Cell Size and Water Permeability as Determining Factors for Cell Viability after Freezing at Different Cooling Rates

Abstract

This work studied the viabilities of five types of cells (two yeast cells, Saccharomyces cerevisiae CBS 1171 and Candida utilis; two bacterial strains, Escherichia coli and Lactobacillus plantarum; and one human leukemia K562 cell) as a function of cooling rate during freezing. The range of investigated cooling rates extended from 5 to 30,000°C/min. Cell viability was classified into three ranges: (i) high viability for low cooling rates (5 to 180°C/min), which allow cell water outflow to occur completely and do not allow any intracellular crystallization; (ii) low viability for rapid cooling rates (180 to 5,000°C/min), which allow the heat flow to prevail over water outflow (in this case, cell water crystallization would occur as water was flowing out of the cell); (iii) high viability for very high cooling rates (>5,000°C/min), which allow the heat flow to be very rapid and induce intracellular crystallization and/or vitrification before any water outflow from the cell. Finally, an assumption relating cell death to the cell water crystallization as water is flowing out of the cell is made. In addition, this general cell behavior is different for each type of cell and seems to be moderated by the cell size, the water permeability properties, and the presence of a cell wall.The freeze-thawing process remains the principal method of cell preservation to date, and the high survival rates achieved by this method are of interest from both the biophysical and practical points of view. This is to ensure that the recovery of entire cell populations is free from the risk of possible subsequent alteration of its genetic composition.Cell cryopreservation, which is commonly used in the food and pharmaceutical industries, requires optimization for each type of microorganism. Moreover, each type of cell has its own protocol for freezing. Numerous researchers have attempted to develop methods that permit 100% preservation of freezethawing of diverse cellular specimens (3, 6), but some microorganisms cannot yet be preserved by freezing.For a better cell preservation some cryoprotectants such as glycerol or dimethyl sulfoxide can be used (8). These molecules improve the cell preservation by minimizing the cell water content (6) and/or supporting the vitrification occurrence (1) and finally by protecting the cell's constitutive macromolecules (2, 5).The freeze-thawing process constitutes a double stress for the cell, i.e., thermal and hyperosmotic stresses, which act simultaneously during cooling (15, 16). The scenario of cell evolution during slow freezing is well known (13). The water surrounding the cell freezes before the cell contents, because the cytoplasm is more concentrated than the growth medium, and because thermodynamically, the component with the largest volume will nucleate first (6, 16). This freezing increases the osmotic pressure of the medium, and the extracellular solutes become concentrated in the remaining liquid extracellular water. Consecutive osmosis will then dehydrate cells as water diffuses from the cy...

Aquaporin-Mediated Improvement of Freeze Tolerance of Saccharomyces cerevisiae Is Restricted to Rapid Freezing Conditions

“…Our results for the effect of different freezing rates on yeast survival in the range of cooling rates that we studied are consistent with the results of other workers (7,8,23,24). The levels of survival (expressed as RGC) generally remained high at cooling rates of 10 to 15°C per min but decreased dramatically at cooling rates of 30 to 35°C per min or higher (Table 1 and Fig.…”

Conservação de grãos de pólen de mamoneira a baixas temperaturas

“…Esta maior eficiência na conservação a ultrabaixas temperaturas pode estar relacionada com a maior velocidade de congelamento, que causa menores danos à célula, por menor desidratação e menos rompimento de membranas (Dumont et al, 2004;Fellows, 2006). Segundo Almeida et al (2002), a técnica da crioconservação, isto é, o armazenamento a -196 ºC, proporciona potencial para uma preservação sem limites de tempo, com a redução do metabolismo a níveis tão baixos que todos os processos bioquímicos são significativamente reduzidos e a deterioração biológica é virtualmente paralisada.…”

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

“…Both penetrate the cell wall and plasmatic membrane, avoiding the intracellular ice formation and altering the plasmatic membrane elasticity, allowing a better accommodation of the cellular volume expansion during water freezing [15]. However, DMSO and GO can also affect the cellular metabolism due to their toxicity [6].…”

“…Besides the loss of cellular structure, slow freezing of external water causes cell dehydration and increase of cytoplasmic ion concentration. This process reduces the freezing temperature of the cytoplasm turning it in a highly viscous concentration mixture with low metabolic activity but with irreversible damages to cell and/or cell proteins [15].…”

“…Cryoprotective agents: 1 % dimethyl sulfoxide (DMSO); 5 % glycerol (GO); 10 % saccharose (SA); 4 % glucose (GU); 6 % polyethylene glycol-6000 (PEG) and 5 % malt extract (ME) GO 56 100 100 100 100 100 0 93 100 87 100 95 DMSO 93 100 100 100 93 98 54 87 100 75 80 86 GU 18 100 100 100 100 100 0 100 93 93 100 96 SA 83 100 100 100 100 100 40 100 100 100 100 100 ME 23 100 100 100 100 100 0 100 87 93 93 93 PEG 0 100 100 100 100 100 0 100 80 80 73 83 Cryoprotectant average 46 100 100 100 99 -16 97 93 88 91 -Cultivation medium and substrates with mycelium: potato dextrose agar disks (PDA); whole oat grains (OG); whole wheat grains (WG), whole rice grains without hulls, known as brown rice (RG) and whole millet grains (MG). Cryoprotective agents: 1 % dimethyl sulfoxide (DMSO); 5 % glycerol (GO); 10 % saccharose (SA); 4 % glucose (GU); 6 % polyethylene glycol-6000 (PEG) and 5 % malt extract (ME) linked intracellular water reduces the differences between intra and intercellular freezing temperatures [15] and controls the cellular dehydration at slow freezing temperatures. Another possibility is that the wheat grain structure is composed of a capillary net [16], which provides physical protection to mycelium.…”