CryoLogyx Ltd, Venture Centre, University of Warwick Science Park, Coventry CV4 7EZ, U.K.
Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, U.K.
Biomacromolecules. 2024 Aug 12;25(8):5352-5358. doi: 10.1021/acs.biomac.4c00760. Epub 2024 Jul 25.
Plaque assays quantify the amount of active, replicating virus to study and detect infectious diseases by application of samples to monolayers of cultured cells. Due to the time taken in thawing, propagating, plating, counting, and then conducting the assay, the process can take over a week to gather data. Here, we introduce assay-ready cryopreserved Vero monolayers in multiwell plates, which can be used directly from the freezer with no cell culture to accelerate the process of plaque determination. Standard dimethyl sulfoxide cryopreservation resulted in just 25% recovery, but addition of polyampholytes (macromolecular cryoprotectants) increased post-thaw recovery and viability in 12- and 24-well plate formats. Variability between individual wells was reduced by chemically induced ice nucleation to prevent supercooling. Cryopreserved cells were used to determine influenza viral plaques in just 24 h, matching results from nonfrozen controls. This innovation may accelerate viral detection and quantification and facilitate automation by eliminating extensive cell culturing.
噬菌斑分析通过将样本应用于培养细胞的单层来定量测定活性、复制的病毒，从而研究和检测传染病。由于解冻、繁殖、接种、计数，然后进行测定所需的时间，该过程可能需要一周以上的时间才能收集数据。在这里，我们引入了即用型冷冻保存的 Vero 单层细胞多微孔板，可直接从冷冻室使用，无需细胞培养，从而加速噬菌斑测定的过程。标准二甲基亚砜冷冻保存仅导致 25%的回收率，但添加多两性电解质（大分子冷冻保护剂）可增加解冻后的回收率和 12 孔和 24 孔板格式的活力。通过化学诱导的冰核形成来减少各孔之间的变异性，以防止过冷。冷冻保存的细胞可在短短 24 小时内确定流感病毒噬菌斑，与未冷冻对照的结果相匹配。这一创新可能通过消除广泛的细胞培养来加速病毒检测和定量，并促进自动化。
