Long-term Culture of Multicellular Spheroids Applicable to an Alternative to Animal Experiments.

我们首次报道了一种简单有效的方法来形成一个由一万个（100x100）肝细胞异质球体组成的二维微阵列。肝细胞异质球体被内皮细胞覆盖在20x20 mm的面积上，使用微制造的玻璃衬底，涂有聚乙二醇（PEG）刷。肝细胞与内皮细胞的共同培养对于稳定肝细胞活力和肝脏特异性功能至关重要，使我们能够获得直径为100 μm的肝细胞球体，其功能相当于小型化的肝脏，可以分泌白蛋白至少三周。这里构建的球形阵列作为组织和细胞生物传感器（TBB和CBB）的平台非常有用，可以通过细胞生理反应检测各种临床、药理学和毒理学活性化合物。该系统可以作为动物实验的替代方案，在各种情况下，依赖动物被证明是不切实际的。此外，通过将球体植入三维支架，从阵列中分离出的球体可能在构建组织工程肝脏方面具有潜在的用途。

We report here for the first time a simple and effective way to form a two-dimensional micro-array of ten thousand (100x100) hepatocyte hetero-spheroids. The hepatocyte hetero-spheroids were underlain with endothelial cells in an area of 20x20.mm, by using micro-fabricated glass substrates, coated with poly (ethylene glycol) (PEG) brushes. Co-cultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver specific function, allowing us to obtain hepatocyte spheroids with a diameter of 100-μm, functioning as a miniaturized liver to secret albumin for at least three weeks. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors (TBB and CBB), detecting a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response. This system can be used as an alternative to animal experiments in a variety of situations in which the reliance on animals would prove impractical. Furthermore, the isolated spheroids from the array may have potential utility in constructing a tissue engineered liver by seeding spheroids into three-dimensional scaffolds.