我们非常感谢玛丽亚Kilfoil提供（开源共享MATLAB的粒子跟踪代码<a href="http://people.umass.edu/kilfoil/">http://people.umass.edu/kilfoil/</a> ），以及由约翰·C·克罗克和Eric提供的较早的IDL代码和大量的文档R.周。这项工作之所以成为可能，由国家癌症研究所（NCI / NIH），K99CA155045和R00CA155045（PI：JPC）资助。

1，培养肿瘤细胞球体<ol><li>将10mL细胞培养级水中0.1克琼脂糖，得到在1％琼脂糖溶液。 </li><li>热的琼脂糖溶液至70℃以上等分40微升的琼脂糖溶液放入井在96孔板中之前（大致14秒在一个标准的微波或通过使用加热板）。 </li><li>培养板在37℃下放置至少1小时，同时用标准技术收获细胞。 </li><li>使用血细胞计数器确定在悬浮液中细胞的浓度。 </li><li>稀释细胞悬液，以1,000个细胞/毫升，加入100微升这种稀释到井含固化琼脂糖床。 </li><li>将样品放置在摇床上过夜的培养箱设定为37℃和5％CO 2。 </li><li>从振动筛取出样品，加入100μl的细胞培养基。 </li><li>孵化球体，直到所需的直径就达到了。例如，9天后，一个SPheroid将是直径约450微米。在此期间，新鲜的生长培养基中可以加入到孔中，但抽吸应该避免，因为这将导致在除去球体。 </li></ol> 2，准备3D肿瘤细胞球体嵌入ECM <ol><li>准备与层流罩内所需材料的工作站。 </li><li>通过添加2份股票探针（2％固体），以25份用无菌水制备羧化物修饰为1μm直径荧光示踪探针的稀释的混合物。 </li><li>从冰箱中取出一瓶3.1毫克/毫升的牛胶原蛋白，并将其放置在冰上。 </li><li>分装125微升胶原蛋白的成空机2 ml小瓶的。 </li><li>加入50微升的稀释示踪探测解决方案，含有胶原蛋白和涡简要分配探头的小瓶。 </li><li>添加235微升含有酚红（这会变黄）为41的总体积适当的细胞培养介质的0毫升和涡去除205毫升（一半的总体积），并放置在新的2ml小瓶前短暂。小瓶1将包含肿瘤球体和2瓶将是一个混合的控制。 </li><li>添加〜2微升的1M NaOH至样品瓶1中以使该溶液回到中性pH（含有酚红的培养基应返回到红色）。请注意，这将导致混合物开始固化，如果它不保持在冰上。短暂涡旋混合，然后立即返回到冰架上。 注意：该球体是隐约可见肉眼之后的文化和从琼脂糖床的去除9天是一个棘手的任务。使用解剖显微镜可能对某些用户有帮助的。在传输过程中保持球体的完整性是至关重要的。 </li><li>使用宽口移液管尖轻轻从井中含有肿瘤球体去除40微升的介质。留住这40微升，同时进行下一个步骤。 </li><li>检查井，看是否球体除去在前面的步骤。如果是，添加包含球体到小瓶1的40微升，如果不是，将40微升回入井，并重复前面的步骤。 </li><li>在60微升的部分将所述混合物分成三个独立的96孔板的孔中之前，轻轻搅拌样品瓶1（不冒险的涡流，它可能会损坏球体）。加入混合物，以确定哪些以及包含该球体后检查各孔用显微镜。 </li><li>添加〜2微升的1M NaOH和40μl细胞培养基至瓶2和aliquotting 60微升该混合物在96孔板和标签作为对照的空井之前涡流。 </li><li>将板置于37℃培养箱中培养，以固化至少1小时。 </li></ol> 3，构建样本点网格，并采取视频在每个点<ol><li>从培养箱转移样品板的显微镜载物台上。允许10分钟的样品等角球ibrate与室温如果加热阶段不可用。 </li><li>观察样品与低功率的物镜，以确保它是完整的，并准备进行成像。确定在所述井中的肿瘤的位置。 </li><li>决定多少个采样点取。典型地，在每个孔20的采样点，分布在周围的旋转椭球体的同心环将产生足够的详细结果。 </li><li>移动台到各个所需的位置，并使用显微镜接口软件记录的x和y坐标（或记录手动坐标如果接口软件不可用）。 </li><li>显微镜切换到高功率的物镜（通常100X），并选择相应的过滤器立方体的示踪剂探头的激发波长。使用步骤3.4中创建点的列表移动到网格中的第一点。 </li><li>调整焦距，以发现井的底部，然后向上移动，找到视角（FOV）包含几个焦点对准TRAC场呃探头。 </li><li>观察的强度直方图和调整曝光强度和时间，得到的最大动态范围的可能，同时确保图像不会变得饱和。 </li><li>获得视频序列的每帧20至30毫秒的帧速率（约800帧或16-24秒的长度，建议提供足够的统计数据强劲MSD计算的需要尽量减少在每个空间网格点的采集时间平衡）和保存用适当的约定。在录音时，请勿触摸显微镜或桌子上。 </li><li>对网格中的每个采样点重复步骤3.6至3.9。 </li><li>每口井在实验中重复步骤3.3至3.10。 </li></ol> 4，分析视频数据来计算流变学特性在每个采样点<ol><li>所有的视频​​数据复制到一个文件夹中的分析（可能在不同的计算机上）。 </li><li>导入图像数据到MATLAB或其他分析软件。 NOTE：关于一套这里通过MATLAB的粒子跟踪程序的大量文档可在<a href="http://people.umass.edu/kilfoil/">http://people.umass.edu/kilfoil</a> 。建议使用一个自定义的调用函数用于自动在不同位置的多个文件的处理。 </li><li>通过分析视频的多个帧来适当地确定选择和拒绝参数（尺寸，带通等），用于识别探针中心位置校准软件。广泛的背景，这些关键步骤是由克罗克和格里尔描述。 </li><li>使用该软件可自动判断所有视频帧的每个探头的位置，然后将这些位置连接成轨迹。 </li><li>使用轨迹数据来计算均方位移（MSD）作为延迟时间的函数，即确保将适当的空间校准因数（每像素微米）为特定的物镜，任何像素组合等（典型地中的元数据）。 </li><li>计算G *（使用GSER的适用性为特定样品24,28,43的限制。另外，用户可能希望从MSD通过简单地比较幂缩放，高原直接解释ECM性能考虑广义Stokes-Einstein关系异质性或其他参数的值，指数。 </li><li>每个FOV在实验中重复步骤4.2至4.6。 </li><li>从步骤3.4粘弹性modulii在感兴趣的特定频率共同注册位置数据。根据所使用的显微镜​​的制造商，这一步可以通过它读取显微镜的元数据或位置数据可以手动制表一个自定义例程来促进。 </li><li>使用3D插值函数来生成ECM的空间流变图。 </li></ol>

The authors declare that they have no competing financial interests.

在这个协议中，我们介绍一个强大和广泛适用的策略，纵向跟踪三维肿瘤模型在流脑刚度局部变化。我们设想，这种方法可能是癌症生物学家和生物物理学家感兴趣的肿瘤的生长和侵袭过程中牵连的基质重塑机械敏感性行为被采纳。的基质降解动力学精确量化可能是特别有价值的研究基质金属蛋白酶，赖氨酰氧化酶或其他有关生化物质在三维肿瘤模型是直接链接到基质重塑的活性。除了 ​​应用...

很明显从文献中的证据，癌细胞，与非恶性哺乳动物上皮细胞，是向周围的细胞外基质（ECM）和其他微环境组分1-9的机械和生物物理特性高度敏感越来越多。优雅的机理研究提供了见解外刚度作为调控的恶性生长行为和形态2,3,10,11一个复杂的机械敏感性的信令合作伙伴的角色。这项工作提供了便利尤其是3D的在恢复生物学相关的组织架构和可以种植在支架材料具有可调力学和光学显微镜成像12-19 体外肿瘤模型的发展。然而，肿瘤和微环境，通过这些癌细胞反过来修改其周围的流变性之间的这种mechanoregulatory对话的另一侧，仍有些更难以研究。例如，在侵入过程中，细胞在肿瘤的周围可经历上皮至间质转变（EMT）和增加基质金属蛋白酶（MMPs）的表达，导致ECM 20-22，这反过来又影响的机械敏感性生长行为的局部降解其他近端肿瘤细胞。通过各种生化过程，癌细胞不断地拨打他们的环境的局部刚度上下，以适应不同的过程在不同的时间。这里描述的方法是由需要该报告在生长过程中的刚性和遵守ECM的局部变化，可与三维肿瘤模型被集成并与生物化学和表型变化的纵向相关性而不终止培养分析工具的动机。 为了寻找一个合适的技术，在此背景下实施的，粒子跟踪microrheology（PTM）的出现作为一个强有力的候选人。这种方法，由梅森和韦茨23,24最初开创，使用嵌入在一个复杂的流体在微米尺度报告的频率依赖的复合粘弹性剪切模量G *（ω）示踪探头的运动。这是一般的做法已经开发出适合在软凝聚态物质，胶体，生物物理学和高分子物理25-31不同应用程序的多个变体。 PTM具有相对于其它方法的某些优点，因为本地粘弹性的读数被并入在培养制备物的时间和保持在原位生长过度长时间的生化无效的示踪探针的非破坏性的视频成像提供。这是相对于黄金标准测量与振荡剪切流变仪本体，这必然需要终止培养，并报告样品的体积宏观流变性而不是复杂的三维肿瘤microenvir内点测量onment。的确许多研究已经说明解释的示踪探测运动中或周围癌或非癌细胞以测量与细胞迁移32，机械应力引起的膨胀球体33，细胞内的流变34,35相关联的变形的测量的效用，并映射的机械应力和应变在工程化组织36，并且孔径和入侵速度37之间的关系。适于microrheology其他技术，例如原子力显微镜（AFM）可以被实现，但是主要用于探测点在样品表面并且还可以构成该复杂纵向测量38培养无菌的问题。 在这里，我们描述了一个全面的协议涵盖为适合传送到ECM与嵌入式荧光探针用于视频粒子跟踪和分析方法可靠地映射空间三维球体瘤生长的方法在文化随时间的变化microrheology。在本实施方式中，三维肿瘤模型是生长在多孔形式与对掺入microrheology测量与其它传统的测定法（ 例如 ，细胞毒性），其中该格式是有利的视图。在这种方法我们培养体外三维使用PANC-1细胞的球状体这代表插图，已知以形成球状体39，但本文所述的所有测量值建立的胰腺癌细胞系是广泛适用的实体瘤的利用各种细胞系，研究适合3D的文化。因为这种方法本质上是基于成像的它非常适合与大视透射光场的报告改变细胞生长，迁移和表型高分辨率microrheology数据配准。 PTM的实施与透射光显微镜集成以这种方式假定在显微镜阶段WH的重现性定位ICH通常可在机动车商业广角落射荧光生物显微镜。下面开发的协议可以与任何合理配备自动荧光生物显微镜来实现。这是一种固有的数据密集型方法，这就需要收购数字视频显微镜数据进行离线处理千兆字节。 在下面的协议，协议1涉及在此所说明利用叠加在琼脂糖，但是可以用各种其他方法，如悬滴40，或旋转式培养41技术被取代肿瘤球状体的初始准备。方案2描述了在胶原蛋白支架嵌入球体虽然可替代地， 在体外三维肿瘤可以由封装或重新悬浮的细胞包埋在细胞外基质12,15，而不是单一的预成形的非粘附球状体生长的过程。随后的协议描述为O过程通过收购和处理视频显微镜数据，分别btaining时间分辨microrheology测量。数据处理是用MATLAB，利用对PTM建立在最初由克罗克和格里尔42，这也为不同的软件平台被广泛开发的算法描述开放源代码例程（见http://www.physics.emory.edu/描述〜周/ IDL /）。

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:177px;">Bovine type 1 collagen</td>
			<td style="width:235px;">BD Biosciences, San Jose, California</td>
			<td style="width:100px;">354231</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PANC-1</td>
			<td>American Type Cell Culture, Manassas, VA</td>
			<td>CRL1469</td>
			<td>or other appropriate cell type</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent Microspheres</td>
			<td>Life Technologies, Carlsbad, California</td>
			<td>906906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences, Bedford MA</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Fisher Bioreagents, Waltham, MA</td>
			<td>C12H18O9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fisher Bioreagents, Waltham, MA</td>
			<td>NC0480985</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well Imaging plates</td>
			<td>Corning Inc., Corning, NY</td>
			<td>3904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Hyclone, Waltham, MA</td>
			<td>SH30243.01</td>
			<td>or appropriate&#160; cell culture media</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Zeiss AxioObsever Microscope</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td>includes high-speed camera and imaging software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB software</td>
			<td>The Mathworks, Natick, MA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal measurement of extracellular matrix rigidity in 3d tumor models using particle-tracking microrheology

机械微环境已被证明作为肿瘤生长行为和信号传导的关键调节剂，它是本身改造和修饰的一组复杂的，双向的机械敏感性交互的一部分。虽然生物相关的3D肿瘤模型的发展，促进了对基材的流变性对肿瘤生长的影响机理研究，在诱发肿瘤的力学环境映射变化的逆问题仍然充满挑战。在这里，我们描述粒子跟踪microrheology（PTM）在与胰腺癌三维模型相结合实施工作，作为一个强大的和可行的方法为纵向监测在肿瘤微环境的物理变化， 原位部分。这里描述的方法结合制备包埋在模型细胞外基质I型胶原的（ECM）的支架体外三维模型的系统与荧光标记探针为均匀分布婆sition和时间依赖性microrheology测量整个样品的体外肿瘤被镀和使用多孔成像板探测平行的条件。借鉴成熟的方法，示踪探测移动视频是通过广义斯托克斯爱因斯坦关系（GSER）上报复杂的与频率相关的粘弹性剪切模量G *（ω）的转化。因为这种方法是基于成像，机械特性也映射到大型透射光的空间领域同时上报3D肿瘤大小和表型的质变。代表性的结果对比显示与局部浸润引起基质降解以及系统校准相关的子区域的机械响应，验证数据列。从常见的实验误差和对这些问题的故障排除不良后果也被提出。在这个协议中实现的96孔培养三维镀格式为conducive到microrheology测量与治疗筛选试验或分子成像，以获得新的见解的治疗方法或生化刺激对机械微环境影响的相关性。

为了验证G *（ω）在内的复杂模型肿瘤微环境局部位置测量的有效性，两个初始的验证实验。首先，我们试图验证我们的测量对大宗振荡剪切流变的“金标准”。我们以1.0毫克/毫升的胶原蛋白的浓度制备的胶原蛋白基质的相同的样品（无细胞）。这些样品用探针本体流变仪（TA Instruments的AR-G2，使用40毫米平行板几何形状），并通过PTM（使用相同的参数，如在本协议），将得到的G测量&#39;（?...

Watch this Scientific Journal Video about Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology at JoVE.com

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

粒子跟踪microrheology可用于非破坏性的量化和空间映射的变化在三维肿瘤模型中的细胞外基质的机械性能。

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

longitudinal-measurement-extracellular-matrix-rigidity-3d-tumor

Research

JoVE Journal

Bioengineering

11.4K 次观看.  University of Massachusetts Boston. 粒子跟踪microrheology可用于非破坏性的量化和空间映射的变化在三维肿瘤模型中的细胞外基质的机械性能。 

视频: Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

使用滑翔分析生物大分子的抗弯刚度测量

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

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 ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

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 ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...