论文中介绍的工作得到了国家科学基金会的支持, 研究和创新领域的新兴前沿 (NSF-EFRI) 方案赠款0938072。本文的内容完全是作者的责任, 不一定代表 NSF 的官方观点。作者还希望感谢 Dr. Samsoon Inayat 为他的工作设计和测试的初步实验设置的化学刺激和 Mr. 温 Raghunathan 为他的工作设计, 制作, 并评价了多端口微流控装置使用这项研究。

<ol>
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作者没有什么可透露的。

这里介绍的方法展示了一个独特的神经刺激模式, 其中视网膜神经元的化学刺激, 注射当地神经递质化学物质进入视网膜的地下体外。这种化学刺激技术在传统的电刺激技术中提供了一些好处, 包括选择性和靶神经元的高聚焦特异性。上面的协议详细说明了如何小体积的神经递质谷氨酸注射在目标视网膜神经元附近使用单玻璃微或自定义微流控流体设备引出在生理学上重要的研资反应。虽然?...

感光性退行性疾病, 如视网膜色素变性和年龄相关的黄斑变性, 是导致视力丧失的可继承原因, 目前是无法治愈的1,2。尽管这些疾病是由各种特定的基因突变引起的, 但感光变性疾病是由视网膜感光细胞的逐渐丧失而引起的, 最终导致失明。光的丢失引发了整个视网膜的广泛重塑, 但存活的视网膜神经元, 包括双极细胞和视网膜, 仍然完好无损, 即使在感光变性的高级阶段也相对正常,3 ,4,5,6,7。
这些疾病的机制和病理特点已经很好的特征3,4,5,6,7 , 但有效的治疗仍然难以捉摸。在过去的三年中, 世界各地的研究人员已经研究了多种治疗方法, 以恢复视力的那些受影响的感光变性疾病, 包括基因治疗8, 干细胞治疗9,视网膜移植10, 人工刺激11,12存活的视网膜神经元。其中, 最有临床意义的是视网膜假体, 这是人工神经装置, 传统上使用了一系列电极, 以电刺激双极细胞或视网膜在特定模式的目标在患者中创建人工视觉感知11。当前世代电子假肢, 例如守卫 II13和阿尔法 IMS14设备, 获得了临床批准, 并且初步研究表明, 他们能改善患者的生活质量通过恢复测量视觉使用两个前 (视网膜的前面) 和视网膜 (视网膜的后面) 植入的设备15,16。世界各地的研究小组正在努力推进视网膜假肢超越这些 first-generation 设备的成功17,18,19,20 ,但已面临困难设计一个电子假肢能够恢复高视力视力低于法律失明水平的病人。最近的研究表明, 实现更高的空间分辨率比目前的代为主假肢是挑战, 因为电荷注入限制, 这就需要使用大的电极来安全地刺激视网膜神经元以空间分辨率为代价,即视觉灵敏度11,21。此外, 电刺激是进一步有限的, 因为它通常刺激所有附近的细胞, 从而引起病人的不自然和混淆的看法, 很大程度上是因为它是一个固有的非自然刺激范例21。然而, 早期的电刺激成功表明, 人工神经可以是一种有效的治疗感光变性疾病。这导致一个假设, 更有效的治疗可能是可以通过刺激视网膜与神经递质化学品, 在化学突触沟通的自然代理人。该方法的目的是探讨化学刺激的治疗可行性, 旨在模拟视网膜神经元之间的突触通信的自然系统, 作为电刺激的仿生替代品。用于视网膜假体
对化学性视网膜假体的治疗性化学刺激的概念的翻译依赖于化学活化的目标视网膜神经元, 少量的本地神经递质, 如谷氨酸, 通过微流控物释放一种装置, 包括大量的 microports 以响应视觉刺激。这样, 一个化学的视网膜假体将本质上是一个仿生的人工感光层, 把光子自然地到达视网膜的化学信号。由于这些化学信号在正常的视网膜信号中使用相同的神经递质, 并通过正常视觉通路所使用的相同突触通路刺激退化视网膜的存活的视网膜神经元, 由此产生的视觉通过一个化学的视网膜假体的知觉可以更自然和可理解相比, 通过一个电动假肢诱发。此外, 由于神经递质被释放的 microports 可以非常小, 并排列在高密度, 不像电极, 一个潜在的化学假体可能能够获得更多的焦点刺激和更高的空间分辨率比电子假肢。因此, 基于这些潜在的优势, 化学性视网膜假体提供了一个非常有前途的替代电气假肢。
然而, 直到最近, 对视网膜的化学刺激也相对较少。而电刺激的视网膜已经很好地表征了几十年的工作通过体外22,23,在体内23,24, 和临床研究13 ,14, 对化学刺激的研究仅限于少数体外工作25,26,27,28。耶齐和芬利森26和 Inayat et al.27分别使用单电极和多阵列 (前) 对视网膜的化学刺激进行了实验, 以记录视网膜神经元的谷氨酸诱发反应。最近, 朗特里et al.28显示了视网膜侧的谷氨酸和视网膜通路的差异刺激, 并记录了视网膜上多个部位的神经元反应。虽然这些工作已经初步确定了化学刺激的可行性, 但进一步的研究对于研究这种方法的许多方面是非常必要的, 除了这些方面, 到目前为止25,26,27,28, 并 fine-tune 的治疗刺激参数在两个体外和在体内动物模型之前, 把这个概念转化为化学视网膜假体如上所述。然而, 目前还没有确定的方法, 以完成化学刺激的视网膜在文献中使用的方法, 在以前的作品没有被描述的细节, 这将是必要的复制研究。因此, 本方法的基本原理是提供一个明确的框架来进行体外对视网膜的化学刺激, 对于那些有兴趣复制我们以前的研究的调查者27, 28或进一步推进这一新生的化学神经概念。
在这里, 我们演示了一种进行体外的方法, 对野生型大鼠 wholemount 视网膜神经元的化学刺激及感光变性大鼠模型的模拟疾病在人。在体外模型中开发这种刺激方法的基本原理是评估各种刺激参数的治疗范围, 并研究在中不可能或难以观察到的神经反应特性. 体内的模型, 特别是在最初的研究集中在评估这种方法的可行性。在这个过程中, 我们显示站点和同时多站点的视网膜的化学刺激通过提供少量的1毫米谷氨酸近靶视网膜神经元通过商业可用的单玻璃 micropipettes 和自定义多口微流控装置, 分别。虽然站点和多站点刺激完成了研究化学调节的治疗可行性的基本目标, 但它们都具有独特的优势。站点刺激, 这可能是完成与商业可用的 pre-pulled 玻璃 micropipettes, 可用于直接注入化学品的视网膜下在一个单一的地点, 并有助于调查, 如果可见的研资局穗率类似于视觉诱发光反应的反应可以在注射部位局部。另一方面, 多站点刺激, 需要专门制作的多端口微流控装置, 可以被用来在多个地点的视网膜表面上注入化学物质, 并有助于研究谷氨酸诱发 RCG反应模式对应于模式刺激研究中的谷氨酸注射模式。

<table>
	<tbody>
		<tr>
			<td>Microelectrode array, perforated layout</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>60pMEA200/30iR-Ti-pr</td>
			<td>http://www.multichannelsystems.com/products/microelectrode-arrays/60pmea20030ir-ti</td>
		</tr>
		<tr>
			<td>MEA amplifier</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MEA1060-Inv</td>
			<td>http://www.multichannelsystems.com/products/mea1060-inv</td>
		</tr>
		<tr>
			<td>Bottom perfusion groundplate for pMEA</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MEA1060-Inv-(BC)-PGP</td>
			<td>http://www.multichannelsystems.com/products/mea1060-inv-bc-pgp</td>
		</tr>
		<tr>
			<td>3-axis Motorized Micromanipulator</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MP-285</td>
			<td>https://www.sutter.com/MICROMANIPULATION/mp285.html</td>
		</tr>
		<tr>
			<td>Micromanipulator Control System</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MPC-200</td>
			<td>https://www.sutter.com/MICROMANIPULATION/mpc200.html</td>
		</tr>
		<tr>
			<td>Gantry style micromanipulator stand with linear slide</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MT-75/LS</td>
			<td>https://www.sutter.com/STAGES/mt75.html</td>
		</tr>
		<tr>
			<td>8-channel Programmable Multichannel Pressure Injector</td>
			<td>OEM: MicroData Instrument, S. Plainfield, NJ 
			Vendor: Harvard Apparatus UK</td>
			<td>PM-8000 or PM-8</td>
			<td>OEM: http://www.microdatamdi.com/pm8000.htm 
			Vendor: https://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/product_11555_10001_39808_-1_HAUK_ProductDetail</td>
		</tr>
		<tr>
			<td>Axopatch 200A Integrating Patch Clamp Amplifier</td>
			<td>Molecular Devices, Sunnyvale, CA</td>
			<td>Axopatch 200A</td>
			<td>Axopatch 200A has been replaced with a newer model Axopatch 200B: 
			https://www.moleculardevices.com/systems/axon-conventional-patch-clamp/axopatch-200b-amplifier</td>
		</tr>
		<tr>
			<td>Patch clamp headstage</td>
			<td>Molecular Devices, Sunnyvale, CA</td>
			<td>CV 201A</td>
			<td>http://mdc.custhelp.com/app/answers/detail/a_id/16554/~/axopatch-200a%3A-selection-cv-headstage</td>
		</tr>
		<tr>
			<td>Vacuum waste kit</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>VMK</td>
			<td>http://alascience.com/product/vacuum-waste-kit/</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Warner Instruments, Hamden, CT</td>
			<td>QSW-A10P</td>
			<td>https://www.warneronline.com/product_info.cfm?id=915</td>
		</tr>
		<tr>
			<td>Pre-pulled 10 &#956;m tip diameter glass micropipettes</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>TIP10TW1</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/make-selection-pre-pulled-glass-pipettes-plain/</td>
		</tr>
		<tr>
			<td>Zoom stereomicroscope</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>SMZ-745T</td>
			<td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ745</td>
		</tr>
		<tr>
			<td>Microscope boom stand with dual linear ball bearing arm</td>
			<td>Old School Industries, Inc., Dacono, CO</td>
			<td>OS1010H-16BB</td>
			<td>http://www.osi-incorp.com/productdisplay/dual-linear-ball-bearing-arm</td>
		</tr>
		<tr>
			<td>Zoom Stereo Microscope with C-LEDS Hybrid LED Stand</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>SMZ-445</td>
			<td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ445</td>
		</tr>
		<tr>
			<td>Inverted microscope system</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>Eclipse Ti-E</td>
			<td>https://www.nikoninstruments.com/Products/Inverted-Microscopes/Eclipse-Ti-E</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1420</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/a1420</td>
		</tr>
		<tr>
			<td>L-Glutamic Acid (Glutamate)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/mm/100291</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>S8761</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/s8761</td>
		</tr>
		<tr>
			<td>60 mm Petri dish (10 mm tall)</td>
			<td>Fischer Scientific, Waltham, MA</td>
			<td>FB0875713A</td>
			<td>60 mm clear petri dish; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875713a</td>
		</tr>
		<tr>
			<td>Jewelers #5 Forceps</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>555227F</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/555227f-jewelers-5-forceps-11cm-straight-titanium/</td>
		</tr>
		<tr>
			<td>Standard Scalpel Blad #24</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>500247</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/500247-standard-scalpel-blade-24/</td>
		</tr>
		<tr>
			<td>Scalpel Handle #4</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>500237</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/500237-scalpel-handle-4-14cm/</td>
		</tr>
		<tr>
			<td>Vannas Tubingen Dissection Scissors</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>503378</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/503378-vannas-tubingen-scissors-8cm-straight-german-steel/</td>
		</tr>
		<tr>
			<td>Nylon mesh kit</td>
			<td>Warner Instruments, Hamden, CT</td>
			<td>NYL/MESH</td>
			<td>https://www.warneronline.com/product_info.cfm?id=1173</td>
		</tr>
		<tr>
			<td>Harp slice grid</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>HSG-5AD</td>
			<td>http://alascience.com/product/standard-harp-slice-grids/</td>
		</tr>
		<tr>
			<td>Ag/AgCl reference electrode pellet</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>P1060</td>
			<td>http://www.multichannelsystems.com/products/p1060</td>
		</tr>
		<tr>
			<td>4 Channel Valve Controlled Gravity Perfusion System</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>VC3-4xG</td>
			<td>http://alascience.com/product/4-channel-valve-controlled-gravity-perfusion-system/</td>
		</tr>
		<tr>
			<td>Zyla 5.5 sCMOS microscope camera</td>
			<td>Andor Technology, Belfast, UK</td>
			<td>Zyla 5.5 sCMOS</td>
			<td>http://www.andor.com/scientific-cameras/neo-and-zyla-scmos-cameras/zyla-55-scmos</td>
		</tr>
		<tr>
			<td>Silver wire (50 &#956;m diameter)</td>
			<td>Fischer Scientific, Waltham, MA</td>
			<td>AA44461G5</td>
			<td>https://www.fishersci.com/shop/products/silver-wire-0-05mm-0-002-in-dia-annealed-99-99-metals-basis-3/aa44461g5</td>
		</tr>
		<tr>
			<td>Tygon microbore tubing (1.6 mm diameter)</td>
			<td>Cole Parmer, Vernon Hills , IL</td>
			<td>EW-06419-01</td>
			<td>https://www.coleparmer.com/i/tygon-microbore-tubing-0-020-x-0-060-od-100-ft-roll/0641901</td>
		</tr>
		<tr>
			<td>Tilting Tool Holder with Steel Cannula</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>TILTPORT</td>
			<td>One each of these were utilized for top perfusion and suction; http://alascience.com/product/tilting-tool-holder-with-steel-cannula/</td>
		</tr>
		<tr>
			<td>Roscolux #26 Light Red Filter Sheet</td>
			<td>Rosco Laboratories Inc., 52 Harbor View, Stamford, CT</td>
			<td>R2611</td>
			<td>Manufacturer: http://us.rosco.com/en/products/catalog/roscolux 
			Vendor: https://www.bhphotovideo.com/c/product/43957-REG/Rosco_RS2611_26_Filter_Light.html</td>
		</tr>
		<tr>
			<td>Smith &#38; Wesson Galaxy Red Flashlight</td>
			<td>Smith &#38; Wesson, 2100 Roosevelt Avenue, Springfield, MA</td>
			<td>4588</td>
			<td>Manufacturer: https://www.smith-wesson.com/ 
			Vendor: http://www.mypilotstore.com/mypilotstore/sep/4588</td>
		</tr>
		<tr>
			<td>MC_Rack Software</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MC_Rack</td>
			<td>http://www.multichannelsystems.com/software/mc-rack</td>
		</tr>
		<tr>
			<td>Labview Software</td>
			<td>National Instruments, Austin, TX</td>
			<td>LabVIEW</td>
			<td>http://www.ni.com/labview/</td>
		</tr>
		<tr>
			<td>NIS-Elements: Basic Research Software</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>NIS-Elements BR</td>
			<td>https://www.nikoninstruments.com/Products/Software/NIS-Elements-Basic-Research</td>
		</tr>
	</tbody>
</table>

methodology for biomimetic chemical neuromodulation of rat retinas with the neurotransmitter glutamate in vitro

感光变性疾病通过视网膜感光细胞的逐渐丧失而导致无法挽回的失明。视网膜假体是一种新兴的治疗感光退行性疾病, 寻求恢复视力的人为刺激幸存的视网膜神经元, 希望能获得可理解的视觉感知的病人。目前的视网膜假体已证明是成功的恢复有限的视力患者使用一系列电极电刺激视网膜, 但面临巨大的物理障碍, 恢复高视力, 自然视力的病人。使用天然神经递质的化学神经是一种仿生替代电刺激, 可以绕过与使用电神经的视网膜假肢相关的基本限制。具体而言, 化学神经有可能恢复更多的自然视力与可比或更好的视觉视力患者通过注射极少量的神经递质, 相同的自然药物的交流使用的视网膜化学突触, 比当前的电子假肢更精细的分辨率。然而, 作为一个相对未被探索的刺激范例, 没有建立的协议为实现视网膜的化学刺激在体外。这项工作的目的是提供一个详细的框架, 以完成化学刺激视网膜的研究者谁希望研究的潜在化学调节的视网膜或类似的神经组织体外。在这项工作中, 我们描述的实验设置和方法, 诱导视网膜神经节细胞 (王国) 穗反应类似于视觉光反应的野生型和感光 wholemount 大鼠视网膜的注射控制体积神经递质谷氨酸进入视网膜空间使用玻璃 micropipettes 和定制多端口微流控装置。这种方法和协议是通用的, 足以适应调节使用其他神经递质或甚至其他神经系统组织。

该协议可用于化学刺激正常, 野生类型的视网膜和感光变性视网膜, 尽管大量的细胞重塑造成的损失的光。在开始实验, 无论是感光器退化或野生类型的视网膜, 记录和刺激设备 (图 1和图 2) 需要准备好, pMEA (图 5) 应该被清除, 以最小化每个电极通道上的噪音 (图 6)。虽然感光变性视网?...

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Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

本议定书描述了一种新的方法, 研究一种形式的化学神经的 wholemount 大鼠视网膜体外与神经递质谷氨酸。化学神经是一种有前途的替代传统的电神经视网膜神经元治疗不可逆转性失明引起的感光变性疾病。

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

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Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

7.7K 次观看.  University of Illinois at Chicago. 本议定书描述了一种新的方法, 研究一种形式的化学神经的 wholemount 大鼠视网膜体外与神经递质谷氨酸。化学神经是一种有前途的替代传统的电神经视网膜神经元治疗不可逆转性失明引起的感光变性疾病。

视频: Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

基于植物的提取胶囊微胶囊化应用

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...