Controlled Release of Cryoprotectants by Near-Infrared Irradiation for Improved Cell Cryopreservation

Abstract

Cryopreservation is essential to store living cells and tissues for future use while maintaining the proper levels of cell functions. The use of cryoprotective agents (CPAs) to inhibit intracellular ice formation during cryopreservation is vital for cell survival, but the addition and removal of CPAs and ice recrystallization during rewarming will cause fatal injury to cells. The conventional CPA loading and unloading methods generate osmotic shocks and cause mechanical injury to biological samples, and the conventional method of rewarming using a water bath also leads to ice recrystallization and devitrification. A new CPA-loaded microparticle-based method for loading and photothermal rewarming under near-infrared (NIR) laser irradiation was proposed to overcome these difficulties. We have successfully achieved the controlled release of CPAs (2 M EG, 2 M PG, and 0.5 M trehalose) with a graphene oxide (GO, 0.04% w/v) core from a 1.5% (w/v) sodium alginate shell to the human umbilical vein endothelial cells (HUVECs) within 60 s using NIR laser irradiation (808 nm Lasever at 5000 mW/cm 2 ) and successfully recovered the CPA-loaded cells with 0.04% (w/v) GO in 8−10 s using the same NIR irradiation. The results show that this method achieved 25% higher viability of HUVECs compared to the conventional method. In short, this study proposes a new approach for achieving controlled CPA loading to cells with a photothermal-induced strategy for cell cryopreservation.

Osmolytes as Cryoprotectants under Salt Stress

“…The potential features of these small zwitterionic organic molecules (osmolytes) as a cryoprotectant have shown their useful applications in dealing with the recent challenges in the cell-based biomedicines (cell transplantation and stem cell therapy) . Especially for cryopreservation of cells, the osmolytes have been found to be a promising biocompatible alternative to commonly used organic solvents which are highly toxic at physiological temperature. − Cold-responsive nanoparticles are being developed , for efficient intracellular delivery of cryopreserving small molecules such as trehalose. ,− …”

Principles and advances of cell cryopreservation

“…程或发现新的CPA渗透现象并探索其机制. 本团队基 于参数精确测定结果, 发现了单步法测定的渗透性参 数不能用于多步法优化 [55] ; 实现了对人脐静脉内皮细 胞CPA添加和去除过程的成功优化 [60] ; 与微流控技术 或表面声波技术结合实现了芯片上小样品细胞悬浮液 低温保护剂的添加和去除 [61,62] (图3B); 发现了受精失 败的卵母细胞与正常卵母细胞的渗透性差异, 为优质 卵母细胞的筛选提供了潜在新方法 [63] ; 结合近红外激 光光热控制释放方法, 实现了CPA的按需释放 [64] (图 3B). 上述研究积累已被应用到宏观体积细胞悬浮液 的CPA处理过程 [65] ; 相关实验系统也被进一步集成, 进而研制了膜式全自动细胞处理仪(图3C).…”

“…本团队针对低温保存全过程 传热传质分析所需关键热物理参数进行了较为系统的 图 3 低温保护剂处理方法、装置及系统. A: 细胞膜渗透性测定方法建立 [51,56] ; B: 小体积细胞悬浮液中CPA的微流控法添加 和去除 [61,62,64] ; C: 大体积细胞悬浮液中CPA的处理原理及仪器化 [65] . 图A修改自文献 [51], 已获得American Society of Mechanical Engineers版权许可; 引自文献 [56], 已获得Royal Society of Chemistry版权许可.…”

Droplet Generation, Vitrification, and Warming for Cell Cryopreservation: A Review

“…The survival rate of blastocysts vitrified on paper containers was not significantly different from that of those vitrified on EM grids, OPS or cryoloop. Cell encapsulation in hydrogels has been widely used in cell therapy, tissue engineering, regenerative medicine, reproductive medicine, and three-dimensional cell culture. − The encapsulation and direct immersion of cells in liquid nitrogen for vitrification and preservation can avoid direct contact between cells and liquid nitrogen. Meiser et al proposed a new vitrification-based method for encapsulating HIPSC.…”