Name: mammalian cell division in 3d matrices via quantitative confocal reflection microscopy
Uploaded: 2017-11-29T14:00:00
Description: Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

这项工作得到了 NIH 赠款 R01CA174388 和 U54CA143868 的支持。作者要感谢约翰·霍普金斯大学的安玛塔拉普拉奖, 支持魏通陈这一材料是根据国家科学基金会研究生研究金资助 No. 1232825 的工作。

所提供的议定书遵循了 HIRB 机构审查委员会的准则。
1. H2B-mCherry 作为细胞有丝分裂标记物的稳定表达
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人胚肾 293T (HEK 293T) 细胞中慢粒子的生成
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<li>在 10 cm 细胞培养皿上 HEK 293T 细胞, 在细胞培养基中的密度为 5 x 106细胞/皿中 (Dulbecco 氏改良的鹰培养基 (DMEM), 含高糖 (4.5 克/升), 丙酮酸钠, 10% 胎牛血清 (FBS) 和1%青霉素-链霉素 (笔/链球菌))。孵育24小...

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由于实验的局限性和技术上的挑战18,19, 先前对3D 单元划分的研究没有效率。在3D 胶原基质中有效研究哺乳动物细胞分裂的关键步骤是: (1) 将荧光标记的有丝分裂标记纳入细胞;(2) 细胞分裂的同步;(3) 利用活细胞成像技术、时间分辨共焦反射显微镜和定量成像分析对3D 矩阵中的分裂事件进行监测。
在2D 基底上的有丝分裂细胞可以根据?...

细胞有丝分裂是细胞生命的重要事件, 其调控在组织和器官发育中起着至关重要的作用。异常有丝分裂涉及自然遗传变异、人类衰老过程和癌症的进展1,2,3,4,5。与正常细胞相比, 肿瘤细胞增殖率增加是癌症的标志之一, 尽管在不同类型的肿瘤中, 甚至在患者中, 细胞行为是相当不均匀的。尽管有希望的临床研究结果, 一些新开发的有丝分裂药物没有证明是有效的在临床医学试验6,7,8,9,10 ,11。必须考虑实验和临床前模型的相关性。许多类型的正常哺乳动物和癌细胞在三维 (3D) 基质中分裂, 如成纤维细胞和纤维细胞在富含胶原蛋白的3D 结缔组织中, 以及3D 基质细胞外基质 (ECM) 中的转移癌细胞。然而, 绝大多数的哺乳动物细胞分裂实验和化验都是在 two-dimensional (2D) 基质上培养的细胞上进行的。一个工程化的3D 矩阵可以更好地概括的组织, 机械性能和生化信号的 3D ECM 的正常和病理组织12,13,14, 15,16,17。
尽管生理相关性和治疗意义18,19, 但在3D 环境中如何调节哺乳动物细胞分裂的研究仍大体未被探索。可能的原因包括在3D 矩阵中研究细胞分裂的技术难点和实验挑战。细胞有丝分裂在整个细胞周期中构成一个小的时间分数20。以前的研究表明, 许多哺乳动物细胞的增殖率, 如人类乳腺腺癌 MCF-7, 人骨肉瘤 U2OS, 和人的肝脏 HepG2, 是低得多的3D 矩阵相比, 他们的对应在2D 基板21, 22。此外, 嵌入在3D 矩阵中的细胞在活细胞成像过程中进出焦点。所有这些因素都有助于在3D 文化中利用成像技术捕获细胞分裂事件的效率极低。
ECM 和细胞之间的相互作用在调节细胞分裂方面起着至关重要的作用。在这里, 我们描述了一个有效的方法来研究哺乳动物细胞分裂3D 胶原基质。该方法包括将有丝分裂标记纳入细胞, 细胞分裂的同步, 以及使用活细胞成像技术、时间分辨共聚焦反射显微镜对3D 矩阵中的分裂事件进行监测, 以及定量成像分析。荧光标记的组蛋白 H2B 首先引入细胞中, 作为分化有丝分裂和相间细胞的标志物。然后, 细胞是同步使用胸苷阻断和 nocodazole 治疗的组合, 其次是机械 shake-off 技术。然后将同步细胞直接封装到3D 胶原基质中。多细胞的细胞分裂事件的监测有效使用 low-magnification 延时实时细胞成像。胶原纤维的形变是细胞-基质相互作用的指示物, 通过高倍的共聚焦反射显微镜进行监测。
我们以前使用这种技术来监测和量化细胞-基质相互作用之前, 期间和之后, 两个转移癌细胞株, 人浸润性导管癌 MDA-MB-231 和人纤维 HT1080 细胞, 在3D 胶原矩阵19。这里提出的方法提供了一个有效的和一般的方法来研究哺乳动物细胞分裂在3D 环境和细胞基质相互作用。在整个论文中, MDA-MB-231 单元格线被用作示例。这项协议提供了新的洞察到分子基础的发展的正常组织和疾病, 也可以允许设计新的诊断和治疗方法。

<table>
	<tbody>
		<tr>
			<td>Human embryonic kidney 293T</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>Physical Sciences Oncology Center, NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>ThermoFisher Scientific</td>
			<td>12100-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life technologies</td>
			<td>11668-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding H2B-mCherry in a lentiviral vector</td>
			<td>Addgene</td>
			<td>plasmid 21217</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>GibcoBRL</td>
			<td>11810-025</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>113375-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Bake</td>
			<td>3722-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV syringe filter unit, 0.45-&#956;m, PVDF, 33 mm</td>
			<td>Millipore</td>
			<td>SLHVM33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon TE2000E epifluorescence microscope</td>
			<td>Nikon</td>
			<td>TE2000E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade 1K CCD camera</td>
			<td>Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR imaging software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1 confocal microscope</td>
			<td>Nikon</td>
			<td>A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammalian cell division in 3d matrices via quantitative confocal reflection microscopy

关于哺乳动物细胞分裂如何在3D 环境中被调控的研究, 尽管其生理相关性和治疗意义仍未得到很大的探索。缺乏探索的可能原因是实验性的限制和技术上的挑战, 使3D 文化中细胞分裂的研究效率低下。在这里, 我们描述了一个 imaging-based 的方法, 以有效地研究哺乳动物细胞分裂和细胞基质相互作用的3D 胶原基质。用荧光 H2B 标记的细胞同步使用胸苷阻断和 nocodazole 治疗的结合, 其次是机械 shake-off 技术。然后将同步细胞嵌入3D 胶原基质中。细胞分裂被监测使用活细胞显微镜。细胞分裂期间和之后的胶原纤维的变形是细胞-基质相互作用的指示物, 可以用定量共焦反射显微镜进行监测和量化。该方法提供了一个有效的和一般的方法来研究哺乳动物细胞分裂和细胞基质相互作用在一个生理相关的3D 环境。这种方法不仅为正常组织和疾病的分子基础的发展提供了新的洞察力, 而且还允许设计新的诊断和治疗方法。

本文的目的是提出一个 imaging-based 的方法来研究哺乳动物细胞分裂过程的3D 矩阵, 并量化的互动之间的细胞和3D 细胞外基质细胞分裂。为了促进细胞有丝分裂的成像, 我们利用慢转导将 H2B-mCherry 纳入 MDA-MB-231 细胞。H2B 结合荧光蛋白被用作有丝分裂标记, 以区分有丝分裂细胞与间期细胞, 并定义在细胞有丝分裂期间的不同阶段19,20<s...

Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

该协议有效地研究了3D 胶原基质中的哺乳动物细胞分裂, 结合细胞分裂的同步, 用活细胞成像技术监测3D 矩阵中的分裂事件, 时间分辨共聚焦反射显微镜和定量成像分析。

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

The overall goal of this procedure is to study mammalian cell division and cell matrix interactions in a physiologically relevant 3D environment. The main advantage of this technique is that it provides an efficient and general approach to study mammalian cell division and cell matrix interactions. This method can help answer key questions in cell biology such as the molecular basis of the development of normal and disease tissue.A day prior to transfection, plate five million HEK-293 T-cells in a 100 milliliter cell culture dish in 10 milliliters of normal cell culture medium. Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day just before starting transfections, remove a vial of the transfection reagent from four degrees Celsius and leave it out to reach room temperature.Then to start, add one milliliter of reduced serum medium to a sterile microfuge tube. To this, add specified amounts of lentiviral vector plasmids used for transfection and mix by pipetting. Incubate the tube at room temperature for five minutes.After five minutes, add 48 microliters of the transfection reagent into this tube and mix gently by pipetting. Subsequently, incubate this mixture at room temperature for at least 15 minutes. Once the incubation is complete, remove the plate with HEK-293 T-cells from the incubator.Add the prepared DNA transfection reagent mixture drop wise onto the cells and gently swirl the plate to ensure even distribution in the plate. Place the plate back into the incubator. Approximately six hours after transfection, remove the plate and aspirate the medium.Add 10 milliliters of fresh culture medium and transfer the plate back into the incubator. At multiple time points, 24, 48, and 72 hours post-transfection, harvest the culture medium containing the lentiviral particles in separate tubes. Next, filter the harvested culture medium through a sterile 0.45 micron filter to remove cellular debris.Replace with fresh culture medium following each harvest. The filtrate containing the lentiviral particles can be used for transduction right away or can be stored at minus 80 degrees Celsius. Plate human breast carcinoma cells MDA-MB-231 in a 35 millimeter culture dish in normal culture medium.Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day, aspirate the culture medium from the plate and add one milliliter of the lentiviral particles together with one milliliter of fresh culture medium. Incubate the cells in the incubator for about 16 hours.After the incubation with viral particles is complete, remove the plate to aspirate the medium. Then add two milliliters of fresh medium and transfer back to the incubator for 24 to 72 hours. Check the expression of H2B-mCherry in these transduced cells using an epifluorescence microscope.To start the synchronization experiments, first plate MDA-MB-231 cells expressing H2B-mCherry at 50 to 60%confluency in a 24-well plate. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, replace the culture medium with 0.5 milliliters of medium containing two millimolar Thymidine.Leave the plate in the incubator for 24 hours. Once the incubation is complete, wash the cells three times with PBS to release them from the Thymidine treatment. Then leave the cells in 0.5 milliliters of normal culture medium for five hours in the incubator.After this, to block these cells in mitosis, add Nocodazole to the culture medium. Leave the cells in the incubator for 12 hours. After the Nocodazole treatment is complete, take out the plate and using an orbital shaker, shake the cells for 45 seconds to one minute to release mitotic cells.Subsequently, transfer the medium containing the extracted mitotic cells into a new centrifuge tube. Then add 0.5 milliliters of fresh medium to each well. After, repeat the shaking and extraction of cells two more times.Pool the total collected medium and then centrifuge it to pellet the mitotic cells. After the spin, resuspend the cell pellet in 0.25 milliliters of fresh cell culture medium. Using a hemocytometer, count the number of cells.At the start of this step, prepare 50 milliliters of 10x DMEM solution and filter sterilize it. Next, determine the volumes of all the components that will be used to make the 3D collagen matrix as specified in the text protocol. Pre-chill all these components on ice to slow down the gelation of collagen.Now, to a pre-chilled 1.5 milliliter centrifuge tube, add the required numbers of cells in medium then 10x DMEM, FBS, deionized water, collagen, and finally sodium hydroxide. Mix carefully using a one milliliter pipette. Once the solution is well mixed, add 500 microliters of the mixture into each well of the 24-well plate.Place the 24-well plate in a 37 degree Celsius incubator. After 30 minutes, take out the plate and add 500 microliters of pre-warmed culture medium on top of the gel. Prior to imaging, turn on the live cell units of the phase contrast and confocal microscopes.To capture dividing cells, place the 24-well plate containing the cells embedded in collagen matrices into the live cell unit of the fluorescence microscope. Using the 10X objective, find the bottom of the plate and then move it up until the microscope focuses at about 500 micrometers from the bottom of the plate. Move the stage around to find cells in different stages of mitosis and select multiple positions for imaging.Set up the timelapse experiment with two-minute intervals. Next, to image the collagen network, configure the confocal microscope to capture only reflected light from the 488 nanometer laser. Then place the plate containing cells in collagen matrices into the live cell unit of this microscope.Find the bottom of the plate and then move the objective lens up until it focuses at about 100 micrometers from the bottom of the plate. Move the stage around to find cells and select multiple positions for imaging. Set up the timelapse experiment with five-minute intervals.Fluorescent images of a dividing H2B-mCherry expressing MDA-MB-231 cell embedded in collagen matrix are shown here. Different phases of mitosis are readily observed as expected. The distribution of collagen fibers in white visualized simultaneously using confocal reflection microscopy changes very little during the course of mitosis.Cell synchronized G2-M phase and embedded in a collagen matrix complete cell division in the first two hours while control asynchronous cells divide randomly. Quantification of the distribution of collagen fibers during interphase and mitotic phases suggest that matrix deformation is minimal during cell division and higher in interphase cells. Once mastered, this technique can be completed in seven days starting from generating the stable cell line to analyzing the video cell division in a 3D matrix.After watching this video, you hopefully have a good understanding of how to synchronize and monitor mammalian cell division in 3D matrices using live cell imaging and how to determine matrix deformation using confocal reflection microscopy and quantitative imaging analysis techniques.

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Unit 1

Cell-Matrix Interactions

SCIENTISTS IN ACTION

Video-rate Scanning Confocal Microscopy and Microendoscopy

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Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

科研

生物工程

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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, ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

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We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

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Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Novel Process for 3D Printing Decellularized Matrices

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...