Maintenance Of Cell Viability Research Articles

Mitochondrial damage during cerebral ischemia

Cerebral ischemia causes a rapid decline in the ability of brain mitochondria to synthesize adenosine triphosphate when they are exposed to oxygen and oxidizable substrates. Ischemia also results in a decreased capacity for energized mitochondria to sequester the abnormally high levels of calcium that are present within ischemic tissue. The degree to which these processes are affected is likely to influence the maintenance of cell viability during cerebral resuscitation. Factors that have been proposed to account for mitochondrial damage during ischemia and reperfusion include intracellular acidosis, Ca2+-induced membrane damage, and free-radical-dependent membrane lipid peroxidation. Ongoing research indicates that measures can be taken to manipulate these factors so that mitochondrial damage may be minimized and cell viability optimized during resuscitation.

Studies on the structural features of sodium alginate entrapped Kluyveromyces marxianus NCYC179 cells used for alcohol production from whey permeate

AbstractAlginate‐entrapped living Kluyveromyces marxianus NCYC179 cells were successfully used in alcohol production from whey permeate. Using 10% whey lactose as substrate, an alcohol production efficiency of 81.5–84.9% and 84.0–88.2% with cell viability maintenance between 82.8–84.3% and 81.0–84.3% of the immobilised cells during the monitoring periods, in batch (six repeat runs) and continuous (23 days of experimenting time) systems, respectively. The immobilised samples remained stable during the course of fermentation with no leakage of K. marxianus cells into the surrounding medium in the case of the batch system. However, slight leakage after 10 days of continuous run in the continuous process was recorded. The scanning electron microscopic studies of the beads containing entrapped yeast cells and sections or crushed samples of the beads carried out at the start and termination of fermentation in both the processes (batch and continuous) revealed that the immobilisation procedure does not influence any morphological change in the entrapped cells during the prolonged periods of fermentation. Moreover, the gel matrix structure was also found to be unaffected by the fermentation conditions employed during the course of present studies.

Molecular dissection of the human transferrin receptor.

Transferrin is the major iron carrier protein in vertebrates and is required for maintenance of cell viability. To deliver iron, transferrin binds to its receptor, the complex is internalized and directed into acidic vacuoles where iron is dissociated and the ligand-receptor complex is recycled back to the plasma membrane. The transferrin receptor is a transmembrane glycoprotein, composed of two disulphide-bonded subunits (each of apparent Mr 90 000). It contains three N-linked glycan units and is post-translationally modified with both phosphate and fatty-acyl groups. The primary structure of the receptor consists of 760 amino acids divided into three domains. Starting from the N-terminal residue the cytoplasmic domain consists of 62 amino acids, followed by 26 predominantly non-polar residues, which constitute the transmembrane domain, and 672 residues form the C-terminal extracellular domain. It does not contain an N-terminal cleavable signal sequence.

Age-dependent pattern of androgen-binding protein secretion from rat Sertoli cells in primary culture.

The pattern and hormonal control of rat androgen-binding protein (rABP) secretion in vitro was investigated using animals of different ages to initiate primary cultures of Sertoli cells. Sertoli cells were isolated from testes of 7- to 31-day-old rats and cultured for periods of up to 30 days in serum-free medium, medium supplemented with insulin, transferrin, and epidermal growth factor (3F), or 3F plus FSH, testosterone, progesterone, hydrocortisone, and vitamin E (8F). The amount of rABP secreted by Sertoli cells during the first 24 h in culture (initial rate) exhibited an age-dependent pattern which reflected the apparent in vivo activity of these cells. Between 7 and 25 days of age, the initial rate of rABP secretion per Sertoli cell increased 20-fold; a further 2-fold increase occurred between 25 and 35 days of age. The pattern of rABP secretion exhibited by Sertoli cells cultured for several weeks was dependent not only on added factors (3F or 8F), but also on Sertoli cell age, expressed as the total of animal age plus time in culture (total age). In cultures of Sertoli cells isolated from very young animals (7-10 days old), the rate of rABP secretion increased until 20 days (total age), but declined thereafter. This early increase in rABP secretion was augmented by, but not dependent on, hormone additions. In contrast, Sertoli cells isolated from older animals always showed decreasing rates of secretion with time in culture. Furthermore, Sertoli cells from very young animals retained the capacity to respond to hormones in vitro with increased secretion of rABP and maintenance of cell viability. This responsiveness decreased with age, similar to the loss of hormone response seen in vivo after puberty. In conclusion, culture conditions were established which permitted the study of FSH-dependent and independent regulation of rABP secretion and of the acquisition of hormone resistance at the time of puberty. The initial rate of rABP secretion in culture (first 24 h) correlates with the age of the animal from which the cultures are obtained. The pattern of rABP secretion during subsequent long term culture is determined by the total age (animal and culture age), with increasing rates of secretion up to 20 days and decreasing rates thereafter. This inherent pattern of rABP secretion as well as loss of responsiveness of the Sertoli cell to hormonal stimulation appear to be programmed early in development.

Dialyzable serum components can support the growth of hybridoma cell lines in vitro

Conditions are defined for the in vitro growth of antibody producing hybrid myeloma cell lines in an immediate environment free of non-dialyzable serum components. Continuous access to low molecular weight serum growth factors was critical to the maintenance of cell viability and antibody production. This requirement was met by use of two-chambered Marbrook vessels, whose geometry allows a small chamber containing metabolically active cells to communicate through a dialysis membrane with a large adjacent reservoir filled with serum-supplemented medium. This tissue culture technique permits the rapid isolation of proteins secreted by cells in vitro, and should permit the further definition of minimal nutritional requirements for the growth of differentiated cells in vitro.

Preservation of aortic heart valves with maintenance of cell viability

The goal of the present study was to develop a procedure for sterilization and infinite preservation of aortic heart valves with maintenance of cell viability. For this purpose the influence of a number of methods of controlled freezing on the viability of the fibroblasts of the aortic valve was tested. Cell viability was assessed quantitatively by the incorporation of [ 3H]-proline by the valve fibroblasts. Controlled freezing at a rate of 1°C/min under protection of 10% dimethylsulfoxide yielded the highest number of viable fibroblasts (88%). This preservation method was also tested for its influence on the structural and functional integrity of the valve matrix, which was found to be preserved throughout sterilization and storage. This study is the first to present quantitative data on cell survival, stress—strain characteristics, and the electronmicroscopic structure of collagenic fibrils after sterilization and controlled freezing of aortic valves.

Importance of capillary perfusion

Perfusion is more critical than oxygen in the maintenance of cell viability. A high hematocrit or high fibrinogen level increases blood viscosity and predisposes to disseminated intravascular coagulation. It is recommended that a hematocrit of about 30 be maintained in periods of circulatory stress such as shock or extracorporeal circulation.

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis.

Liver parenchymal cells were isolated from adult rats by digesting liver slices or perfusing liver with collagenase. The cell yields were 1.5 X 10(7) and 1.0 X 10(8) cells/g liver from slices and perfused liver, respectively, and in both cases the cell viabilities and attachment efficiencies were over 90% and 60%, respectively. The cells were viable for more than one week when cultured in Williams medium E with 10% fetal bovine serum, and addition of insulin and dexamethasone enhanced the maintenance of cell viability. Various biochemical functions or freshly isolated cells and cultured cells were compared in this medium. In freshly isolated cells, induction of tyrosine transaminase [EC 2.6.1.5] by dexamethasone was low and none of the hormones examined stimulated protein synthesis; but when the cells had been cultured for a few days, induction of tyrosine transaminase became prominent, and insulin and dexamethasone stimulated protein synthesis and glucagon inhibited their effect. About half the synthesized proteins were secreted into the medium and among these proteins, albumin, transferrin, fibrinogen, and lipoproteins were identified immunochemically and electrophoretically. It was also shown that the polysomes in freshly isolated cells were almost completely disaggregated, but that in cells after a few days culture they were reaggregated. These results showed that freshly isolated cells have impaired functions, but that after culture for a few days the cells recover various liver functions and thus become more suitable for use in biochemical studies on liver functions.

Phagocytic release of lysosomal enzymes from guinea pig neutrophils—regulation by corticosteroids, autonomic neurohormones and cyclic nucleotides

The effects of various pharmacologic agents on the capacity of guinea pig neutrophils to phagocytize serum-treated zymosan particles and release lysosomal enzymes were determined. Neutrophils (10 7) and zymosan were incubated in Krebs-Ringer phosphate (KRP) medium containing 7.5 mM glucose, pH 7.4, at 37° in the absence and presence of corticosteroids, cyclic nucleotides, and adrenomimetic and cholinomimetic agents. Methylprednisolone hemisuccinate, triamcinolone acetonide, dexamethasone acetate, paramethasone acetate and hydrocortisone hemisuccinate reduced particle uptake by and discharge of lysosomal enzymes from guinea pig neutrophils. Aldosterone hemisuccinate and deoxycorticosterone acetate were inactive. Adrenomimetic (e.g. epinephrine) agents inhibited particle uptake by and lysosomal enzyme secretion from neutrophils, and cholinomimetic (e.g. acetylcholine) agents accelerated lysosomal enzyme release but had no effect on phagocytosis. Cyclic 3',5'-adenosine monophosphate (cyclic AMP) and one of its analogs inhibited particle ingestion by and lysosomal enzyme release from neutrophils; and this inhibition was potentiated by theophylline. Cyclic 3',5'-guanosine monophosphate (cyclic GMP), in contrast to the actions of corticosteroids, adrenomimetic agents and cyclic AMP, accelerated lysosomal enzyme secretion but had no effect on particle uptake. Cyclic GMP did not affect release of cytoplasmic lactate dehydrogenase, thus indicating maintenance of cell viability during release of lysosomal enzymes. Cytochalasin B, an agent which blocked phagocytic uptake of zymosan, did not interfere with inhibition of lysosomal enzyme secretion by corticosteroids, adrenomimetic agents and cyclic AMP or acceleration of this event by cholinomimetic agents or cyclic GMP. These studies indicate that guinea pig neutrophils are capable of releasing lysosomal enzymes during phagocytosis of zymosan and that certain agents can modulate lysosomal enzyme secretion and/or phagocytosis.

Effect of streptozotocin on red-blood-cell-reduced glutathione: modification by glucose, nicotinamide, and epinephrine.

Previous studies had shown that administration of streptozotocin to rats produces both diabetes and hemolysis and that both could be ameliorated by prior injections of diazoxide. Thus, it appeared pertinent to define the effect of streptozotocin on the red cell. In the present studies, streptozotocin administered in vivo to rats produced a rapid fall in red-cell-reduced glutathione. This effect was duplicated in vitro in incubated human red cells. Furthermore, it was demonstrated that glucose loading prior to bleeding modified the in-vitro red-cell GSH response to streptozotocin and that preincubation of red cells from fasted individuals with glucose, nicotinamide, and epinephrine (but not nicotinic acid) protected against the subsequent effect of streptozotocin on RBC GSH. The pattern of the RBC GSH response under each of these conditions is that which occurs in response to challenge with an oxidant, that is, with appropriate protection, oxidation stress produces an acute rise rather than falll in gsh. further, when glucose was present through both preincubation and test periods (i.e., in presence of streptozotocin) a third pattern of GSH response was observed--no change. The data are compatible with the postulate that the cytotoxic action of streptozotocin is dependent, in part, on an oxidant effect, and that glucose may protect through at least two mechanisms, that adrenergic stimulation can enhance protective mechanisms against redox insults and so contribute to maintenance of cell viability.

Differential thermolability of exonuclease and endonuclease activities of the recBC nuclease isolated from thermosensitive recB and recC mutants.

The recBC nuclease (also called exonuclease V) has been partially purified from Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and recB270 recC271 mutations. Of the multiple activities associated with the enzyme, only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and endonucleolytic degradation of single-stranded DNA is no more thermosensitive than that catalyzed by the wild-type enzyme. These results suggest that the defects in genetic recombination, DNA repair, and the maintenance of cell viability observed in recBC mutants in vivo result primarily from the specific loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.

15] Specific fractionation and manipulation of cells with chemically derivatized fibers and surfaces

The specific fractionation of cells has not assumed the importance in cytology that fractionation of molecules has in chemistry, perhaps because of its inherent difficulty, and because other methods of obtaining certain pure lines of cells are available. Although a number of physical methods for the fractionation of eukaryotic tissues and cells are in current use, there are few methods utilizing the chemical properties and specificities of the cell as a means for both cell fractionation and cell manipulation. The main requirements of a chemical method for the manipulation of cell populations are specificity, general applicability, high yield and maintenance of cell viability. Two main approaches to a chemically specific method of cell fractionation suggest themselves. The first, which to the authors' knowledge has not yet been implemented, would rely upon the development of a spectroscopically detectable marker that interacted with an appropriately specific reagent within a living cell followed by physical separation of the marked cell. This faces a number of difficulties and therefore several workers have resorted to a second approach utilizing specific interaction with cell surface components.

Stimulation of Phagocytic Release of Neutral Protease from Human Neutrophils by Cholinergic Amines and Cyclic 3′,5′-Guanosine Monophosphate

Abstract The purpose of this study was to elucidate the actions of cholinergic agents and cyclic 3′,5′-guanosine monophosphate (cyclic GMP) on phagocytic release of a lysosomal neutral protease from human neutrophils in the presence of aggregated IgG-rheumatoid factor complex. Neutrophils (5 × 106) and complex were incubated in a balanced salt solution, pH 7.4, at 37°C for 15 min in the absence and presence of various cholinergic agents and cyclic GMP analogs. Acetylcholine, acetyl β-methylcholine, carbamylcholine, and pilocarpine, but not choline, provoked a 74% to 94% increase in enzyme release at 10-7 M. Atropine, but not hexamethonium, blocked the accelerating action of acetylcholine on enzyme release. Cyclic GMP as well as three of its analogs also enhanced release of neutral protease from phagocytosing human neutrophils (71% to 136% increase at 10-7 M). Guanosine 5′-monophosphate or triphosphate did not provoke enzyme release and atropine did not block the effect of cyclic GMP. Neither the cholinergic agents nor cyclic GMP affected release of cytoplasmic lactate dehydrogenase, thus indicating maintenance of cell viability during lysosomal enzyme release. The data suggest that human neutrophils have muscarinic receptors that respond to cholinergic agents by acceleration of lysosomal enzyme release, perhaps via a mechanism involving intracellular cyclic GMP.

An autoradiographic study of H 3-proline utilization by aging mouse skeletal tissues—II. Cartilage cell compartments

In an autoradiographic study the uptake, turnover and distribution of H 3-proline was assessed at the femoral epiphyseal plate and articular cartilage of 5 to 52 weeks old mice. Ninety-nine shortlived BNL mice were given H 3-proline in a single dose of 2 μCi/gm body weight (Sp. Act. = 1 Ci/mM) and killed between 15 min to 14 days later. NTB-3 autoradiograms which were exposed for 16 days were prepared and stained with hematoxylin. Grain counts were made over epiphyseal and articular cartilage cell compartments and the data statistically evaluated. The results showed significant uptake and utilization of H 3-proline by both cell types, although the activity of epiphyseal cartilage was initially higher. H 3-proline was utilized extensively for matrical precursor formation, as well as, for a variety of intracellular substances necessary for cellular viability. The uptake and turnover of H 3-proline was diminished in both cell compartments by 26 weeks of age, revealing peak activity shifts. At 52 weeks the uptake by epiphyseal cartilage cells was insignificant, whereas, articular cartilage activity was retained. The significantly diminished grain counts observed over epiphyseal plate cells with increasing age, reflect release of physiological demands for matrical precursor formation, closure of the plates and termination of longitudinal growth. The continued significant H 3-proline uptake exhibited by articular cartilage was a reflection of the physiological requirement for maintenance of normal joint function throughout the life-span of the organism. The maintenance of cell viability against accumulating age changes is essential to the integrity of the articular cartilage.

Maintenance of Cell Viability

An early distinction between the early and reversible changes of cellular ischemia and the irreversible changes of cell necrosis is essential to a rational program for the treatment of coronary artery disease, to a definition of the permissible limits of nonperfusion of the anoxic arrested heart at open-heart surgery, and to a determination of the maximal allowable transport time for cardiac transplantation. The factors in the myocardial cellular environment that support cellular viability and help maintain homeostasis are reviewed. Reversible surgically induced anoxic arrest is contrasted with the irreversible arrest and cell necrosis which often attend the myocardial anoxia of coronary occlusion. The enhancement of glycolysis, other changes in carbohydrate metabolism, and the depressed oxidation and lipid storage associated with alterations in lipid metabolism are outlined and their interrelationships discussed. The effect of the myocardial tissue acidosis which accompanies ischemic metabolism is an important factor in determining the reversibility of the effects of ischemia. The probable role of myocardial acid hydrolases in pathological ischemic autolysis is described. A sequence of cellular events induced by ischemia and leading to ultimate cell death is outlined. Promising areas for future investigation into the pathogenesis of myocardial infarction and into improved methods of treatment are suggested.