Monitoring of freezing patterns within 3D collagen-hydroxyapatite scaffolds using infrared thermography.

The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities. In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to -25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions ('in air' or 'in bulk CPA'). For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 ('scaffold in a bulk CPA') vs. 7.71 ± 0.43 ('bulk CPA', P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 ('scaffold in a bulk CPA') vs. 7.68 ± 0.34 ('bulk CPA', P < 0.0001)). At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen 'in air' were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 µg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively). Moreover, when frozen 'in air' in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend. The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus 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.

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars.

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 Me2SO) 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 Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-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% Me2SO/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 -80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions.

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.

Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging.

Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sperm when exposed to freezing. Trehalose uptake likely occurs during freezing-induced membrane phase transitions. Stallion sperm was freeze-dried in various formulations consisting of reducing or nonreducing sugars combined with albumin as bulking agent. Chromatin stability was studied during storage at 37°C, using the flow cytometric sperm chromatin structure assay and microscopic assessment of chromatin dispersion and DNA fragmentation after electrophoresis. Freeze-drying did not affect sperm chromatin, irrespective of the formulation that was used. DNA fragmentation index (DFI) values ranged from 5 to 8%. If sperm was freeze-dried without protectants or in a combination of glucose and proteins, DNA damage rapidly accumulated during storage at 37°C, reaching DFI values of respectively 95 ± 4 and 64 ± 42% after 1 month. DFI values of sperm freeze-dried with sucrose or trehalose ranged between 9-11% and 33-52% after 1 and 3 months storage, respectively. In conclusion, freeze-drying sperm with disaccharides results in uptake during freezing, which greatly reduces chromatin degradation during dried storage.

Spectroscopic monitoring of transport processes during loading of ovarian tissue with cryoprotective solutions.

There is an increasing demand for female fertility preservation. Cryopreservation of ovarian cortex tissue by means of vitrification can be done ad-hoc and for pre-pubertal individuals. Obtaining a homogeneous distribution of protective agents in tissues is one of the major hurdles for successful preservation. Therefore, to rationally design vitrification strategies for tissues, it is needed to determine permeation kinetics of cryoprotective agents; to ensure homogeneous distribution while minimizing exposure time and toxicity effects. In this study, Fourier transform infrared spectroscopy (FTIR) was used to monitor diffusion of different components into porcine ovarian cortex tissue. Water fluxes and permeation kinetics of dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG) were investigated. Diffusion coefficients derived from FTIR data, were corroborated with differential scanning calorimetry and osmometer measurements. FTIR allowed real-time spectral fingerprinting of tissue during loading with mixtures of protective agents, while discriminating between different components and water. Exposure to vitrification solutions was found to cause drastic initial weight losses, which could be correlated with spectral features. Use of heavy water allowed distinguishing water fluxes associated with dehydration and permeation, both of which were found to precede permeation of cryoprotective agents. Overall, DMSO and EG were found to permeate faster than GLY and PG. In mixtures, however, solutes behave differently. The non-invasive spectroscopic method described here to study permeation of vitrification solution components into ovarian tissue can be applied to many other types of engineered constructs, tissues, and possibly organs.

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.

Hydrogen Bonding Interactions and Enthalpy Relaxation in Sugar/Protein Glasses.

In this study, hydrogen bonding interactions and enthalpy relaxation phenomena of sugar and sugar/protein glasses have been studied using Fourier transform infrared spectroscopy and differential scanning calorimetry. The sugar OH band in Fourier transform infrared spectra was used to derive the glass transition temperature, Tg, and the wavenumber-temperature coefficient (WTC) of the OH band. A study on mixtures of sucrose and albumin revealed that the glass transition temperature and strength of hydrogen bonds increased with increasing percentages of albumin. WTCg and Tg derived from sucrose/albumin glasses showed a negative linear correlation. The Lu-Weiss equation was used to fit Tg data of sucrose/albumin mixtures. An inflection point was observed at a 1:1 mass ratio, which coincided with an inflection of the OH-stretching band denoting a change in hydrogen bonding interactions. Enthalpy relaxation, which is seen as an endothermic event superimposed on the glass transition in differential scanning calorimetry thermograms, increases with increasing storage temperature. Activation energies of enthalpy relaxation of sucrose and sucrose/albumin glasses were determined to be 332 and 236 kJ mol-1, respectively. Addition of albumin to sucrose increases the Tg, average strength of hydrogen bonding, heterogeneity, and the enthalpy relaxation time, making the glass more stable during storage at room temperature.

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.

Stallion Sperm Cryopreservation Using Various Permeating Agents: Interplay Between Concentration and Cooling Rate.

In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min-1. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cooling rates for cryopreservation. Sperm cryosurvival was experimentally determined through flow cytometric assessments on membrane intactness and using computer-assisted analysis of motility. Sperm could withstand exposure to 1500 mM concentrations prefreeze for all CPAs tested. The overall highest cryosurvival rates were obtained with DMF, followed by GLY and EG, whereas the use of PG and DMSO resulted in poor cryosurvival rates. Cryosurvival with DMF increased with increasing concentration, reaching a plateau at 500 mM, whereas for GLY and EG, an optimum concentration between 250 and 500 mM resulted in maximal survival. An optimal cooling rate was only observed at low CPA concentrations, whereas at higher concentrations, cryosurvival rates were not affected by the cooling rate. In the case of DMF, survival remained relatively high in the investigated range of concentrations and cooling rates, whereas with GLY and EG, a much narrower combination of CPA concentration and cooling rate resulted in optimal cryosurvival. Sperm cryopreserved with DMF showed altered motility characteristics indicating hyperactivation, which was not observed with GLY and EG. Optimal cooling rates that were predicted from calculated dehydration curves did not match experimentally determined optimal cooling rates.