Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.
Cryologyx Ltd, 71-75 Shelton Street, London, WC2H 9JQ, UK.
Biomater Sci. 2023 Nov 21;11(23):7639-7654. doi: 10.1039/d3bm01046e.
Cell culture plays a critical role in biomedical discovery and drug development. Primary hepatocytes and hepatocyte-derived cell lines are especially important cellular models for drug discovery and development. To enable high-throughput screening and ensure consistent cell phenotypes, there is a need for practical and efficient cryopreservation methods for hepatocyte-derived cell lines and primary hepatocytes in an assay-ready format. Cryopreservation of cells as adherent monolayers in 96-well plates presents unique challenges due to low volumes being susceptible to supercooling, leading to low recovery and well-to-well variation. Primary cell cryopreservation is also particularly challenging due to the loss of cell viability and function. In this study, we demonstrate the use of soluble ice nucleator materials (IN) to cryopreserve a hepatic-derived cell line (HepG2) and primary mouse hepatocytes, as adherent monolayers. HepG2 cell recovery was near 100% and ∼75% of primary hepatocytes were recovered 24 hours post-thaw compared to just 10% and 50% with standard 10% DMSO, respectively. Post-thaw assessment showed that cryopreserved HepG2 cells retain membrane integrity, metabolic activity, proliferative capacity and differentiated hepatic functions including urea secretion, cytochrome P450 levels and lipid droplet accumulation. Cryopreserved primary hepatocytes exhibited reduced hepatic functions compared to fresh hepatocytes, but functional levels were similar to commercial suspension-cryopreserved hepatocytes, with the added benefit of being stored in an assay-ready format. In addition, normal cuboidal morphology and minimal membrane damage were observed 24 hours post-thaw. Cryopreserved HepG2 and mouse hepatocytes treated with a panel of pharmaceutically active compounds produced near-identical dose-response curves and EC values compared to fresh hepatocytes, confirming the utility of cryopreserved bankable cells in drug metabolism and hepatotoxicity studies. Cryopreserved adherent HepG2 cells and primary hepatocytes in 96 well plates can significantly reduce the time and resource burden associated with routine cell culture and increases the efficiency and productivity of high-throughput drug screening assays.
细胞培养在生物医学发现和药物开发中起着至关重要的作用。原代肝细胞和肝细胞衍生的细胞系是药物发现和开发的特别重要的细胞模型。为了实现高通量筛选并确保细胞表型的一致性，需要在可进行测定的即用型格式下，对肝细胞衍生的细胞系和原代肝细胞进行实用且高效的冷冻保存方法。由于体积小，易发生过冷，导致回收率低且孔间差异大，因此以贴壁单层形式在 96 孔板中进行细胞冷冻保存会带来独特的挑战。由于细胞活力和功能丧失，原代细胞的冷冻保存也特别具有挑战性。在这项研究中，我们展示了使用可溶性冰核材料 (IN) 来冷冻保存肝衍生细胞系 (HepG2) 和原代小鼠肝细胞作为贴壁单层。与使用标准的 10％ DMSO 相比，HepG2 细胞的回收率接近 100％，解冻后 24 小时约有 75％的原代肝细胞被回收，而分别只有 10％和 50％。解冻后评估显示，冷冻保存的 HepG2 细胞保持膜完整性、代谢活性、增殖能力和分化的肝功能，包括尿素分泌、细胞色素 P450 水平和脂滴积累。与新鲜的肝细胞相比，冷冻保存的原代肝细胞的肝功能降低，但功能水平与商业悬浮冷冻保存的肝细胞相似，并且具有以可进行测定的即用型格式储存的额外优势。此外，解冻后 24 小时观察到正常的立方体形貌和最小的细胞膜损伤。用一组具有药理活性的化合物处理冷冻保存的 HepG2 和小鼠肝细胞，与新鲜的肝细胞相比，产生了几乎相同的剂量反应曲线和 EC 值，证实了可冷冻保存的细胞库在药物代谢和肝毒性研究中的实用性。96 孔板中冷冻保存的贴壁 HepG2 细胞和原代肝细胞可显著减少与常规细胞培养相关的时间和资源负担，并提高高通量药物筛选测定的效率和生产力。
