Engineering zonal human liver functions in vitro using microfluidics

描述(申请人提供)：肝功能的区带异质性在体内和体外都得到了很好的证实，这是肝脏的一个重要特征，与区带药物毒性有关。然而，肝脏内的各种因子梯度如何相互作用来调节人类肝脏的带状肝功能，以及肝基质细胞在这种调节中扮演的角色还不是很清楚。由于不同物种肝功能的显著差异，诱导和研究调节原代人肝细胞带状结构的因素对于阐明与动物的异同至关重要，从而使体外和体内研究得以连续进行。然而，目前还缺乏人肝分区的体外模型，尤其是那些评估因子梯度和基质相互作用对原代人肝细胞分区功能的长期影响的模型。为了解决上述认识上的差距，我们建议研究肝相关因子梯度和肝基质细胞对原代培养人肝细胞长期功能的影响。我们将利用微流体使有限的原代人类肝细胞的使用量降至最低，并在多细胞肝培养上创建独立的、复杂的和重叠的可溶性梯度。我们将评估我们的设备产生的梯度(O2，激素)对肝功能的影响，这些功能被认为是在体内分区的，无论有没有肝基质细胞。体外结果将与体内已知的机制进行比较。最终，创造方法和设计规则来制造不同种类和稳定的人类肝细胞群体，将允许在设计用于 药物筛选、临床应用(如生物人工肝设备)和干细胞分化。

DESCRIPTION (provided by applicant): Zonal heterogeneity in hepatic functions is well established both in vivo and in vitro, and is an important feature of the liver with implications fr zonal drug toxicity. However, how various factor gradients within the liver interact to modulate zonal hepatic functions in the human liver, and what role liver stromal cells play in such modulation are not well understood. Due to significant species-specific differences in liver functions, inducing and studying factors that regulate zonation in primary human liver cells is critical to elucidate similarities and differences with their animal counterparts for enabling a continuum of in vitro and in vivo investigations. However, in vitro models of human liver zonation are lacking, especially those that evaluate the long-term effects of factor gradients and stromal interactions on zonal functions in primary human hepatocytes. In order to address the aforementioned gap in knowledge, here we propose to investigate the effects of liver- relevant factor gradients and liver stromal cells on long-term functions of primary human hepatocytes in culture. We will utilize microfluidics to enable minimal use of limited primary human liver cells, and create independent, complex and overlapping gradients of soluble gradients onto multicellular liver cultures. We will assess the effects of gradients (O2, hormones) generated by our device on liver functions thought to be zonated in vivo, in the presence or absence of liver stromal cells. Results in vitro will be compared to mechanisms known in vivo. Ultimately, creating approaches and design rules for making a heterogeneous and stable population of human liver cells will allow recapitulation of this important liver feature in systems designed for drug screening, clinical use (i.e. bioartificial liver devices), and stem cell differentiation.