这项工作得到了美国国立卫生研究院拨款R01DK120986（KPM）的支持。

肠道炎症对已建立的小鼠结肠样的上皮影响

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贡献作者没有什么可透露的。

类器官的发展彻底改变了科学界在 体外 研究器官系统的方式，能够在培养皿中部分概括动物或人类的细胞结构和功能。此外，源自患有疾病的人类的类器官系统为个性化医疗提供了一种有前途的工具，可以指导治疗决策。在这里，我们描述了一种运行良好的加密隔离协议，并介绍了允许在电镀前清理隔离中多余碎屑的关键步骤。此外，我们还解释了一个在这个过程中经常被忽视的关键细?...

炎症性肠病 （IBD） 是一种慢性、缓解和复发性疾病，其特征是炎症发作和临床静止。IBD 的病因是多因素的，但该疾病的关键特征包括屏障功能缺陷和肠上皮通透性增加，此外还有上皮区间内激活的促炎信号级联反应 1,2。几种体外和体内模型已被用于概括IBD期间的上皮反应，包括炎症的细胞培养和小鼠模型3。然而，所有这些系统都有重要的缺点，限制了它们在IBD4期间概括上皮变化的能力。大多数用于研究IBD的细胞系都是转化的，具有形成单层的能力，并且可以分化3种，但本质上的繁殖方式与宿主中未转化的肠上皮细胞不同。几种不同的小鼠炎症模型用于研究IBD，其中一些包括敲除模型，感染模型，化学炎症模型和T细胞转移模型5,6,7,8。虽然每个人都可以研究IBD的某些病因方面，例如遗传易感性，屏障功能障碍，免疫失调和微生物组，但他们研究疾病的多因素性质的能力有限。
肠道类器官，包括类肠类和结肠类，在过去十年中已被建立为一种有用的体外模型，不仅可以研究肠道干细胞的动力学，还可以研究它们在肠道稳态和疾病中的屏障完整性和功能的作用。这些实体为我们理解IBD9的发病机制做出了重大贡献，并为个性化医疗开辟了新的机会。结肠样或干细胞衍生的自组织组织培养物已经从小鼠和人体组织发展而来，其过程允许位于肠隐窝内的干细胞繁殖并无限期维持10。体内干细胞生态位依赖于细胞外因子来支持其生长，特别是典型的Wnt信号传导和骨形态发生蛋白信号通路11。这些因素的添加促进了结肠样细胞的健康和寿命，但也推动培养物走向干细胞样状态，这种状态不反映体内上皮细胞结构，该结构由自我更新和终末分化的细胞组成12,13。虽然肠上皮的功能取决于干细胞区室和分化细胞之间的持续串扰，但在结肠样培养系统中同时具有两者的能力相当有限。尽管存在这些限制，类器官培养系统仍然是研究体外上皮内在特性的金标准。尽管如此，可能需要考虑替代文化策略来回答手头的科学问题。
已经表明，连续7天葡聚糖硫酸钠（DSS）方案的小鼠发生上皮炎症和屏障功能障碍14。此外，线粒体生物发生失败和肠上皮内的代谢重编程，已被证明在人类IBD中很明显，也已捕获在结肠炎15的DSS模型中。然而，我们的初步数据表明，线粒体生物发生失败的特征并未保留在源自DSS处理动物隐窝的结肠中（补充图1）。因此，在检查小鼠肠道炎症期间炎症如何驱动上皮变化时，必须使用替代培养方法。在这里，我们概述了我们开发的一个协议，该协议描述了1）如何从整个结肠组织中分离隐窝以建立鼠结肠样，2）如何最终分化该细胞群以反映 体内的细胞群，以及3）如何在此 体外 模型中诱导炎症。为了研究发炎上皮内的药物相互作用，我们开发了一种方案，从鼠结肠样体中建立2-二脉（2D）单层，可以用炎症介质进行基础治疗，并用药物治疗进行顶端治疗。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4-&#956;M transparent transwell, 24-well</td>
			<td>Greiner Bio-one</td>
			<td>662-641</td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#956;M cell strainer</td>
			<td>VWR</td>
			<td>76327-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-010</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>B-27 supplement&#160;</td>
			<td>Invitrogen</td>
			<td>12587-010</td>
			<td>Stock Concentration (50x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Chopsticks Electrode Set for EVO</td>
			<td>World Precision Instruments</td>
			<td>STX2</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel GFR Membrane Mix</td>
			<td>Corning</td>
			<td>354-230</td>
			<td>Stock Concentration (100%); Final Concentration (100%)</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-5G</td>
			<td>Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Thermo Fisher</td>
			<td>11960-069</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline without Calcium and Magnesium</td>
			<td>Gibco&#160;</td>
			<td>14190-144</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (ETDA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>Stock Concentration (0.5 M); Final Concentration (30 mM)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Bio-Techne</td>
			<td>S11150H</td>
			<td>Stock Concentration (100%); Final Concentration (1%)</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>InvivoGen</td>
			<td>ant-ga-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>Gentamicin Reagent</td>
			<td>Gibco/Fisher</td>
			<td>15750-060</td>
			<td>Stock Concentration (50 mg/mL); Final Concentration (250 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>GlutaMAX-1</td>
			<td>Fisher Scientific</td>
			<td>35050-061</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Stock Concentration (1 M); Final Concentration (10 mM)</td>
		</tr>
		<tr>
			<td>hIFN&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF</td>
			<td>Stock Concentration (1000 ng/&#181;L); Final Concentration (10 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hIL-1&#946;</td>
			<td>R&#38;D Systems</td>
			<td>201-LB</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (20 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hTNF&#945;</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (40 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide&#160;</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media)</td>
		</tr>
		<tr>
			<td>Hygromycin B Gold</td>
			<td>InvivoGen</td>
			<td>ant-hg-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>L-WRN Cell Line</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>mEGF</td>
			<td>Novus</td>
			<td>NBP2-35176</td>
			<td>Stock Concentration (0.5 &#181;g/&#181;L); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma&#160;</td>
			<td>A9165-5G</td>
			<td>Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>Stock Concentration (0.1 ng/&#181;L); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Microplates, 24 well tissue culture treated, sterile</td>
			<td>Greiner Bio-one</td>
			<td>5666-2160</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>Stock Concentration (0.25 &#181;g/&#181;L); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>Tryp LE Express</td>
			<td>Thermo Fisher</td>
			<td>12604-013</td>
			<td>Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA)</td>
		</tr>
		<tr>
			<td>UltraPure Water&#160;</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Y-27632 dihyddrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120129</td>
			<td>Stock Concentration (10 mM); Final Concentration (10 &#181;M); Solvent (UltraPure Water)</td>
		</tr>
	</tbody>
</table>

studying the epithelial effects of intestinal inflammation in vitro on established murine colonoids

肠上皮在人体健康中起着至关重要的作用，在宿主和外部环境之间提供了屏障。这种高度动态的细胞层提供了微生物和免疫群体之间的第一道防线，并有助于调节肠道免疫反应。上皮屏障的破坏是炎症性肠病（IBD）的标志，对于治疗靶向具有重要意义。三维结肠培养系统是研究IBD发病机制中肠道干细胞动力学和上皮细胞生理学的非常有用的 体外 模型。理想情况下，从动物发炎的上皮组织中建立结肠样体对于评估遗传和分子对疾病的影响最有益。然而，我们已经表明， 体内 上皮变化不一定保留在由急性炎症小鼠建立的结肠样中。为了解决这一限制，我们开发了一种方案，用通常在IBD期间升高的炎症介质混合物来治疗结肠样。虽然该系统可以普遍应用于各种培养条件，但该方案强调对分化结肠和源自已建立的结肠的二维单层的治疗。在传统培养环境中，结肠富含肠道干细胞，为研究干细胞生态位提供了理想的环境。然而，该系统不允许分析肠道生理学的特征，例如屏障功能。此外，传统的结肠样不能提供研究终末分化上皮细胞对促炎刺激的细胞反应的机会。这里介绍的方法提供了一个替代的实验框架来解决这些限制。二维单层培养系统也为 离体治疗药物筛选提供了机会。这种极化细胞层可以用细胞基底侧的炎症介质处理，并伴有假定的治疗方法，以确定它们在IBD治疗中的效用。

3D肠结肠样培养系统是研究上皮对肠粘膜稳态的内在贡献的宝贵工具。所描述的协议提供了有关如何在8周龄时从C57BL / 6J（WT）小鼠中分离隐窝并建立可用于多个下游应用的长期结肠样培养系统的详细说明。在基底膜基质中分离和电镀隐窝后，当通过明场显微镜观察时，隐窝在结构上显得致密和多细胞。它们可以是球形的，也可以是细长的圆柱形，类似于通常在 体内 肠道结构中观察到的单?...

Watch this Scientific Journal Video about Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids at JoVE.com

作者聚焦:研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

我们描述了一个协议，详细说明了小鼠结肠隐窝的分离以开发三维结肠。然后，可以在接受炎症激发或被引导建立上皮单层之前对已建立的结肠样蛋白进行终末分化以反映宿主上皮的细胞组成。

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

通过我们的研究，我们的目标是探索如何从传统的类结肠培养系统修饰小鼠结肠，以研究肠道生理学的某些方面及其在发炎疾病状态下的改变。当使用仅反映干细胞生理学的传统鼠结肠样培养系统时，研究炎症性肠病等疾病状态下的屏障功能和肠道生理学是有限的。我们的协议是有利的，因为它为读者提供了多种技术来研究炎症介质如何影响肠上皮，通过最终分化它们或创建2D单层系统。未来，我们的实验室将专注于理解如何改变人类结肠培养系统，以研究肠道生理和疾病的不同方面，特别是炎症性肠病。

studying-epithelial-effects-intestinal-inflammation-vitro-on

Research

JoVE Journal

Biology

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

890 次观看.  UPMC Children’s Hospital of Pittsburgh. 通过我们的研究，我们的目标是探索如何从传统的类结肠培养系统修饰小鼠结肠，以研究肠道生理学的某些方面及其在发炎疾病状态下的改变。当使用仅反映干细胞生理学的传统鼠结肠样培养系统时，研究炎症性肠病等疾病状态下的屏障功能和肠道生理学是有限的。我们的协议是有利的，因为它为读者提供了多种技术来研究炎症介质如何影响肠上皮，通过最终分化它们或创?...

视频: 作者聚焦:研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

科研

生物学

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning方法小鼠结肠黏膜显微切割

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响