Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

Vitrification as a prospect for cryopreservation of tissue-engineered constructs

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation

Vitrification, relaxation and free volume in glycerol-water binary liquid mixture:Spin probe ESR studies

Glass transition and relaxation of the glycerol-water (G-W) binary mixture system have been studied over the glycerol concentration range of 5-85 mol% by using the highly sensitive technique of electron spin resonance (ESR). For the water rich mixture the glass transition,sensed by the dissolved spin probe, arises from the vitrified mesoscopic portion of the binary system. The concentration dependence of the glass transition temperature manifests a closely related molecular level cooperativity in the system. A drastic change in the mesoscopic structure of the system at the critical concentration of 40 mol is confirmed by an estimation of the spin probe effective volume in a temperature range where the tracer reorientation is strongly coupled to the system dynamics.

Role of molecular reorientation in the vitrification of molecular liquids: A Monte Carlo study of the liquid and glassy states of 2,2-dimethylbutane

The liquid and the glassy phases of 2,2-dimethylbutane have been investigated by isothermal isobaric ensemble Monte Carlo simulation. Thermodynamic Properties and radial distribution functions for both the liquid and the glass have been obtained. The radial distribution functions have been classified into three types based on the accessibility of the group. It has been shown that the structure of the Iiquid and the glass can be understood in terms of the above classification of the radial distribution functions. Molecular reorientation plays an important role in the structural rearrangement accompanying glass formation. As much as 35% of the contribution to the increase in the intermolecular interaction energy on vitrification is due to the reorientation of the neighbouring pairs of molecules. The observed changes in the dimerisation energy and the bonding energy distribution function are consistent with the observed structural changes.

Importance of orientational rearrangement during vitrification of hydrocarbons: dependence on molecular shape

On the basis of Monte Carlo calculations of 2,2-dimethylpropane (neopentane), n-pentane, and 2,2-dimethylbutane (neohexane) at several temperatures, thermodynamic properties and radial distribution functions as well as dimerization and bonding energy distribution functions are reported for both liquid and glassy states. Changes in the radial distribution functions on cooling depend on whether the groups are accessible (peripheral) or inaccessible. Peaks in the radial distribution functions corresponding to peripheral groups do not shift to lower distances on cooling and at times display a large increase in the intensity of the first peak. The peaks due to inaccessible groups, on the other hand, shift to lower distances on cooling. The magnitude of the reorientational contribution in determining the resulting structure of the glass is estimated for the different hydrocarbon molecules investigated. The reorientational contribution is highest for neopentane (26%) followed by isopentane (24%), neohexane (22%), and n-pentane (0%). It appears that molecular geometry has an important role in determining the magnitude of the reorientational contribution to the structure of the glass.

On mechanical properties of metallic glass and its liquid vitrification characteristics

A systematic survey of the available data such as elastic constants, density, molar mass, and glass transition temperature of 45 metallic glasses is conducted. It is found that a critical strain controlling the onset of plastic deformation is material-independent. However, the correlation between elastic constants of solid glass and vitrification characteristics of its liquid does not follow a simple linear relation, and a characteristic volume, viz. molar volume, maybe relating to the characteristic size of a shear transformation zone (STZ), should be involved.

The formation mechanism of low leaching glassy slag during plasma arc vitrification treatment of fly ash

In this paper, the glass formation theory is applied to study the formation mechanism of the low leaching glassy slag during the process of plasma waste treatment. The research shows that SiO2 acts as network former to form a 3-dimensional Si-O tetrahedral network in which heavy metals are bonded or encapsulated, so the Si-O tetrahedron protect heavy metals against leaching from the vitrified slag or acid corrosion. For given chemical compositions of waste, the formation ability of the vitrified slag can be represented by the ratio of the whole oxygen ions to the whole network former ions in glass (O/Si) which is appropriate in the range of 2~3. A plasma arc reactor is used to conduct the vitrification experiments of two kinds of fly ashes with additives in which effects of various parameters including arc power, cooling speed, treatment temperature are studied. The chemical compositions of fly ashes are analyzed by X-ray fluorescence (XRF) spectrometry. The experimental results show that both cooling speed and O/Si have important influence on the formation of the vitrified slag, which is qualitatively in accordance with the predictions of the glass formation theory.

Cryopreservation of Kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols

The cryopreservation of oocytes has been only marginally successful with any of the current protocols, including slow cooling, rapid cooling and vitrification. We wished to test the hypothesis that oocytes from a single mouse strain would freeze successfully by 1 of the 3 mentioned protocols. Unfertilized Kunming mouse oocytes obtained 14 h after PMSG/hCG administration were randomly assigned to be cryopreserved after slow cooling, ultra rapid cooling and vitrification. Oocytes were thawed by straws being placed into 37 degrees C water, and their morphological appearance and in vitro fertilization capability were compared with that of oocytes that had not undergone cryopreservation. Survival of oocytes was indicated by the absence of darkened ooplasm or by broken membranes or zona pellucida. Functional integrity was evaluated by the formation of a 2-cell embryo after IVF. Survival rate of slow cooled oocytes did not differ from that seen in vitrified oocytes (55.1 vs 65.9%) but was significantly lower in the rapidly cooled oocytes (24.2%; P<0.01). The results of NF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P>0.05). It appears that Kunming mouse oocytes can be successfully cryopreserved using the slow cooling method with 1,2-propanediol and vitrification, which contains both permeating and nonpermeating cryoprotectants. (C) 1997 by Elsevier Science Inc.

Vitrification of Yunnan Yellow Cattle oocytes: work in progress

The objective of this study was to provide a simple cryopreservation method for oocytes from Yunnan Yellow Cattle and facilitate preservation efforts in this native Chinese breed, which is threatened by agricultural modernization. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 22-24 h, then selected for cryopreservation. Vitrification in open pulled straws (OPS) or in microdrops on a cooled metal surface (solid surface vitrification, SSV) was compared. The OPS vitrification solution consisted of 20% ethylene glycol (EG) and 20% DMSO. The SSV solution was a mixture of 35% EG, 5% polyvinyl-pyrrolidon (PVP) and 0.4 M trehalose. Vitrified and warmed oocytes were either fertilized in vitro or parthenogenetically activated. The rates of cleavage and development to blastocysts of fertilized oocytes following OPS versus SSV were not statistically different (38.3 and 12.5% versus 35.8 and 6.0%, respectively). The corresponding rates of parthenogenetic development to blastocysts were also not different (8.2 versus 3.5%, respectively). Development to blastocysts of non-vitrified controls following fertilization was significantly higher than that of the vitrified oocytes (22.6%, P < 0.05). These results demonstrate for the first time, that although both OPS and SSV procedures reduced embryonic development, Yunnan Yellow Cattle oocytes are capable of developing to blastocysts following cryopreservation. (C) 2002 Elsevier Science Inc. All rights reserved.

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO + 10% PG + 10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6 +/- 16.7% (mean +/- S.D.) and 77.8 +/- 15.5%, were achieved by the straw vitrifying method (20.5% DMSO + 15.5% acetamide + 10% PG, thawing at 43 degrees C and washing in 0.5 M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5 M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation. (C) 2007 Elsevier Inc. All rights reserved.

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification

Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5%) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.

Recent insights on the role of cryoprotective agents in vitrification

In recent efforts to produce cryoprotective solutions which cause either complete, or almost complete, vitrification of the cell or tissue material, increasingly complex cocktails of solutes have invariably been used. Why some of these solutes are so much more effective in suppressing ice formation than other, related solutes has never been clear. To begin to compare and contrast the role of the solute in aiding vitrification we have examined the nature of the hydrogen bonding interactions between the solute and water and between the solute molecules themselves, via proton nuclear magnetic resonance experiments. These experiments, carried out on neat samples of the solutions, show marked differences between solutes such as ethylene glycol, 1,2-propanediol, 1,3-propanediol, the family of butanediol isomers, dimethylsulfoxide, and so on, at fixed concentration. The solutions also show marked trends in the NMR chemical shift as a function of concentration in any given solution. Thus it appears that, from the point of view of the physical suppression of ice in aqueous solutions, cryoprotective agents which can act as moderately strong bases are optimum. The mechanism by which the solute promotes glass formation was also investigated in a separate series of NMR experiments using more dilute solutions of the solute in water. These experiments indicate that the role of the solute is twofold in that it must (i) effectively suppress the anomalous structuring which occurs in supercooled water and is responsible for the rapid nucleation of ice and (ii) provide a decrease in molecular mobility at low temperatures such that the nucleation probability is decreased and glass formation occurs at a relatively high temperature. It is argued that both such effects can be brought about by the strong hydrogen bonding interactions between water and solutes such as 2,3-butanediol.