Microfluidic and Biological Engineering, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Lab Chip. 2017 Dec 19;18(1):153-161. doi: 10.1039/c7lc01114h.
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. The process is reversible upon increasing the pH, with the rate constant for the shrinkage being -0.12 min. 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.
球状体是一种三维（3D）细胞培养物，旨在弥合使用整个动物和细胞单层之间的差距。微流控技术被认为是一种能够主动控制 3D 细胞培养物化学环境的使能技术。尽管已经开发出了各种平台来处理球状体培养物，但开发用于球状体的分析系统仍然是一个主要挑战。在这项研究中，我们设计了一种用于组织透明化和成像的微流控大规模集成（mLSI）芯片平台。为了能够在 mLSI 芯片上处理直径在数百微米范围内的球状体并进行培养，我们首先开发了一种通用的快速原型制作程序，该程序允许气动膜阀（PMV）的尺寸扩大。所提出的原型制作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。半圆形通道轮廓对于通常处于常开配置的常用 PMV 是必需的。对于光刻模具上这种通道轮廓，高度限制为数十微米，这阻碍了 3D 组织模型在 mLSI 芯片上的应用。该原型制作技术用于生产用于微型化、自动化和整合组织透明化方法 CLARITY 所涉及步骤的 mLSI 芯片，包括球状体固定、丙烯酰胺水凝胶渗透、温度引发的水凝胶聚合、脂质提取以及线粒体蛋白 COX-IV 和代谢酶 GAPDH 的免疫荧光染色。对球状体培养腔室中的液体进行精确的流体控制，允许在球状体组织内独家实施局部水凝胶聚合反应。水凝胶嵌入的球状体根据周围缓冲溶液的 pH 值进行膨胀和收缩。从 pH8.5 到 5.5 的 pH 跃变使水凝胶嵌入的球状体体积缩小 108%，速率常数为 0.36min。当 pH 值增加时，该过程是可逆的，收缩的速率常数为-0.12min。水凝胶嵌入的球状体中的 pH 重复循环会引起渗透压流。在 10 分钟的总时间间隔内在芯片上进行 30 个循环，将水凝胶嵌入的球状体（直径 200μm）的透明化时间从 14 天缩短至 5 小时。因此，我们开发了一种物理化学方法来缩短水凝胶嵌入组织的透明化时间。虽然渗透泵机制是降低组织透明化时间的电泳力的替代方法，但将 CLARITY 方法集成到芯片上可以实现具有 3D 组织培养物的高通量成像。
