Understanding and manipulating lactate metabolism in single cells

今天开出的许多药物都是在动物细胞培养中生产的蛋白质。几十年的研究已经检验了通过改变细胞获得的营养物质、细胞本身的基因工程和其他策略来提高这些培养物生产率的方法。然而，仍然存在限制蛋白质生产总量的挑战。这些挑战之一是代谢废物乳酸的积累，乳酸是导致剧烈运动后肌肉酸痛的相同废物。动物细胞培养物中乳酸的浓度随着时间的推移而变化。早期，它由于快速新陈代谢而积累，但后来细胞利用它来制造能量和氨基酸。这就是所谓的乳酸开关。不同批次的细胞在不同的时间经历乳酸开关，而触发开关的触发机制还不是很清楚。然而，就蛋白质药物的生产而言，乳酸开关是与较高蛋白质产量相关的积极特征。拟议的工作旨在创造一套工具来理解和操纵乳酸开关。第一个目标是创建一种非侵入性指示器，允许测量单个细胞中的乳酸浓度。这一工具将使我们能够测量细胞之间的差异程度，这是目前标准技术无法实现的。我们将利用它来了解不同的培养条件如何影响细胞中的乳酸积累，试图确定在每种情况下有多大的可变性。总体而言，这些信息可以用来选择不同细胞之间差异较小的制造条件。第二个目标是开发工具，允许我们改变与乳酸生产和消费相关的单个酶的表达水平。有了这些工具，我们将能够通过外部信号控制哪些酶与乳酸开关相关的假说。一旦确定了酶，我们就可以使用系统来控制乳酸开关何时被翻转，确保培养中的所有细胞确实从乳酸生产切换到消费，并确保开关在最佳时间翻转，以确保高蛋白产量。最后，我们将合并这两套工具来创建细胞，这些细胞可以感知自己的乳酸浓度，当乳酸浓度太高时，调节自己的乳酸代谢基因。这将产生不会产生太多乳酸的细胞，这应该会增加它们产生的蛋白质数量。“自我调节细胞”也将成为未来如何利用其他感兴趣的特征来实现这一点的模型。因此，这个项目可能会改变为制造目的而开发电池的方式。

Many of the medicines prescribed today are proteins that are manufactured in animal cell cultures. Decades of research has examined ways to increase the productivity of these cultures by changing the nutrients the cells are fed, genetic engineering of the cells themselves and other strategies. However, there are still challenges that limit the overall amount of protein produced. One of these challenges is the accumulation of the metabolic waste product, lactic acid, the same waste product that causes your muscles to be sore after strenuous exercise. The concentration of lactate in animal cell cultures changes over time. Early on, it accumulates due to rapid metabolism, but later the cells use it to make energy and amino acids. This is called the lactate switch. Different batches of cells undergo the lactate switch at different times and the trigger for flipping the switch is not well understood. However, with respect to the production of protein medicines, the lactate switch is a positive trait associated with higher protein production.The proposed work seeks to create a set of tools for understanding and manipulating the lactate switch. The first goal is to create a non-invasive indicator that allows measurement of lactate concentration in individual cells. This tool would allow us to measure the extent of variation between cells, something that is currently not possible with standard techniques. We will use it to understand how different culture conditions affect lactate accumulation in the cells to try and identify how much variability there is in each scenario. Overall, this information can be used to choose manufacturing conditions with less difference between cells.The second goal is to develop tools that allow us to change the expression level of individual enzymes associated with lactate production and consumption. With these tools, we will be able to test hypotheses about which enzymes are associated with the lactate switch by controlling them with an external signal. Once the enzymes are identified, we could use the system to control when the lactate switch is flipped, making sure that all cells in the culture do switch from lactate production to consumption and making sure the switch flips at the best time to ensure high protein production. Finally, we will merge the two sets of tools to create cells that sense their own lactate concentration and when it gets too high, regulate their own lactate metabolism genes. This will create cells that do not make too much lactate, which should increase the amount of protein that they produce. The 'self-regulating cells' will also serve as a model for how to do this with other traits of interest in the future. This project could, therefore, change the way cells are developed for manufacturing purposes.