Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants.,Biomacromolecules

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Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants.
Biomacromolecules ( IF 5.5 ) Pub Date : 2020-06-05 , DOI: 10.1021/acs.biomac.0c00591
Kathryn A Murray ₁ , Matthew I Gibson _{1,

2}

Affiliation

The storage and transport of cells is a fundamental technology which underpins cell biology, biomaterials research, and emerging cell-based therapies. Inspired by antifreeze and ice-binding proteins in extremophiles, macromolecular (polymer) cryoprotectants are emerging as exciting biomaterials to enable the reduction and/or replacement of conventional cryoprotective agents such as DMSO. Here, we critically study post-thaw cellular outcomes upon addition of macromolecular cryoprotectants to provide unambiguous evidence that post-thaw culturing time and a mixture of assays are essential to claim a positive outcome. In particular, we observe that only measuring the viability of recovered cells gives false positives, even with non-cryoprotective polymers. Several systems gave apparently high viability but very low total cell recovery, which could be reported as a success but in practical applications would not be useful. Post-thaw culture time is also shown to be crucial to enable apoptosis to set in. Using this approach we demonstrate that polyampholytes (a rapidly emerging class of cryoprotectants) improve post-thaw outcomes across both measures, compared to poly(ethylene glycol), which can give false positives when only viability and short post-thaw time scales are considered. This work will help guide the discovery of new macromolecular cryoprotectants and ensure materials which only give positive results under limited outcomes can be quickly identified and removed.

中文翻译：

解冻后培养和总细胞回收率的测量对于评估新型大分子冷冻保护剂至关重要。

细胞的储存和运输是支撑细胞生物学、生物材料研究和新兴细胞疗法的基础技术。受极端微生物中的抗冻剂和冰结合蛋白的启发，大分子（聚合物）冷冻保护剂正在成为令人兴奋的生物材料，能够减少和/或替代传统冷冻保护剂（如 DMSO）。在这里，我们批判性地研究了添加大分子冷冻保护剂后的解冻后细胞结果，以提供明确的证据，证明解冻后培养时间和混合检测对于获得阳性结果至关重要。特别是，我们观察到，即使使用非冷冻保护聚合物，仅测量回收细胞的活力也会产生假阳性。一些系统的存活率明显很高，但总细胞回收率非常低，这可以被报告为成功，但在实际应用中没有用处。解冻后的培养时间也被证明对于细胞凋亡的启动至关重要。使用这种方法，我们证明，与聚乙二醇相比，聚两性电解质（一类快速新兴的冷冻保护剂）可改善解冻后的结果。当仅考虑活力和短解冻时间尺度时，这可能会给出误报。这项工作将有助于指导新的大分子冷冻保护剂的发现，并确保能够快速识别和去除仅在有限结果下产生积极结果的材料。

更新日期：2020-07-13

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