Engineering Circadian Biology into Induced Pluripotent Stem Cell Organ-on-a-Chip Models

芯片上器官(OOAC)设备是一种新兴的工具，用于研究器官如何发挥作用，以及在实验室环境中测试新的和重新定位的药物。每个芯片都含有中空通道，其中包含人体细胞的组合，这些细胞相互作用，模仿我们器官的功能。机械力可以用来复制细胞在我们的组织和器官中经历的物理力，例如心脏和血管的流体流动和拉伸。最终，OOAC系统可能被用来减少动物在研究中的使用，并最终完全取代动物的常规使用。人体内的所有组织都有一个内部生物生物钟，根据白天或晚上的时间协调我们组织和器官中的细胞过程，每24小时创造一次生物功能循环。重要的是，药物的有效性和毒性往往随着给药时间的不同而不同，这意味着一种药物可能在一天中的某些时间最有效，产生的副作用最少。然而，对于大多数治疗方法来说，生物时间对疗效的影响还没有被研究或利用。这在一定程度上是因为药物开发的早期阶段，涉及简单的细胞系统，然后是动物模型，不能复制人类的生物钟。因此，新药第一次遇到相关的人体生物钟是在开发过程多年的I期临床试验期间。这导致了药物发现计划在后期阶段代价高昂的失败，以及在开发的早期阶段不必要地使用动物。目前，昼夜节律没有被纳入OOAC技术，也没有办法在这些设备中建立细胞的生物时间，限制了该技术的治疗和商业潜力。因此，这个项目的总体目标是将生物钟整合到OOAC系统中，允许监测细胞的“一天中的时间”，并在不同的时间点测试药物，以反映我们的器官的昼夜。在这个项目中，我们将从诱导多能干细胞(IPSCs)中开发能够‘报告’生物钟时间的细胞系。这涉及到使用基因编辑技术CRISPR将发光标签与控制时钟的关键基因联系起来。然后，可以无创地诱导和监测细胞的昼夜节律。IPSC可以分化为体内所有类型的细胞，使我们能够开发一个新的人类IPSC来源的分化时钟报告细胞库。在这个项目中，我们将把IPSCs转化为内皮细胞，这些细胞排列在我们的血管和骨骼肌细胞中。这些细胞将在OOAC设备中生长，并将提供刺激，以设置时钟的时间，从而导致时钟基因的表达，通过发光测量，将在24小时内振荡。我们将使用各种刺激来设置时钟的时间，包括化学因素，如生长因子和糖皮质激素，但也包括机械刺激，如流体流动和拉伸，这是OOAC设备不可或缺的。最后，我们将看看已知的药物是否对细胞有不同的影响，这取决于它在24小时昼夜节律期间的给药时间。将生物时间纳入OOAC系统的能力将使该系统能够更好地复制我们体内器官的功能，并增强它们被用作实验室系统的能力，以取代动物在研究中的常规使用。

Organ-on-a chip (OOAC) devices are an emerging tool for studying how organs function and for testing new and repurposed drugs in a laboratory environment. Each chip contains hollow channels containing combinations of human cells that interact with each other to mimic the functioning of our organs. Mechanical forces can be applied to replicate the physical forces that cells experience in our tissues and organs, for example fluid flow and stretch for the heart and blood vessels. Ultimately OOAC systems may be used to reduce the use of animals in research and ultimately to replace routine use of animals altogether.All tissues in the body have an internal biological circadian 'clock' which co-ordinates cellular processes in our tissues and organs according to the time of day or night, creating cycles of biological function every 24h. Importantly drug effectiveness and toxicity often varies according to the time of administration, which means that there may be times of day when a drug is most effective and produces the least side effects. However, for the majority of treatments, the influence of biological time on therapeutic effectiveness has not been studied or exploited. This is partly because the early stages of a drug development, involving simple cell systems and then animal models do not replicate the human circadian clock. Accordingly, the first time a novel drug encounters a relevant human circadian clock is during phase I clinical trials many years into the development process. This results in costly late-stage failure of drug discovery programmes and the unnecessary use of animals in earlier stages of development. Currently circadian rhythms are not incorporated into OOAC technology and there is no means of establishing the biological time of cells within these devices, limiting the therapeutic and commercial potential of the technology. The overall aim of this project is, therefore, to incorporate the circadian clock into OOAC systems, allowing the cellular 'time of day' to be monitored and for drugs to be tested at different time points that reflect day and night for our organs.In this project we will develop cell lines from Induced Pluripotent Stem Cells (iPSCs) that are able to 'report' the time of the circadian clock. This involves using the gene editing technique CRISPR to link a luminescent tag to key genes that control the clock. Cellular circadian rhythms can then be induced and monitored non-invasively. iPSCs can be differentiated into all cell types in the body allowing us to develop a library of novel human iPSC-derived differentiated clock reporter cells. In this project we will turn the iPSCs into endothelial cells which line our blood vessels and skeletal muscle cells. The cells will be grown in OOAC devices and stimuli will be provided that 'set the time' of the clock resulting in the expression of clock genes, measured by luminescence, that will oscillate over a 24hr period. We will use a variety of stimuli to 'set the time' of the clock including chemical factors, such as growth factors and glucocorticoids, but also mechanical stimuli, such as fluid flow and stretch which are integral to OOAC devices. Finally we will look at whether known drugs have a different effect on the cells depending on when it is administered during the 24hr circadian rhythm. The ability to incorporate biological time into OOAC systems will make the systems better able to replicate the functioning of organs in our bodies and enhance their ability to be used as laboratory systems to replace the routine use of animals in research.