Centre for Biological Engineering, School of Mechanical, Electrical and Manufacturing Engineering, Loughborough University, Loughborough LE11 3TU, UK.
Guy Hilton Research Centre, School of Pharmacy and Bioengineering, Keele University, Staffordshire, Stoke-on-Trent ST4 7QB, UK.
Int J Mol Sci. 2020 Nov 13;21(22):8579. doi: 10.3390/ijms21228579.
Mesenchymal stem cells (MSCs) can differentiate into multiple different tissue lineages and have favourable immunogenic potential making them an attractive prospect for regenerative medicine. As an essential part of the manufacturing process, preservation of these cells whilst maintaining potential is of critical importance. An uncontrolled area of storage remains the rate of change of temperature during freezing and thawing. Controlled-rate freezers attempted to rectify this; however, the change of phase from liquid to solid introduces two extreme phenomena; a rapid rise and a rapid fall in temperature in addition to the intended cooling rate (normally -1 °C/min) as a part of the supercooling event in cryopreservation. Nucleation events are well known to initiate the freezing transition although their active use in the form of ice nucleation devices (IND) are in their infancy in cryopreservation. This study sought to better understand the effects of ice nucleation and its active instigation with the use of an IND in both a standard cryotube with MSCs in suspension and a high-throughput adhered MSC 96-well plate set-up. A potential threshold nucleation temperature for best recovery of dental pulp MSCs may occur around -10 °C and for larger volume cell storage, IND and fast thaw creates the most stable process. For adhered cells, an IND with a slow thaw enables greatest metabolic activity post-thaw. This demonstrates a necessity for a medical grade IND to be used in future regenerative medicine manufacturing with the parameters discussed in this study to create stable products for clinical cellular therapies.
间充质干细胞（MSCs）可分化为多种不同的组织谱系，具有良好的免疫原性，因此成为再生医学的有吸引力的前景。作为制造过程的重要组成部分，在保持潜能的同时保存这些细胞至关重要。储存过程中一个不受控制的区域是冷冻和解冻过程中温度变化的速率。控速冷冻机试图对此进行纠正；然而，从液态到固态的相变除了在冷冻保存的过冷事件中预期的冷却速率（通常为-1°C/min）之外，还会引入两个极端现象；温度的快速上升和快速下降。成核事件众所周知会引发冷冻转变，尽管在冷冻保存中，它们以冰核形成装置（IND）的形式被积极使用，但仍处于起步阶段。本研究旨在更好地了解成核及其在悬浮 MSC 的标准冷冻管和高通量贴壁 MSC 96 孔板设置中的主动引发的影响。牙髓 MSC 最佳回收的潜在临界成核温度可能在-10°C 左右，对于更大体积的细胞储存，IND 和快速解冻可创造最稳定的过程。对于贴壁细胞，具有缓慢解冻的 IND 可在解冻后实现最大的代谢活性。这表明，在未来的再生医学制造中，需要使用医疗级 IND，并采用本研究中讨论的参数来为临床细胞疗法创造稳定的产品。
