Spheroids are three-dimensional (3D) cell cultures that aim to bridge the gap between the use of whole animals and cellular monolayers. Microfluidics is regarded as an enabling technology to actively control the chemical environment of 3D cell cultures. Although a wide variety of platforms have been developed to handle spheroid cultures, the development of analytical systems for spheroids remains a major challenge. In this study, we engineered a microfluidic large-scale integration (mLSI) chip platform for tissue-clearing and imaging. To enable handling and culturing of spheroids on mLSI chips, with diameters within hundreds of microns, we first developed a general rapid prototyping procedure, which allows scaling up of the size of pneumatic membrane valves (PMV). The presented prototyping method makes use of milled poly(methylmethacrylate) (PMMA) molds for obtaining semi-circular microchannels with heights up to 750 μm. Semi-circular channel profiles are required for the functioning of the commonly used PMVs in normally open configuration. Height limits to tens of microns for this channel profile on photolithographic molds have hampered the application of 3D tissue models on mLSI chips. The prototyping technique was applied to produce an mLSI chip for miniaturization, automation, and integration of the steps involved in the tissue clearing method CLARITY, including spheroid fixation, acrylamide hydrogel infiltration, temperature-initiated hydrogel polymerization, lipid extraction, and immuno-fluorescence staining of the mitochondrial protein COX-IV, and metabolic enzyme GAPDH. Precise fluidic control over the liquids in the spheroid culturing chambers allowed implementation of a local hydrogel polymerization reaction, exclusively within the spheroid tissue. Hydrogel-embedded spheroids undergo swelling and shrinkage depending on the pH of the surrounding buffer solution. A pH-jump from 8.5 to 5.5 shrinks the hydrogel-embedded spheroid volume by 108% with a rate constant of 0.36 min⁻¹. The process is reversible upon increasing the pH, with the rate constant for the shrinkage being −0.12 min⁻¹. Repetitive cycling of the pH induces an osmotic flow within the hydrogel-embedded spheroid. Thirty cycles, performed in a total time interval of 10 minutes on-chip, reduced the clearing time of a hydrogel-embedded spheroid (with a diameter of 200 μm) from 14 days to 5 hours. Therefore, we developed a physicochemical method to decrease the clearing time of hydrogel-embedded tissues. While the osmotic pump mechanism is an alternative to electrophoretic forces for decreasing tissue clearing times, the integration of the CLARITY method on chip could enable high throughput imaging with 3D tissue cultures.

中文翻译：

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在微流控芯片上快速清除球状体†

椭球是三维（3D）细胞培养物，旨在弥合整个动物的使用与细胞单层之间的差距。微流体技术被认为是一种主动控制3D细胞培养物化学环境的技术。尽管已经开发了各种各样的平台来处理球体培养，但是球体分析系统的开发仍然是一个重大挑战。在这项研究中，我们设计了一种用于组织清除和成像的微流控大规模集成（mLSI）芯片平台。为了能够在直径在几百微米以内的mLSI芯片上处理和培养球状体，我们首先开发了一种通用的快速原型制作程序，该程序可以扩大气动隔膜阀（PMV）的尺寸。提出的原型方法利用铣削的聚（甲基丙烯酸甲酯）（PMMA）模具来获得高度高达750μm的半圆形微通道。半圆形通道轮廓是常开配置下常用PMV运转所必需的。在光刻模具上，此通道轮廓的高度限制为数十微米，这已阻碍了3SI组织模型在mLSI芯片上的应用。原型技术用于生产mLSI芯片，用于组织清除方法CLARITY的微型化，自动化和集成，包括球体固定，丙烯酰胺水凝胶浸润，温度引发的水凝胶聚合，脂质提取和免疫荧光染色线粒体蛋白COX-IV和代谢酶GAPDH的合成。对球状培养室中液体的精确流体控制允许仅在球状组织内进行局部水凝胶聚合反应。嵌入水凝胶的球体会发生溶胀和收缩，具体取决于周围缓冲溶液的pH值。pH值从8.5跃升至5.5会使水凝胶包埋的球状体体积缩小108％，速率常数为0.36分钟^-1。当增加pH时，该过程是可逆的，收缩的速率常数为-0.12min ^-1。pH的重复循环会在包埋水凝胶的球体内引起渗透流。在芯片上以10分钟的总时间间隔执行的30个循环将嵌入水凝胶的球体（直径为200μm）的清除时间从14天减少到5小时。因此，我们开发了一种物理化学方法来减少水凝胶包埋组织的清除时间。尽管渗透泵机制是电泳力的替代品，可减少组织清除时间，但CLARITY方法在芯片上的集成可以实现3D组织培养的高通量成像。