Freeze-drying of mammalian cells using trehalose: Preservation of DNA integrity

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

Storage stability of liposomes stored at elevated subzero temperatures in DMSO/sucrose mixtures.

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

Water fraction distributions in solutions composed of DMSO and sucrose, determined from the OH-stretching band in FTIR spectra.

<p>In panel A–B, the 3900–2700 cm⁻¹ spectral region is shown for two DMSO/sucrose solutions (S4: 10% v/v DMSO with 0.5 M sucrose, S2: 10% v/v DMSO with 1 M sucrose). Contributions of different water fractions were fitted, as Gaussian profiles centered initially at 3139 cm⁻¹ (fully hydrogen bonded water), 3241 cm⁻¹ (symmetrically hydrogen bonded water), 3389 cm⁻¹ (asymmetrically hydrogen bonded water) and 3533 cm⁻¹ (weakly hydrogen bonded water). The relative shifts in peak positions (C,D) and relative band areas (E,F) of these contributions were determined as a function of the DMSO concentration, in combination with either 0.5 M sucrose (C,E) or 1 M sucrose (D,F).</p

Glass transition temperatures of solutions composed of different contents of DMSO and sucrose.

<p>The onset temperature of glass transition was determined using DSC. This was done for 0–20% (v/v) DMSO solutions without supplements (white circles), as well as supplemented with 0.5 M sucrose (grey circles) or 1 M sucrose (black circles). For liposome storage experiments, performed at −80°C (dotted line), four different formulations were selected (labeled S1–4). Solution S1 was composed of 5% DMSO with 1 M sucrose (T_g: −68°C), S2 of 10% DMSO with 1 M sucrose (T_g: −77°C), S3 of 5% DMSO with 0.5 M sucrose (T_g: −84°C) and S4 of 10% DMSO with 0.5 M sucrose (T_g: −101°C).</p

Parameters describing molecular mobility in glasses were determined by fitting DSC data on enthalpy relaxation versus storage.

<p>This was done for solution S1 (5% v/v DMSO, 1 M sucrose, white circles), S2 (10% v/v DMSO, 1 M sucrose, black circles), S3 (5% v/v DMSO with 0.5 M sucrose, white triangles) and S4 (10% v/v DMSO with 0.5 M sucrose, black triangles). In panel A, for S1–4, the natural logarithm of the relaxation time is plotted versus the difference between the storage temperature and T_g. In the panel B, data are presented in Arrhenius plots for deriving activation energies. The dotted line indicates −80°C.</p

CF leakage rates of PC liposomes with trapped CF in DMSO/sucrose solutions at different subzero temperatures.

<p>The difference between the storage temperature and T_g was plotted against the CF leakage rate. Cryoprotective solutions that were tested: S1 (5% DMSO, 1 M sucrose, white circles), S2 (10% DMSO, 1 M sucrose, black circles), S3 (5% DMSO with 0.5 M sucrose, white triangles), and S4 (10% DMSO with 0.5 M sucrose, black triangles).</p

Enthalpy relaxation behavior of DMSO/sucrose solutions (S1–4) at different temperatures below the T_g of the solutions.

<p>DSC pans with solution were maintained ~0–15°C below T_g for up to 300 min, after which thermograms were recorded (A–D). Enthalpy relaxation is evident as an endothermic event (oriented upwards) on top of the glass transition event, increasing with storage duration while decreasing if further from T_g. The area of this event, ΔH_relaxation, was determined and plotted versus the storage duration (E–G), for the indicated storage temperatures. Solution S1 was composed of 5% v/v DMSO, 1 M sucrose (A,E), S2 of 10% v/v DMSO, 1 M sucrose (B,F), S3 of 5% DMSO, 0.5 M sucrose (C,G), and S4 of 10% v/v DMSO with 0.5 M sucrose D,H).</p

Storage stability of PC liposomes with trapped CF, frozen in DMSO/sucrose solutions and stored at different temperatures.

<p>Cryoprotective solutions tested were: S1 (5% DMSO, 1 M sucrose, white circles), S2 (10% DMSO, 1 M sucrose, black circles), S3 (5% DMSO with 0.5 M sucrose, white triangles), and S4 (10% DMSO with 0.5 M sucrose, black triangles) HEPES buffered solution without further supplements (grey squares) served as a control. Samples were stored at −150°C (A), −80°C (B) and −25°C (C) for up to 3 months. As a measure for storage stability, CF-retention (i.e. protection against membrane leakiness) was assessed and plotted versus the storage duration. The insets in the panels (A) and (B) show the CF-retention after 90 d at −150°C and −80°C, respectively (no significant differences in CF-retention were found among the formulations). Data points representing mean values ± standard deviations were calculated from four measurements (control samples were measured once).</p

‘In air’ cryopreservation of mesenchymal stromal cells on 3d collagen-hydroxyapatite scaffolds

Abstract: ‘In air’ cryopreservation of mesenchymal stromal cells on 3d collagen-hydroxyapatite scaffolds

Effect of ‘in air’ freezing on post-thaw recovery of <i>Callithrix jacchus</i> mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen ‘in air’ and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of ‘ready-to-use’ TECs