The aim of this study was to investigate preservation of biomolecular structures, particularly DNA, in freeze-dried fibroblasts, after loading with trehalose via freezing-induced uptake. Cells were freeze-dried with trehalose alone or in a mixture of albumin and trehalose. Albumin was added to increase the glass transition temperature and storage stability. No viable cells were recovered after freeze-drying and rehydration. FTIR studies showed that membrane phase behavior of freeze-dried cells resembles that of fresh cells. However, one day after rehydration membrane phase separation was observed, irrespective of the presence or absence of trehalose during freeze-drying. Freeze-drying did not affect the overall protein secondary structure. Analysis of DNA damage via single cell gel electrophoresis (‘comet assay’) showed that DNA damage progressively increased with storage duration and temperature. DNA damage was prevented during storage at 4 °C. It is shown that trehalose reduces DNA damage during storage, whereas addition of albumin did not seem to have an additional protective effect on storage stability (i.e. DNA integrity) despite the fact that albumin increased the glass transition temperature. Taken together, DNA in freeze-dried somatic cells can be preserved using trehalose as protectant and storage at or below 4 °C.
Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6M concentrations compared to only 1M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.
Cryopreservation of biological materials is predominantly done using liquid nitrogen, and its application involves high maintenance costs and the need for periodical refilling of liquid nitrogen. Stable storage in mechanical freezers at −80°C would eliminate these issues and allow for shipment of frozen specimens using dry ice. In this work, the possibility of increasing the storage temperature of cryopreserved samples to −80°C by using combinations of DMSO and sucrose has been studied. Preservation efficacy was studied by measuring stability of liposomes encapsulated with carboxyfluorescein during storage at −150, −80 and −25°C for up to three months. Thermal and molecular mobility properties of the different DMSO-sucrose formulations were measured using differential scanning calorimetry, whereas hydrogen bonding interactions of the formulations were probed by Fourier transform infrared spectroscopy. It was found that addition of sucrose to DMSO solutions increases the Tg, and decreases molecular mobility in the glassy state at a particular temperature. Although it was expected that storage above or close to Tg at −80°C would affect liposome stability, stability was found to be similar compared to that of samples stored at −150°C. Higher molecular mobility in the glassy state could not be associated with faster CF-leakage rates. Distinct differences in storage stability at −25°C, far above Tg, were found among the sucrose/DMSO formulations, which were explained by the differences in permeability of sucrose and DMSO resulting in different levels of osmotic stress in the formulations.
Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me 2 SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me 2 SO-and serum-free biopreservation strategies due to safety concerns over Me 2 SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me 2 SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at À 80 � C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me 2 SO-and serumfree cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.
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