Saleheen Amirus, Acharyya Debalina, Prosser Rebecca A, Baker Christopher A
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, USA.
Anal Methods. 2021 Mar 21;13(11):1364-1373. doi: 10.1039/d0ay02291h. Epub 2021 Mar 1.
Ex vivo brain slice cultures are utilized as analytical models for studying neurophysiology. Common approaches to maintaining slice cultures include roller tube and membrane interface techniques. The rise of organ-on-chip technologies has demonstrated the value of microfluidic perfusion culture systems for sampling and analysis of complex biology under well-controlled in vitro or ex vivo conditions. A number of approaches to microfluidic brain slice culture have been developed, however these typically involve complex design, fabrication, or operational parameters in order to meet the high oxygen demands of brain slices. Here, we present proof-of-principle for a novel approach to microfluidic brain slice culture. In this system, which we term a microfluidic bubble perfusion device, principles of droplet microfluidics were employed to generate droplets of perfusion media dispersed between bubbles of carbogen gas, and brain tissue slices were perfused with the resulting monodispersed droplets and bubbles. The challenge of tissue immobilization in the flow system was addressed using a two-part cytocompatible carbohydrate-based tissue adhesive. Best practices are discussed for perfusion chamber designs that maintain segmented flow throughout the course of perfusion. Control of droplet and bubble volumes was possible across the range of ca. 4-15 μL, bubble generation frequency was well controlled in the range ca. 1-7 bubbles per min, and bubble duty cycle was well controlled across the range ca. 20-80%. Murine hypothalamic tissue slices containing the suprachiasmatic nuclei were successfully maintained for durations of 8-10 hours, with tissue remaining viable for the duration of perfusion as assessed by Ca imaging and propidium iodide (PI) staining.
体外脑片培养被用作研究神经生理学的分析模型。维持脑片培养的常见方法包括滚管法和膜界面技术。芯片器官技术的兴起证明了微流控灌注培养系统在体外或离体条件下对复杂生物学进行采样和分析的价值。已经开发了多种微流控脑片培养方法，然而，这些方法通常涉及复杂的设计、制造或操作参数，以满足脑片对高氧的需求。在此，我们展示了一种新型微流控脑片培养方法的原理验证。在这个我们称之为微流控气泡灌注装置的系统中，利用液滴微流控原理产生分散在二氧化碳气体气泡之间的灌注培养基液滴，并用产生的单分散液滴和气泡灌注脑组织切片。使用一种基于碳水化合物的两部分细胞相容性组织粘合剂解决了流动系统中组织固定的挑战。讨论了在整个灌注过程中维持分段流动的灌注室设计的最佳实践。在约4-15 μL的范围内可以控制液滴和气泡的体积，气泡产生频率在约每分钟1-7个气泡的范围内得到很好的控制，气泡占空比在约20-80%的范围内得到很好的控制。含有视交叉上核的小鼠下丘脑组织切片成功维持了8-10小时，通过钙成像和碘化丙啶（PI）染色评估，组织在灌注期间保持存活。
