The effect of cryopreservation on the cytogenetic characteristics of a cell subline of skin fibroblasts from the Indian muntjac

After thawing cells, previously cryopreserved in the presence of dimethyl sulfoxide (DMSO), a decrease in their viability and increase in unscheduled DNA synthesis was observed. In 7 days, these parameters restored to the control level. Cryopreservation without DMSO resulted in the decrease in both cell viability and replicative and unscheduled DNA synthesis. In 14 days, these characteristics were seen to return to the normal level. Cryopreservation of cells without DMSO and their preservation in liquid nitrogen induced the frequency of chromosomal aberrations, mostly chromosomal breaks. The frequency of chromosomal aberrations increased with the duration of cell preservation in liquid nitrogen. The normal level was achieved following 7 days after cell thawing. Cells treated with DMSO only (without cryopreservation) display an increased number of chromosomal and chromatid breaks and translocations. Nonrandom distribution of chromosomal aberrations was observed, with particular chromosomes being involved in the appearance of dicentrics and translocations. The data obtained indicate that cryoprotective activity of DMSO is probably associated with the cell repair systems. The detected antimutagenic and mutagenic activity of DMSO may presumably reflect various conditions for its interaction with cells (with or without cryopreservation), as well as it may be specific for the muntjac cell line used in the present work.     

Effects of pulsed electric fields on inactivation and metabolic activity of Lactobacillus plantarum in model beer

AIMS: Inactivation and sublethal injury of Lactobacillus plantarum at different pulsed electric field (PEF) strengths and total energy inputs were investigated to differentiate reversible and irreversible impacts on cell functionality. METHODS AND RESULTS: Lactobacillus plantarum was treated with PEF in model beer (MB) to determine critical values of field strength and energy input for cell inactivation. Below critical values, metabolic activity and membrane integrity were initially reduced without loss of viability. Above critical values, however, irreversible cell damage occurred. Presence of nisin or hop extract, during PEF treatment, resulted in an additional reduction of cell viability by 1;5 log cycles. Also, addition of the hop extract resulted in an additional two log cycles of sublethal injury. Partial reversibility of membrane damage was observed using propidium iodide (PI) uptake and staining. Inoculated MB containing hops was stored after PEF to evaluate the efficacy of such treatment for beer preservation. CONCLUSION: Cells were inactivated only above critical values of 13 kV x cm(-1) and 64 kJ x kg(-1); below these values cell damage was reversible. Storage experiments revealed that surviving cells were killed after 15 h storage in MB containing hops. SIGNIFICANCE AND IMPACT OF THE STUDY: Both reversible and irreversible cell damage due to PEF treatment was detected, depending on specific treatment conditions. The combination of PEF and hop addition is a promising nonthermal method of preservation for beer.     

Study of the optimal conditions of preservation of cephalosporin C-producing fungus Acremonium chrysogenum

The influence of various preservation conditions of viability and antibiotic activity of Acremonium chrysogenum 281A and 305A strains producing cephalosporin C was studied. Cryogenization of the culture in the form of suspension of the vegetative mycelium in 20 per cent glycerol solution at a rate of 1 degree/min showed to be advantageous over lyophilization and L-drying. Cryogenization under such conditions provided rather high viability of the culture and preservation of its initial antibiotic activity for the period of its storage for at least 1.5 years in liquid nitrogen at a temperature of -196 degrees C.     

Lysosomal enzyme release in hypothermically perfused dog kidneys

This study investigated lysosomal disruption during hypothermic perfusion preservation of kidneys and its possible relationship to viability. The percentage of free and bound enzyme activity was analyzed for three lysosomal enzymes in homogenates made from perfused canine kidney cortex tissue, including beta-glucuronidase, cathepsin-D, and aryl sulfatase. All three enzymes displayed characteristic increases in free enzyme activity (47-68%) throughout 5 days of perfusion preservation. The increased activity obtained at 5 days of preservation was found to indicate "severe" tissue damage, as shown by a similar increase obtained in renal cortex tissue exposed to warm ischemia (37 degrees C) for 4 hr or longer. Aryl sulfatase was found to be the most sensitive indicator of severe damage. Pretreatment of kidney donors with methylprednisolone, a lysosomal stabilizer, was also studied in kidneys exposed to 5 days of perfusion. Pretreatment was found to reduce the percentage of free lysosomal enzyme activity following 5 days (nonviable) of perfusion to those levels normally obtained following 3-day (viable) perfusion. This indicates that methylprednisolone may be useful in modulating the severe disruption of lysosomes induced by long-term preservation. It is concluded that extensive disruption of lysosomes occurs during hypothermic perfusion preservation and may represent one cause for loss of organ viability.     

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.     

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4^oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4^oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 ^oC). We find that there exists an optimum temperature (-4^oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.     

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.     

Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria

Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.     

Effect of cultivation conditions on trehalose content and viability of brewing yeast following preservation via filter paper or lyophilization methods

The effects of variations in cultivation conditions on trehalose concentration and the viability of brewing yeasts following preservation by filter paper or lyophilization methods were evaluated. In case of filter paper preservation, the cultivation period had no affect on yeast viability, while agitation and aeration during cultivation had a positive effect regarding viability of the bottom-fermenting strains, Rh and Frank. For effective preservation, it was necessary to harvest yeast cells from the stationary phase during cultivation. For lyophilization preservation, the yeast strains tested showed a negative effect on viability, independent of strain or cultivation method. No significant correlation was found between trehalose concentration and yeast viability following either filter paper or lyophilization preservation. However, the filter paper preservation method was suitable for both bottom and top brewing yeast strains with regard to feasibility, viability, and maintenance of the yeast’s specific character.     

Evaluation of Analytical Techniques to Predict Viability after Cryopreservation

Preservation of plant germplasm is important to safeguard biodiversity and to store elite plants. Cryopreservation is one of the possible preservation techniques. Research for a cryopreservation protocol is often inefficient because of slow or poor regrowth of plant material. Therefore, at least one technique, that allows a quick and accurate prognosis of viability after cryopreservation, is required. We evaluated five techniques: electrolyte leakage, triphe-nyltetrazoliumchloride (TTC) staining (visual and spectrophotometrical analysis), malondialdehyde concentrations in plant tissue and a mathematical model that relates ‘water content’ to the weight of encapsulated plant material. Electrolyte leakage and TTC-staining (if visually analysed) are efficient to predict viability. Our mathematical model allows us to save time and plant material in order to develop an efficient encapsulation—dehydration protocol. All other techniques were rejected because of the high variability of the results. This is due to the variability of biochemical activity in plant tissue and the small amount of tissue used in the experiments.     

Kinematic study on the effect of pH on bull sperm function

Since the mammalian spermatozoa became capable of motion, during the epididymal transit, the spermatozoon swims in a liquid medium and it is completely dependent on the environmental conditions. Some reports have suggested an influence of pH on sperm kinetic characteristics, but no study has objectively described how motility changes in a different environmental pH. In this study, we evaluated the effect of different environmental pHs (5.5, 6, 6.5, 7, 7.5, 8, and 8.5) on kinetic parameters, sperm viability, mitochondrial activity, and sperm morphology of bull semen immediately and 1 h after dilution. The results showed higher values for sperm motility characteristics, viability, and mitochondrial activity at pH 7 and 7.5. Values of pH lower than 6.5 and higher than 8 resulted in suboptimal motility, with a decrease in most parameters. At pH 8 and 8.5, a discrepancy between viability and total and progressive motility was found, with a significant amount of spermatozoa that were live but immotile. This reduction seemed related to a decrease in mitochondrial activity, possibly due to the increase in pH. The flow cytometric evaluation of sperm viability assessed by calcein AM was very consistent with the amount of spermatozoa with membrane integrity, evaluated in fluorescence by propidium iodide/SYBR-14 stain. Thus, the calcein AM stain could be used as viability stain instead the classic propidium iodide/SYBR-14 stain because this could allow the addiction of other functional stains without a overlapping of the fluorescent signal in the flow cytometer.     

Select antioxidants improve the function of extended boar semen stored at 10 degrees C

The objective was to determine the effects of antioxidant addition to extender on viability, acrosome integrity and penetrability in vitro of boar spermatozoa preserved at 10 degrees C. Washed spermatozoa were resuspended at 1 x 10(8) cells/mL in modified Modena solution containing 20% (v/v) boar seminal plasma and 5 mM antioxidant (glutathione, cysteine or hypotaurine). Control aliquots were the same suspension without added antioxidants. Sperm suspensions were then chilled to 10 degrees C with a computerized cooling program. Sperm viability after 7 and 14 d was higher in the presence of glutathione or cysteine, whereas hypotaurine did not improve the survival rate. Percentage of chlortetracycline (CTC) fluorescence pattern as intact live cells was higher in spermatozoa preserved with glutathione or cysteine at 7 and 14 d of preservation. When the preservation period was prolonged until 57 d, survival rate was higher with cysteine than controls. When spermatozoa were preserved with cysteine and then inseminated in an IVF system, penetration rate was not different until 15 d of preservation and higher than controls at 15-29 d, whereas no sows became pregnant after AI with spermatozoa preserved for 21-23 d. Therefore, glutathione and cysteine can improve the viability and functional status of boar spermatozoa during liquid preservation and boar spermatozoa penetrated in vitro even after preservation in the presence of cysteine at 10 degrees C for 29 d.     

Preservation of basidiomycete strains on perlite using different protocols

For preservation of 31 basidiomycete strains on perlite in cryovials we used five different perlite protocols to compare their applicability in laboratories with different equipment, namely a viability of the controlled freezing device or the electric deep-freezer and liquid nitrogen supply. The viability of the strains, macromorphological characteristics and the production of laccase were tested after 48 h, six months and one year of storage in the respective device. Our results indicated that the different response to the freezing/thawing process is an intrinsic feature of the respective strain. Nevertheless, the highest viability and preservation of laccase production in our tested strains was found when we used pre-freezing to −80 °C at a freezing rate of 1 °C/min in a programmable IceCube 1800 freezer or in freezing container Mr. Frosty before storage in liquid nitrogen or at ultra-low temperature freezer at −80 °C, respectively. The two abovementioned protocols enable all tested strains to survive three successive freezing/thawing cycles without substantial reduction of growth rate. The majority of the strains also do not lose laccase production. Our results showed that direct immersion of the strains into liquid nitrogen or placing them into −80 °C without pre-freezing is not suitable for basidiomycete cryopreservation.     

The influence of various storage conditions on cell viability in amniotic membrane

Up to now freeze-dried, gamma-sterilised or glycerol-preserved amniotic membranes (AMs) have widely been used in the field of ophthalmology and wound care (e.g. leg ulcers, burns). After some preservation processes in use, like freeze-drying or glycerol-preserving, the cells in the AM are no longer viable. Within this study we evaluated the influence of different short-term and long-term storage conditions on cell viability in AM. Therefore AMs from cesarean section placentae were washed and biopsied to evaluate the microbiological status and to determine the viability of the tissue. Additionally, viability under various storage conditions was examined by assessment of mitochondrial activity. Preservation included temperatures above and below 0°C as well as various media compositions. As expected, cell viability in amnion decreases during storage, in fact the effect was more pronounced when stored frozen, but the higher viability of amnion obtained by storage above 0°C with medium is associated with the limitation to a short period of storage of about 28 days. The evaluated preservation methods are the basis for future non-clinical in-vivo studies in which the possible benefit of amnion as a viable biomaterial in wound healing will be investigated. A part of this work was presented at the World Congress on Tissue Banking in Rio in May 2005 and was honoured by the Poster Award Commission.     

Use of specific collagenases for the isolation of rat liver cells with preserved lipase activities

The heparin-releasable neutral lipase (EC 3.1.1.3) from rat liver is inactivated by the common preparations of collagenases (EC 3.4.24.3) used for the isolation of liver cells. We show that two collagenases purified from Clostridium histolyticum allow both the complete preservation of this lipolytic activity and a good viability of liver cells isolated by the usual perfusion protocol.     

Effects of microwave exposure and Gemcitabine treatment on apoptotic activity in Burkitt’s lymphoma (Raji) cells

Abstract

We investigated the effects of 1.8?MHz Global System for Mobile Communications (GSM)-modulated microwave (MW) radiation on apoptotic level and cell viability of Burkitt’s lymphoma (Raji) cells with or without Gemcitabine, which exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase). Raji cells were exposed to 1.8?GHz GSM-modulated MW radiation at a specific absorption rate (SAR) of 0.350?W/kg in a CO₂ incubator. The duration of the exposure was 24?h. The amount of apoptotic cells was analyzed using Annexin V-FITC and propidium iodide (PI) staining with flow cytometer. The apoptotic activity of MW exposed Raji cells was increased significantly. In addition, cell viability of exposed samples was significantly decreased. Combined exposure of MW and Gemcitabine increased the amount of apoptotic cells than MW radiation alone. Moreover, viability of MW?+?Gemcitabine exposed cells was lower than that of cells exposed only to MW. These results demonstrated that MW radiation exposure and Gemcitabine treatment have a synergistic effect on apoptotic activity of Raji cells.     

Characteristics of human embryonal hepatocytes cultivated in vitro]

A simple and reproducible method for cultivation of human embryo (7--12-week) hepatocytes in a monolayer is presented. The culture showed the capacity for limited growth, displayed a viability for 4 to 6 weeks without being subcultured, and retained some specific characteristics: synthesis of serum protein (albumin and alpha-fetoprotein), a significant amount of glycogen, a high activity of monaminooxidase, and a characteristics response to a high concentration of glucocorticoids. These properties are useful as markers for genetic experiments or for selection.     

Effect of various parameters on viability and growth of bacteria immobilized in sol–gel-derived silica matrices

Immobilized bacteria are being extensively used for metabolite production, biocatalysts, and biosensor construction. However, long-term viability and metabolic activity of entrapped bacteria is affected by several conditions such as their physiological state, the presence of high-osmolarity environments, porous structure and shrinkage of the matrix. The aim of this work was to evaluate the effect of various parameters on bacteria immobilized in sol–gel-derived silica matrices. With this purpose, we evaluated the stress of immobilization over bacteria cultures obtained from different growing states, the effect of cell density and bacteria capability to proliferate inside matrices. Best results to attain longer preservation times were obtained when we immobilized suspensions with an optimized bacterial number of 1 × 10⁷ cfu/gel in the presence of LB medium using aqueous silica precursors. Furthermore, the impact of osmotic stress with the subsequent intracellular trehalose accumulation and the addition of osmolites were investigated. Shorter preservation times were found for bacteria immobilized in the presence of osmolites while trehalose accumulation in stressed cells did not produce changes on entrapped bacteria viability. Finally, nutrient addition in silica matrices was studied indicating that the presence of a carbon source without the simultaneous addition of nitrogen was detrimental for immobilized E. coli. However, when both carbon and nitrogen sources were present, bacteria were able to survive longer periods of time.     

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

Introduction

Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37 °C for up to 20 h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4 °C regardless of the preservation solution. At temperatures above 20 °C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20 °C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20 °C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20 °C. Only ECGM maintains HUVEC viability at temperatures above 20 °C.     

Benign and Malignant Renal Cells Are Differentially Inhibited during Prolonged Organ Preservation

The worry of potential residual renal cancer cells in donor kidney after resection of small renal cancer impedes the extensive use of such controversial donor source. To explore the impacts of organ preservation process on the survival of renal cancer cells, we detected cell proliferation and viability of benign and malignant renal cell lines and clinical renal samples after treated with simulated organ preservation process. It was found that the viability and proliferation of malignant renal cells are inhibited much more than that of benign renal cells during prolonged organ preservation. The inhibition of proliferation in benign renal cells is fully reversible, while in malignant renal cancer cells is not fully reversible after a certain time. So potential residual renal cancer cells could be partly inhibited and eliminated by organ preservation process.