDA-MB-231，用2.4毫克/毫升的内胶原凝胶和= 2×10 6个细胞/ ml的细胞接种密度，作为一个例子。 如图2中 ，典型地经过培养几天，细胞违反内外凝胶接口，并开始侵入外凝胶。该细胞群主要传播径向向外。 外凝胶的聚合条件可被修改以研究对细胞迁移特性的矩阵的密度和机械性能的作用。 图3...

Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

机械性能和细胞外基质的组织强烈地影响细胞的三维迁移。 的体外方法来研究在生物物理变量的环境中，在人口和单个细胞水平的时空细胞迁移行为，进行说明。

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

concentric-gel-system-to-study-biophysical-role-matrix

Research

JoVE Journal

Bioengineering

8.5K 次观看.  FOM Institute AMOLF. 机械性能和细胞外基质的组织强烈地影响细胞的三维迁移。 的体外方法来研究在生物物理变量的环境中，在人口和单个细胞水平的时空细胞迁移行为，进行说明。 

视频: Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

标记细胞外囊泡以监测软骨外植体的迁移和摄取

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

微凝胶-细胞外基质复合材料支持人类神经结构的嵌入式3D打印

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...