Introduction

Primary cultured hepatocytes are an important model for early safety evaluations of newly developed drugs. Many factors, however, hinder the wider applications of this technology, especially the difficulty to maintain these cells in long-term culture. To date, creating a stable supply of human or animal hepatocytes with proper hepatic function in vitro has not been achieved. Furthermore, frequently harvesting hepatocytes from living donors for use in culture is highly invasive and simply not feasible. We have previously reported that canine bone marrow-derived mesenchymal stem cells (cBMSCs) can be effectively converted into induced hepatocyte-like cells (iHep cells); however, these cells had reduced function in comparison to mature hepatocytes. In recent studies, spheroid formation-based three-dimensional (3D) culture has been noted to greatly increase hepatocyte function; nevertheless, no reports have described the use of this technology for culturing canine hepatocytes. Therefore, in this study, we aimed to establish a 3D spheroid culture using converted canine iHep cells to investigate their function as hepatocytes.

Methods

The iHep cells were prepared by introducing two genes, namely, the Forkhead box A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α), into cBMSCs seeded onto an ultra-low attachment microplate to induce spheroid formation. Thereafter, the hepatic functions of these spheroids were evaluated using immunocytochemistry, as well as qualitative and quantitative PCR.

Results

Notably, albumin was observed in the iHep spheroids and the expression of hepatic genes, such as albumin and drug metabolism CYP genes, could also be detected. Another interesting finding was evident upon further comparing the quantified albumin gene and CYP2E1 gene expressions in the two-dimensional and three-dimensional culture systems; notably, a 100- to 200-fold increase in gene expression levels was observed in the three-dimensional spheroids when compared to those in conventional monolayers.

Conclusions

Upon incorporating three-dimensional technology, we managed to achieve iHep spheroids that are closer in gene expression to living liver tissue compared to conventional monolayer cultures. Thus, we are one step closer to creating a sustainable in vitro hepatocyte model. Furthermore, we believe that this system is capable of maintaining the stable drug metabolizing capacity of canine hepatocytes in vitro, which might be useful in improving current drug assessment studies.

中文翻译：

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骨髓间充质干细胞来源犬肝细胞样细胞的三维球体培养

介绍

原代培养的肝细胞是新开发药物早期安全性评价的重要模型。然而，许多因素阻碍了该技术的更广泛应用，尤其是难以在长期培养中维持这些细胞。迄今为止，在体外创造了具有适当肝功能的人或动物肝细胞的稳定供应尚未实现。此外，经常从活体供体中采集肝细胞用于培养是高度侵入性的，根本不可行。我们之前曾报道，犬骨髓来源的间充质干细胞（cBMSCs）可以有效地转化为诱导肝细胞样细胞（iHep细胞）；然而，与成熟的肝细胞相比，这些细胞的功能有所降低。在最近的研究中，已经注意到基于球体形成的三维 (3D) 培养可大大提高肝细胞功能。然而，没有报道描述使用该技术培养犬肝细胞。因此，在本研究中，我们旨在使用转化的犬 iHep 细胞建立 3D 球体培养物，以研究它们作为肝细胞的功能。

方法

iHep 细胞的制备方法是将两个基因，即 Forkhead box A1 ( Foxa1 ) 和肝细胞核因子 4 同源框 alpha ( Hnf4α )，引入到接种到超低附着微孔板上的 cBMSCs 中以诱导球体形成。此后，使用免疫细胞化学以及定性和定量 PCR 评估这些球体的肝功能。

结果

值得注意的是，在 iHep 球体中观察到白蛋白，并且还可以检测到肝基因的表达，例如白蛋白和药物代谢CYP基因。在进一步比较二维和三维培养系统中量化的白蛋白基因和 CYP2E1 基因表达后，另一个有趣的发现是显而易见的。值得注意的是，与传统单层相比，在三维球体中观察到基因表达水平增加了 100 到 200 倍。

结论

结合 3D 技术后，我们成功实现了与传统单层培养物相比，基因表达更接近活肝组织的 iHep 球体。因此，我们离创建可持续的体外肝细胞模型又近了一步。此外，我们相信该系统能够在体外维持犬肝细胞稳定的药物代谢能力，这可能有助于改进当前的药物评估研究。