Preservation Of Viable Cells Research Articles

Use of polyethyleneoxide and hydroxyethylstarch as blood plasma substitutes in the cryopreservation of testis interstitium cells in mice

Purpose of the study: to investigate the impact of hydroxyethyl starch (HES) and polyethylene oxide (PEO) on the indicators of preservation of murine testis interstitial cells (IC) under cryopreservation.Materials and methods. To isolate IC the enzymes were used: 0.2 mg/ml collagenase and 0.1 mg/ml DNase. The obtained cell suspension was cryopreserved in the solutions that contained 0; 0,7; 1,4; 2,1; 2,8 M of dimethyl sulfoxide (DMSO) and/or 10%, 20% fetal cow serum, 10 mg/ml PEO or HES. The samples (1 ml) were cooled at a rate of 1 °C/min to -80 °C then stored in liquid nitrogen (-196 °C). They were warmed at 37 °C in the water bath. Cryopreservation solution was removed. The number of cells and their preservation were assessed before and after with the assistance of Goryaev’s camera. Viability of IC, Leydig cell preservation and preservation of metabolic activity were measured with trypan blue dye, histochemical staining for 3β-hydroxysteroid dehydrogenase activity.Results. It was shown that 1,4 M DMSO without supplements favored IC preservation. Addition to the cryopreservation solution 10% and 20% of fetal cow serum or 10 mg/ml HES increased total preservation of IC by more than 10% and Leydig cell cryopreservation by an average 15%. HES 10 mg/ml may decrease DMSO concentration to 0,7 M. This combination had the best indicators of total preservation of IC, preservation of viable cells and Leydig cells: 75,8 (53,3; 93,3), 55,6 (45,1; 69,4), 57,1 (40,2;70,3) %, respectively. PEO was ineffective.Conclusion. High-molecular weight synthetic polymers such as HES can substitute protective properties of blood serum under cryopreservation and allow decreasing effective concentration of permeable cryoprotective such as DMSO.

INFLUENCE OF ULTRAVIOLET RADIATION ON THE VIABILITY OF VEGETATIVE CELLS AND CYSTS OF AZOTOBACTER CHROOCOCUM 2.1

Objective. Study the influence of ultraviolet radiation (UV radiation) on the preservation of cysts and vegetative cells of Azotobacter chroococcum 2.1 in the digest medium and seeds of cucumber, including for the use of the polysaccharide-protein complex.
 Methods. Microbiological, microscopic, physical, statistical.
 Results. Significant negative effect of UV radiation on the viability of A. chroococcum 2.1 was shown. Cysts of A. chroococcum 2.1 are more resistant to UV radiation than vegetative cells. The influence of UV radiation on the viability of azotobacter depends on the time during which the bacterial cells were irradiated. In the digest medium, the number of cysts of A. chroococcum 2.1 was higher than the vegetative cells by 88 % at the minimum exposure time. Under the action of UV radiation for 60 seconds, all vegetative cells in the medium died, while a small number of cysts remained. On bacterized seeds, due to the folded surface of the shell, viable cysts of azotobacter, unlike vegetative cells, was observed even for irradiation within 30 minutes. To reduce the negative effects of UV radiation, the polysaccharide-protein complex (PPC) has been used, ant it contributed in lengthening the viability of microorganism cells. The highest survival of bacterial cells was observed in the variant with A. chroococcum 2.1 in the form of cysts with the addition of PPC. Under such conditions, vegetative cells are detected only in the variant with the addition of PPC.
 Conclusion. UV radiation negatively affects the survival of A. chroococcum 2.1 both on the digest medium and on the seeds of the cucumber, while vegetative cells are more likely to die than cysts. To improve the survival of A. chroococcum 2.1 seeds in the form of cysts and vegetative cells, it is advisable to use the polysaccharide-protein complex. The obtained data are the basis for further research on the possibilities of preservation of viable cells of bacteria of the genus Azotobacter on the seed under the influence of unfavourable environmental factors.

Effect of storage temperature on cultured epidermal cell sheets stored in xenobiotic-free medium.

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic–free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.

Comment on the loss of immunogenicity of tumor cells caused by freezing

The freezing of cells to temperatures below about 1 3 0 ° C permits their extended storage in a state of "suspended animation" for decades if not longer. Such long-term storage of viable biological material offers potential benefits in a variety of research and clinical situations. The thesis advocated by Bartlett et al. (1977) is a good example. They suggest that the storage of human tumor tissue in the frozen state might permit its later use as an autologous vaccine in the immunotherapy of cancer. Unfortunately, the data of Bartlett et al., who used a model system of vaccines consisting of BCG and irradiated L10 hepatoma cells, seem to invalidate their own thesis. They reported that L10 cells that had been frozen and stored in liquid nitrogen (LN2) at --196 ° C possessed little or no capacity to prevent the progressive growth of challenge tumor cells, despite the fact that about 50% of the frozen-thawed cells excluded trypan blue. Bartlett et al. conclude that there was "clear evidence that tumor cells which were 'viable' after storage in LN 2 were significantly less immunogenic than cells used without prior freezing". A reasonable extension of that conclusion, if correct, would be that frozen-thawed cells are not suitable for immunotherapy. I believe there are reasons for suggesting that the conclusion of Bartlett et al., and its corollary, may be unduly pessimistic. Those authors have sought to use one of the major potential advantages of cryobiology, namely, the preservation of viable cells in the frozen state. I believe they have underestimated the present level of mechanistic understanding of low temperature biology, however. In this specific instance, this misreading of cryobiology might preclude its future application to a clinical situation in which freezing would be a requisite for the therapy.

Instrument for cutting animal tissues into small pieces (< 3 mm3) with preservation of viable cells

This instrument is designed to cut animal tissue into minute pieces (about 3 mm3) with the minimum of crushing of cells. Cutting is by a continuous scissor-like shearing action and the instrument is self-sharpening. A steel disk with a spiral groove cut in one face is rotated horizontally below a vertical rectangular steel plate. Cutting takes place when tissue in a specially shaped container is pressed by a sliding weight into the groove of the spiral and upon rotation is carried against the sharp toothed edge of the stationary rectangular vertical steel plate. The cut pieces in the spiral groove are removed by a gear wheel in continuous mesh, and fall into a catch tray. The size of the pieces cut is governed by three factors; (i) the width and depth of the spiral groove, (ii) the weight pressure and (iii) the spacing of the buttress-shaped teeth along the cutting plate edge.