Effects Of L-carnitine On Water Freezing Point And Its Applications In Cell Cryopreservation

Cryopreservation is an essential and effective technology for the long-term storage of cells.At the cryogenic temperatures(-80? or-196?),cells can be cryopreserved and then be recovered at the physiological temperature after a proper thawing process.During the freeze-thaw process,cells could be damaged due to ice formation.To solve this problem,cryoprotectants have been developed to protect cells from cryoinjuries,and now they have become the key to successful cryopreservation.Organic solvents(e.g.,dimethyl sulfoxide,glycerol),saccharides(e.g.,glucose,trehalose)and polymers(polyethyleneglycol,dextran)have been commonly used as cryoprotectants in cell cryopreservation.However,organic solvents are intrinsically cytotoxic,while saccharides and polymers suffer from poor cell permeability results.These problems significantly limit their successful clinical applications.In this work,we presented that a natural zwitterionic molecule—L-carnitine—could serve as a promising cryoprotectant.We tested the thermodynamic parameters of the aqueous solutions of various cryoprotectants by the differential scanning calorimetry.Results showed that compared with current cryoprotectants,L-carnitine possessed the strongest ability to inhibit ice formation and bind water molecules,and the biggest osmotic coefficient.It is hypothesized that DMSO,glycerol and trehalose could inhibit ice crystallization through hydrogen binding,whereas zwitterionic Lcarnitine could inhibit ice formation by stronger electrostatically induced hydration.To explore the potential of L-carnitine as a cryoprotectant,we cryopreserved several different cell lines(GLC-82 cells,MCF-7 cells,NIH-3T3 cells and SRBCs)using L-carnitine as the sole cryoprotectant.L-carnitine was highly biocompatible and nontoxic,and could protect cells from solute injury during the freeze and thaw process.Most importantly,L-carnitine showed high cryopreservation efficiency with the ultrarapid freezing protocol,and the surface attachment and proliferation abilities of the post-thaw cells were similar to the fresh cells.These findings shed light on current cryopreservation technologies,and hold the potential to benefit the patients in various cell-based clinical applications.