由美国国立卫生研究院资助 R01 EY035278 （MR） 支持。

猪的眼睛是从当地的屠宰场获得的。不需要伦理委员会的批准。
1.晶状体解剖（图1）
<ol><li>从猪眼中去除所有周围组织，从巩膜中去除多余的肉，直到只剩下视神经。使用弯曲的镊子和小解剖剪刀完成此过程。在解剖过程中使用神经作为锚来固定眼睛。</li>
	<li>使用手术刀在角膜缘做一个短的圆周切口，然后在赤道处再做一个切口。 注意: 按此顺序执行此步骤以避免损坏镜头和胶囊。</li>
	<li>将显微剪刀插入角膜缘处的切口，并用细钝头镊子提起角膜，同时绕角膜圆周切割，取出角膜。</li>
	<li>使用钝头镊子抬起去除虹膜，然后用微型剪刀剪掉。</li>
	<li>将解剖剪刀插入赤道切口，然后围绕整个赤道圆周切割，直到巩膜一分为二。</li>
	<li>切口完成后，切除巩膜的后部。用镊子轻轻取出玻璃体，留下最少的残留物，以免损坏晶状体。如果需要，切开玻璃体，使后体远离晶状体和前节。</li>
	<li>使用显微剪刀从前到后在巩膜上做一个经向切口。</li>
	<li>从穿过巩膜的新经线切口开始，使用显微剪刀将小带从晶状体上切开。利用晶状体的重量或解剖盘的边缘，在将晶状体和巩膜稍微分开时轻轻拉伸小带，让显微剪刀在小晶状体和睫状体之间、穿过小带和圆锥体周围切割。如果操作正确，这将隔离镜头而不会损坏镜头胶囊。</li>
	<li>如果需要，使用镊子取出胶囊，在赤道处刺穿胶囊，然后用两把镊子剥离胶囊。</li>
	<li>将镜头放入磷酸盐缓冲盐水 （PBS） 中。在进行机械测试之前，目视检查镜头是否有任何损坏。</li>
</ol>2. 带/不带透镜囊的镜头压缩（图 2）
注意:除步骤 2.1 和 2.4 外，此处的所有步骤均由计算机控制。
<ol><li>获得或构建具有 50 克力容量称重传感器的平行板压缩装置，能够测量 1 μm 量级的位移。</li>
	<li>对电动载物台进行编程并加载单元以执行下面描述的加载方案（例如， 补充文件 1）。</li>
	<li>用 PBS 几乎填充一个 1 5/8 英寸 x 1 5/8 英寸的方形盒子，并将其放在压缩平台上。</li>
	<li>将上板降低到与下板接触的位置，以确定运动的下限和绝对间隙高度。</li>
	<li>将上板抬高 ~15 毫米。</li>
	<li>将镜头放在盒子的中心，注意赤道平面是水平的。</li>
	<li>降低靠近镜头上表面但不与镜片上表面接触的上板。</li>
	<li>使用接触阈值为 3 mN 的力反馈启动运动以移动上板与镜头接触。</li>
	<li>在确定接触、记录时间、上板相对于下板的位置以及 500 Hz 的力后开始数据记录。</li>
	<li>应用预处理负载，其中镜头以 1%/s 的速率压缩其初始高度的 2.5% 三次，然后压缩 5% 三次，然后压缩 7.5% 三次。</li>
	<li>预处理后保持上板位置恒定1分钟。</li>
	<li>以 1%/s 的速率施加 15% 的压缩，然后以相同的速率卸载。</li>
	<li>继续卸载运动，直到上板从底板上移动了另外 2% 的卸载透镜厚度，以确保透镜与上板脱粘。</li>
</ol>3. 透镜模量的估算
<ol><li>根据仪器在接触点的间隙估计镜头的厚度。或者，使用图像分析来测量测试前拍摄的照片的厚度。</li>
	<li>使用赫兹模型计算弹性模量 E ，用于平行板之间球体的压缩（公式 [1]; 补充文件2）。 <img alt="Equation 1" src="/files/ftp_upload/66040/66040eq01.jpg" style="margin: auto;">（一） 其中 R 是接触点的曲率半径（假设等于透镜厚度的一半）; F 是称重传感器报告的压缩力; <img alt="Equation 2" src="/files/ftp_upload/66040/66040eq02.jpg" style="margin: auto;"> 是泊松比（假设等于0.5，对应于不可压缩材料）; u 是上级从接触点向下接近。请注意，弹性模量和泊松比是材料属性，分别表示透镜的固有刚度和透镜的相对压缩性。 注意:这种方法忽略了晶状体胶囊的任何作用，但大致考虑了晶状体的尺寸，允许物种之间的比较。</li>
</ol>

自动压缩猪晶状体的生物力学表征

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作者没有利益冲突需要声明。

镜片压缩是一种用于估计镜片刚度的通用方法。上述程序允许比较不同物种和不同尺寸的镜片。所有变形都根据透镜尺寸进行归一化，弹性模量的计算大致考虑透镜尺寸。有效模量远高于之前报道的猪透镜4、7、11、19 的模量，至少部分原因是使用了厚度而不是曲率半径:猪透镜的极曲半径明显大于厚?...

晶状体是眼睛中发现的透明且灵活的器官，它可以通过改变屈光度来聚焦在不同的距离上。这种能力被称为适应。由于睫状肌的收缩和松弛，屈光力会改变。当睫状肌收缩时，晶状体增厚并向前移动，增加其屈光力1,2。屈光率的增加使镜头能够聚焦在附近的物体上。随着人类年龄的增长，晶状体会变得更硬，这种适应能力会逐渐丧失;这种情况被称为老花眼。变硬的机制仍然未知，至少部分原因是与晶状体的生物力学表征相关的困难。
已经采用了多种方法来估计晶状体刚度和生物力学性能。这些方法包括透镜旋转3,4,5，声学方法6,7,8，光学方法，如布里渊显微镜9，压痕10,11和压缩12,13。压缩是最容易获得的实验技术，因为它可以使用简单的仪器（例如，玻璃盖玻片14,15）或单个电动载物台进行。我们之前已经展示了如何从压缩测试中严格估计晶状体的生物力学特性16.这个过程在技术上具有挑战性，需要专门的软件，而对相对刚度测量感兴趣的镜头研究人员不容易使用。因此，在本研究中，我们专注于在考虑透镜尺寸的同时估计透镜弹性模量的可用方法。弹性模量是与其变形性相关的固有材料特性:高弹性模量对应于较硬的材料。
该测试本身是平行板压缩测试，因此可以在合适的商业机械测试系统上进行。在这里，构建了一个由电机、线性平台、运动控制器、称重传感器和放大器组成的定制仪器。这些都是使用定制软件控制的，该软件还定期记录时间、位置和负载。猪镜片不适应，但易于获得且价格低廉17.开发了以下方法来逐步压缩眼睛晶状体并量化其弹性模量。这种方法可以很容易地复制，并且在晶状体刚度的研究中很有用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>Fisherbrand</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Galil COM Libraries</td>
			<td>Galil Motion Control</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Precision Scalpel Handle&#160;</td>
			<td>Fisherbrand</td>
			<td>12-000-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Stage</td>
			<td>McMaster-Carr</td>
			<td>6734K4 0.125&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>Load Cell</td>
			<td>FUTEK</td>
			<td>LSB200-FSH03869</td>
			<td></td>
		</tr>
		<tr>
			<td>Load Cell Amplifier</td>
			<td>FUTEK</td>
			<td>IAA300-FSH03931</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The Mathworks, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobe</td>
			<td>Surgical Design&#160;</td>
			<td>22-079-740</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature Self Opening Precision Scissors&#160;</td>
			<td>Excelta&#160;</td>
			<td>63042-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Motion Controller</td>
			<td>Galil Motion Control</td>
			<td>DMC-31012</td>
			<td></td>
		</tr>
		<tr>
			<td>Motor</td>
			<td>Galil Motion Control</td>
			<td>BLM-N23-50-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Hemastats&#160;</td>
			<td>Fine Science&#160;</td>
			<td>NC9247203</td>
			<td>stainless steel, 14cm&#160;</td>
		</tr>
	</tbody>
</table>

automated compression testing of the ocular lens

人工晶状体的生物力学特性对于其作为可变功率光学元件的功能至关重要。这些特性随着人类晶状体年龄的增长而发生巨大变化，导致近视力丧失，称为老花眼。然而，这些变化的机制仍然未知。晶状体压缩提供了一种相对简单的方法，用于定性地评估晶状体的生物力学刚度，并且当与适当的分析技术相结合时，可以帮助量化生物力学特性。迄今为止，已经进行了各种镜片压缩测试，包括手动和自动，但这些方法应用生物力学测试的关键方面不一致，例如预处理、加载速率和测量间隔时间。本文描述了一种全自动镜头压缩测试，其中电动载物台与相机同步，以在整个预编程的加载协议中捕获镜头的力、位移和形状。然后可以从这些数据中计算出特征弹性模量。虽然这里使用猪镜片进行演示，但该方法适用于任何物种的镜片压缩。

压缩六个猪晶状体，首先在胶囊完好无损的情况下，然后在小心地取出胶囊后。封装镜片的厚度值分别为 7.65 ± 0.43 mm，解封镜片的厚度值分别为 6.69 ± 0.29 mm（平均值±标准差）。典型的加载历史记录如 图 3 所示。赫兹模型很好地拟合了由此产生的力-位移曲线（即，它们的力与升至1.5次方的位移成正比; 图4）。封装和解封膜透镜都是如此。
<p class="j...

Watch this Scientific Journal Video about Advancing Understanding of Age-Related Lens Stiffness Changes at JoVE.com

作者聚焦:增进对与年龄相关的晶状体僵硬变化的理解 

我们提出了一种使用压缩测试表征人工晶状体有效弹性模量的自动化方法。

人工晶状体的自动压缩测试

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

我们试图了解人们如何随着年龄的增长而失去近视力。我们知道这在一定程度上是由于晶状体刚度的变化，但我们仍然不知道为什么晶状体会随着年龄的增长而变硬。目前的实验方法依赖于粗略的方法来加载镜头，例如在镜头上放置显微镜盖玻片。这些方法很繁琐，并且缺乏对加载速率的适当控制。我们已经确定晶状体形状是老花眼的主要驱动因素，并且我们知道形状主要由生物力学参数驱动。该协议为测量透镜刚度提供了一种完全客观、可重复的方法。自动化使我们能够控制加载速率，这是表征粘弹性材料（如透镜）时的关键参数。我们现在有一个客观的、可重复的协议来测量晶状体的生物力学特性。这将使我们能够严格表征晶状体如何随着年龄的增长而变硬，以及特定蛋白质如何改变晶状体的生物力学特性。

automated-compression-testing-of-the-ocular-lens

Research

JoVE Journal

Bioengineering

Automated Compression Testing of the Ocular Lens

2.2K 次观看.  The Ohio State University. 我们试图了解人们如何随着年龄的增长而失去近视力。我们知道这在一定程度上是由于晶状体刚度的变化，但我们仍然不知道为什么晶状体会随着年龄的增长而变硬。目前的实验方法依赖于粗略的方法来加载镜头，例如在镜头上放置显微镜盖玻片。这些方法很繁琐，并且缺乏对加载速率的适当控制。我们已经确定晶状体形状是老花眼的主要驱动因素，并且我们知道形状?...

视频: 作者聚焦:增进对与年龄相关的晶状体僵硬变化的理解 

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

科研

生物工程

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

癌症细胞球体机器人生产与药物测试的双水相系统

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

的发展<em&gt;体外</em&gt;眼平台，试验用隐形眼镜

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

用人工晶状体担架模拟透镜体的力学性能

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

鼠头的生物力学试验

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

用于小型细胞处理的自动逆流离心系统

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

机械测试的注入性量化

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

通过高通量微流体压缩系统对多细胞生物进行机械刺激

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...