Meier Florian, Freyer Nora, Brzeszczynska Joanna, Knöspel Fanny, Armstrong Lyle, Lako Majlinda, Greuel Selina, Damm Georg, Ludwig-Schwellinger Eva, Deschl Ulrich, Ross James A, Beilmann Mario, Zeilinger Katrin
Boehringer Ingelheim Pharma GmbH and Co.KG, Nonclinical Drug Safety Germany, D-88397 Biberach an der Riss, Germany.
Bioreactor Group, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, D-13353 Berlin, Germany.
Int J Mol Med. 2017 Dec;40(6):1759-1771. doi: 10.3892/ijmm.2017.3190. Epub 2017 Oct 16.
Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures.
人诱导多能干细胞（hiPSC）是一种很有前景的细胞来源，可从中分化出不同的体细胞类型用于体外研究或临床应用。现有的hiPSC向肝细胞分化的方案主要基于细胞的二维培养。在本研究中，作者使用两种不同的三维培养系统研究了hiPSC来源的肝细胞样细胞的生成：一种是无三维支架的微球培养系统，另一种是三维中空纤维灌注生物反应器。将这些三维系统中的分化结果与传统二维培养中的结果进行比较，并以原代人肝细胞作为对照。基于特定mRNA表达、蛋白质分泌、抗原表达和代谢活性进行评估。与二维分化系统相比，三维培养系统中甲胎蛋白的表达较低，而细胞色素P450 1A2或3A4的活性较高。与二维分化系统相比，在三维生物反应器中分化的细胞白蛋白和肝细胞核因子4α的表达增加，α-1-抗胰蛋白酶的分泌也增加，这表明其成熟度更高。相比之下，无三维支架的微球培养提供了一种简单且可靠的方法来生成特定大小的微球用于筛选应用，而生物反应器培养模型提供了一种在类似生理条件下进行复杂研究的工具。总之，本研究介绍了两种用于干细胞向肝细胞分化的三维培养系统，每种系统都展示了其在各自应用中的优势以及与二维培养相比的益处。
