Cryoprotective mechanism

The cryopreservation technique has been established for some cells7 As a result, these cryopreserved cells have been used in many studies. However, in the case of neurons, the appropriate cryopreservation process has not yet been established because of their complex structure, low mitotic activity, and uncertainties regarding the cryoprotection mechanism. Thus, primary culture cells are used for studying neurons. 

The difference in the recovery of cortical neurons in the presence of these additives might be a result of the cryopreservation process that is explained as follows DMSO is taken up into the cell and allows vitrification of the intracellular solution, leading to the stabilization of the cell membrane. However, in the case of trehalose, the molecules may be too large to permeate into the cell they may only contact the membrane extracellularly and protect the cell from freezing damage. To clarify the details of this cryoprotection mechanism, we need to study the process of cell freezing. 

Even though osmotic treatments have been proven to be a useful tool in fruit and vegetable cryoprotection, the changes in mechanical properties, caused by the process itself, have to be taken into account. 

Chiralt, A., Martinez-Navarrete, N., Martinez-Monzo, J., Talens, P., Morata, G., Ayala, A., and Fito, P. 2001b. Changes in mechanical properties throughout osmotic processes Cryoprotectant effect. J. Food Engineer. 49, 129-135. 

The presence of a covalent acyl-enzyme intermediate in the catalytic reaction of the serine proteases made this class of enzymes an attractive candidate for the initial attempt at using subzero temperatures to study an enzymatic mechanism. Elastase was chosen because it is easy to crystallize, diffracts to high resolution, has an active site which is accessible to small molecules diffusing through the crystal lattice, and is stable in high concentrations of cryoprotective solvents. The strategy used in the elastase experiment was to first determine in solution the exact conditions of temperature, organic solvent, and proton activity needed to stabilize an acyl-enzyme intermediate for sufficient time for X-ray data collection, and then to prepare the complex in the preformed, cooled crystal. Solution studies were carried out in the laboratory of Professor A. L. Fink, and were summarized in Section II,A,3. Briefly, it was shown that the chromophoric substrate -carbobenzoxy-L-alanyl-/>-nitrophenyl ester would react with elastase in both solution and in crystals in 70 30 methanol-water at pH 5.2 to form a productive covalent complex. These... 

Thus, the results of present research testify to the fact that candidates to stem hemopoietic cells, obtained from two different sources human fetal liver and cord blood, demonstrate quite a high viability after cryopreservation with various methods using cryoprotectants of different effect mechanisms. Revealed differences in cryosensitivity of CD 34 cells in respect of CD 45 cells may be explained by various ratios of cells, expressing these antigens. So, in fetal liver among nucleated CD 45 - cells about 35% express CD 34 ... 

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

The mechanisms of denaturation during frozen storage and of the cryoprotective effects have been discussed and a hypothetical model has been presented. 

Cryo- and Lyoprotectants and Bulking Agents Various mechanisms are proposed to explain why excipients serve as cryo- or lyoprotectants. The most widely accepted mechanism to explain the action of cryoprotection is the preferential exclusion mechanism [177]. Excipients that will stabilize proteins against the effects of freezing do so by not associating with the surface of the protein. Such excipients actually increase the surface tension of water and induce preferential hydration of the protein. Examples of solutes that serve as cryoprotectants by this mechanism include amino acids, polyols, sugars, and polyethylene glycol. 

Numerous compounds can provide general cryoprotection to proteins, when used at concentrations of several hundred millimolar. These include sugars, polyols, amino acids, methylamines, and salting-out salts (e.g., ammonium sulfate) [59,61,68-70]. Based on the results of freeze-thawing experiments with LDH and PFK and a review of the literature on protein freezing. Carpenter and Crowe [59] have proposed that this cryoprotection can be explained by the same universal mechanism that Timasheff and Arakawa have defined for solute-induced protein stabilization in nonfrozen, aqueous solution (reviewed in [4,70,78,79]). 

This argument does indeed support the contention that on a per-mole basis PEG is much more effective than sucrose at increasing protein chemical potential. And for cases where relatively high concentrations of PEG (e.g., >1% wt/vol) are needed to confer cryoprotection, the Timasheff mechanism may be applicable. However, it seems unlikely that a PEG concentration of 0.01% (wt/vol) would have a significant effect on the thermodynamics of the system. This is because the actual parameter of interest is the transfer free energy of the native versus denatured protein from water into cryoprotectant solution. The difference between the values for the two states determines the magnitude of the effect on the free energy... 

Cryoprotectants are added to the culture medium in order to protect cells. Glycerine, the first cryoprotectant to be used, was discovered in the 1940s, and it was used for the cryopreservation of bovine sperm. More recently, it has been discovered that other substances such as dimethyl sulfoxide (DMSO), saccharides," and proteins also function as cryoprotectants. However, the mechanism of the cryopreservation process in each case remains unclear. In the present study, we compared DMSO and trehalose, which are cryoprotectants of different molecular sizes that may differ in their ability to pass through the cell membrane. 

The H-bond networks of hquid water and ice are very dense and extend over a very large region. All water molecules are part of these networks and nearly all of them are tetra-coordinated. This is not so in macromolecules where many different situations can be encountered, following the density of hydrophilic groups this macromolecule exhibits and also the state of hydration of the macromolecule, that is the number of H2O molecules that are embedded in it. Some recent experiments conveyed information on the structures of the H-bond networks in these systems. They are experiments on hydration mechanisms, lyo or cryoprotection, and folding of proteins. These relatively few mechanisms that have been recently studied in some detail are described in this section. They give what could be called a preview on this H-bond network developed by H2O molecules in macromolecules. 

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