Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.
Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.
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DOI：
10.1039/c5lc00237k
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发表时间：
2015-05-21
期刊：
影响因子：
6.1
通讯作者：
Shuler ML
中科院分区：
文献类型：
--
作者：
Esch MB;Prot JM;Wang YI;Miller P;Llamas-Vidales JR;Naughton BA;Applegate DR;Shuler ML
利用体外细胞培养模型更准确地预测药物性肝损伤对制药工业具有重要价值。为此，我们开发了一种低成本的肝细胞培养装置，其在3D原代肝细胞培养物上产生流体流动，所述3D原代肝细胞培养物由多种肝细胞类型组成，包括肝细胞和非实质细胞（成纤维细胞、星状细胞和枯否细胞）。我们测试了14天的流体流动下的细胞培养的性能，发现肝细胞产生的白蛋白和尿素的水平相比，静态培养物升高。当用P450诱导剂激发时，肝细胞也对P450（CYP 1A 1和CYP 3A 4）酶活性的诱导作出反应，尽管我们没有发现静态和流体培养物之间的显著差异。当用10 μM细菌脂蛋白（LPS）攻击时，非实质细胞也有类似的反应，产生白细胞介素8（IL-8）。为了以廉价的方式产生流体流动，我们使用了摇摆平台，该摇摆平台以±12°之间的角度倾斜细胞培养装置，导致周期性变化的流体静压降和双向流体流动（平均流速为650 μL/min，最大剪切应力为0.64达因/cm 2）。代谢活性的增加与以下假设一致：与单向流体流动类似，源自人组织的原代肝细胞培养物响应于双向流体流动而增加其代谢活性。由于双向流动极大地改变了剪切敏感的其他细胞类型的行为，双向流动增加原代肝细胞的代谢活性的发现也支持了这样的理论，即代谢活性的这种增加可能是由气体和代谢物交换水平的增加或可溶性生长因子的积累而不是剪切敏感引起的。我们的研究结果表明，双向重力驱动介质流的装置操作支持原代人肝细胞混合物的14天培养，具有增强代谢活性的益处。我们的设备操作模式使我们能够在流体细胞培养条件下以较低的设备制造和操作成本评估药物。
Predicting drug-induced liver injury with in vitro cell culture models more accurately would be of significant value to the pharmaceutical industry. To this end we have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop and bidirectional fluid flow (average flow rate of 650 μL/min, and a maximum shear stress of 0.64 dyne/cm2). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures derived from human tissues increase their metabolic activity in response to bidirectional fluidic flow. Since bidirectional flow drastically changes the behavior of other cells types that are shear sensitive, the finding that bidirectional flow increases the metabolic activity of primary liver cells also supports the theory that this increase in metabolic activity is likely caused by increased levels of gas and metabolite exchange or by the accumulation of soluble growth factors rather than by shear sensing. Our results indicate that device operation with bi-directional gravity-driven medium flow supports the 14-day culture of a mix of primary human liver cells with the benefits of enhanced metabolic activity. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.
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