Cold-Responsive Nanoparticle Enables Intracellular Delivery and Rapid Release of Trehalose for Organic-Solvent-Free Cryopreservation

Abstract

Conventional cryopreservation of mammalian cells requires the use of toxic organic solvents (e.g., dimethyl sulfoxide) as cryoprotectants. Consequently, the cryopreserved cells must undergo a tedious washing procedure to remove the organic solvents for their further applications in cell-based medicine, and many of the precious cells may be lost or killed during the procedure. Trehalose has been explored as a nontoxic alternative to traditional cryoprotectants. However, mammalian cells do not synthesize trehalose or express trehalose transporters in their membranes, and the lack of an approach for the efficient intracellular delivery of trehalose has been a major hurdle for its use in cell cryopreservation. In this study, a cold-responsive polymer (poly(Nisopropylacrylamide-co-butyl acrylate)) is utilized to synthesize nanoparticles for the encapsulation and intracellular delivery of trehalose. The trehalose-laden nanoparticles can be efficiently taken up by mammalian cells. The nanoparticles quickly and irreversibly disassemble upon cold treatment, enabling the controlled and rapid release of trehalose from the nanoparticles inside cells. The latter is confirmed by an evident increase in cell volume upon cold treatment. This rapid cold-triggered intracellular release of trehalose is crucial to developing a fast protocol to cryopreserve cells using trehalose. Cells with intracellular trehalose delivered using the nanoparticles show comparable postcryopreservation viability compared to that of cells treated with DMSO, eliminating the need for the tedious and cell-damaging washing procedure required for using the DMSO-cryopreserved cells in vivo. This cold-responsive nanoparticle may greatly facilitate the use of trehalose as a nontoxic cryoprotectant for banking cells and tissues to meet their high demand by modern cell-based medicine.

Cryopreservation of Agronomic Plant Germplasm Using Vitrification-Based Methods: An Overview of Selected Case Studies

“…Gold and zinc nanoparticles (NPs) are being studied as promising structures that can help protect animal [ 89 ] and human cells [ 90 , 91 ] during cryopreservation due to their small size and high thermal conductivity [ 92 ]. Nanoparticles can transport heat flux and cause thermal equilibrium with the environment within 10–200 ps [ 93 ].…”

Sand-mediated ice seeding enables serum-free low-cryoprotectant cryopreservation of human induced pluripotent stem cells

“…Cryopreservation of hiPSCs is notably more difficult than cryopreservation of human adult stem cells (e.g., tissue-derived stem cells). Adult stem cells can be cryopreserved as single cells by traditional slow-freezing protocols with post-thaw viability up to 90% [ 52 , 53 ]. However, hiPSCs are more sensitive to stresses during cell cryopreservation, as hiPSCs grow in colonies with cell-cell and cell-matrix interactions and may undergo anoikis-induced apoptosis when dissociated into single cells.…”

“…Of note, various methods have been explored to improve cryopreservation outcome including microencapsulation of cells in alginate hydrogel [ 23 , 60 ], nano-warming with magnetic nanoparticles [ [61] , [62] , [63] , [64] ], supplement of nontoxic CPAs like sugars into the cryopreservation medium [ [65] , [66] , [67] , [68] ], and intracellular delivery of the sugars using cold-responsive nanoparticles [ 53 ]. All these methods may be combined with our sand-mediated ice seeding, which has the potential to further reduce or even eliminate the DMSO needed for cryopreservation of the hiPSCs with high functional survival.…”

Advances in cell cryopreservation take a cue from nature

“…He and his colleagues created nanoparticles that can act as delivery vesicles to efficiently transport trehalose sugar into mammalian cells. 2 Once subjected to cold temperatures, the nanoparticles disassemble and release the trehalose inside cells. Importantly, Dr.…”