Morgan Katie, Bryans Anna, Brzeszczyński Filip, Samuel Kay, Treskes Philipp, Brzeszczyńska Joanna, Morley Steven D, Hayes Peter C, Gadegaard Nikolaj, Nelson Leonard J, Plevris John N
Hepatology Laboratory, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom.
Scottish National Blood Transfusion Service, Advanced Therapeutics, The Jack Copland Centre, Edinburgh, United Kingdom.
Tissue Eng Part A. 2020 Oct;26(19-20):1064-1076. doi: 10.1089/ten.TEA.2019.0241.
Fully differentiated HepaRG™ cells are the hepatic cell line of choice for study in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG-P) that differentiates into a coculture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethyl sulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG-Ps toward a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. It has been shown that tissue culture substrates play an important role in the development and maturity of a cell line, and this is particularly important for progenitor cells, which retain some form of plasticity. Oxygen plasma treatment is used extensively to modify cell culture substrates. There is also evidence that patterned rather than planar surfaces have a positive effect on proliferation and differentiation. In this study, we compared the effect of standard tissue culture plastic (TCP), oxygen plasma coated (OPC), and nanopatterned substrates (NPS) on early differentiation and function of HepaRG-P cells. Since NPS were OPC we initially compared the effect of TCP and OPC to enable comparison between all three culture surfaces using OPC as control to asses if patterning further enhanced early differentiation and functionality. The results show that HepaRG-P's grown on OPC substrate exhibited earlier differentiation, proliferation, and function compared with TCP. Culturing HepaRG-P's on OPC with the addition of NPS did not confer any additional advantage. In conclusion, OPC surface appeared to enhance hepatic differentiation and functionality and could replace traditional methods of differentiating HepaRG-P cells into fully differentiated and functional HepaRGs earlier than standard methods. Impact statement We show significantly earlier differentiation and function of HepaRG progenitor cells when grown in dimethyl sulfoxide-free medium on oxygen plasma substrates versus standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, although one pattern (DSQ120) showed comparable early differentiation and function.
完全分化的HepaRG™细胞是毒理学研究和药物试验中首选的肝细胞系。它们源自一种成肝细胞样祖细胞（HepaRG-P），该祖细胞可分化为肝细胞样细胞和胆管细胞样细胞的共培养物。这个过程需要2周的增殖，随后使用二甲基亚砜（DMSO）进行2周的分化，既耗时又昂贵。确定一种加速HepaRG-P细胞向成熟谱系分化的方法将节省时间和金钱。在不使用DMSO的情况下做到这一点，将消除DMSO诱导CYP途径导致毒理学结果混淆的可能性。已经表明，组织培养底物在细胞系的发育和成熟中起着重要作用，这对于保留某种形式可塑性的祖细胞尤为重要。氧等离子体处理被广泛用于修饰细胞培养底物。也有证据表明，有图案的表面而非平面表面对增殖和分化有积极影响。在本研究中，我们比较了标准组织培养塑料（TCP）、氧等离子体涂层（OPC）和纳米图案化底物（NPS）对HepaRG-P细胞早期分化和功能的影响。由于NPS是OPC，我们最初比较了TCP和OPC的效果，以便使用OPC作为对照来评估图案化是否进一步增强了早期分化和功能，从而能够在所有三种培养表面之间进行比较。结果表明，与TCP相比，在OPC底物上生长的HepaRG-P细胞表现出更早的分化、增殖和功能。在添加NPS的OPC上培养HepaRG-P细胞并没有带来任何额外的优势。总之，OPC表面似乎增强了肝脏分化和功能，并且可以比标准方法更早地将HepaRG-P细胞分化为完全分化且有功能的HepaRGs，从而取代传统方法。影响声明我们发现，与标准组织培养塑料相比，HepaRG祖细胞在无二甲基亚砜的培养基中于氧等离子体底物上生长时，其分化和功能明显更早出现。进一步研究表明，氧等离子体底物的纳米图案化相比光滑氧等离子体并没有带来任何额外优势，不过一种图案（DSQ120）显示出类似的早期分化和功能。
