Trypsin-dissociated Cells Research Articles

Dissociation enzyme effects on the potassium currents of inner hair cells isolated from guinea-pig cochlea

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. Some enzymes using for IHCs dissociation affect these inactivation kinetics. IHCs were dissociated from guinea-pig cochlea by 1 mg/ml trypsin or 0.25 mg/ml protease VIII, and the properties of the K + currents were compared using conventional whole-cell voltage-clamp recordings. TEA-sensitive potassium currents showed fast inactivation kinetics in both trypsin-dissociated cells and protease VIII-dissociated cells. The time constant of the inactivation phase in trypsin-treated cells was similar to that in protease VIII-treated cells. However, the rate of inactivation (compared by the ratio between the steady-state current and initial peak current) in protease VIII-treated cells was larger than that in trypsin-treated cells. In protease VIII-dissociated cells, the time constant of recovery from inactivation elucidated by paired-pulse protocol was 3.5 ms. Papain is another enzyme that is sometimes used for dissociating IHCs, so effects of papain were observed. Extracellular papain application (8 unit/ml) demonstrated a slight increase of the outward potassium currents.

Cell surface properties of amphibian embryonic cells

Gastrula and neurula embryos of Cynops pyrrhogaster were dissociated into cell suspensions with ethylenediaminetetraacetic acid (EDTA), trypsin, and solution. The cells were cultured in Niu-Twitty's balanced salt solution and their aggregates were examined histologically. The results indicated that the capacity of amphibian embryonic cells for aggregate formation, sorting out, and notochord differentiation was not suppressed by EDTA and alkali treatments. Trypsin treatment, however, virtually suppressed the cell's capacity for aggregate formation. When aggregate formation resulted from trypsin-dissociated cells, the aggregates showed the sorting out and no differentiation of notochord.

Variations in the response of enzyme-dissociated rat pituitary cells to thyrotropin releasing hormone

While exploring the interaction between thyrotropin releasing hormone (TRH) and normal rat anterior pituitary cells in monolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase in either TSH or prolactin (PRL) release. The dissociated cells were cultured for 3 days, then washed to remove serum proteins and exposed to 10 −6M TRH for 3 hours. TSH and PRL secretion from stimulated and unstimulated cultures was determined by radio-immunoassay and normalized using cell protein. When such trypsin-dissociated cells were exposed to 0.5 mM dibutyryl cyclic AMP the release of both TSH and PRL doubled indicating that the intracellular secretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and presumably at the level of a TRH surface receptor. Previous studies have shown that such trypsin-dissociated cells respond to LHRH and a crude hypothalamic extract with a dose dependent increase in LH, FSH and ACTH release. This rules out a non-specific effect of trypsin. When pituitary cells were dissociated with a non-trypsin technique, the unstimulated release of both TSH and PRL was comparable to that found with the trypsin-dissociated cultures. However, these cultures did respond to TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsin suggests the possibility that a protein moiety may be closely associated with the function of the receptor.

Studies on heart. XVII Effects of frog heart inotropic substances on beating of rat myocardial cells in culture.

The effects of frog heart inotropic substances on beating phenomena in cultured myocardial cells of neonatal rats were investigated. The beating rate and percentage of trypsin-dissociated cells were determined in Eagle minimum essential medium (MEM) supplemented with 10% bovine serum at 2 days in culture. The attachment and the spreading of cells were observed in Eagle MEM supplemented with 0.5% bovine albumin. The positive inotropic glycoprotein, Fr. A from calf heart, and its cyanogen bromide peptide, Fr. A-1, significantly increased the beating rate and percentage of single cells in their dosages to be effective on frog heart assay as well as epinephrine. Another positive inotropic lipoprotein, Fr. I-B from calf thymus, also increased the rate, but did not effect on the percentage like acetylcholine. Negative inotropic dipeptide, anserine, decreased the rate but not the percentage of single cells. In the beating rate of cell-cluster, Fr. A, Fr. A-1 and Fr. I-B showed accelerating effects in their concentrations of 10-8M, 10-7M and 10-9M, respectively. The effect of Fr. I-B on cell-cluster was transitory 5 to 20 min after sample addition, and those of Fr. A and Fr. A-1 were relatively continuous up to 40 min. The chronotropic actions on cell-cluster by these three samples were not affected by the addition of propranolol. Fr. A significantly increased the attached cells and Fr. I-B greatly decreased the spreading cells, but both of them did not promote the spreading process of myocardial cells in their doses of improving the beating properties.

Scanning electron microscopy of aggregating embryonic neural retina cells

Scanning electron microscopy of in vitro reaggregation of trypsin-dissociated neural retina cells from 10-day chick embryos revealed that filopodial projections participate in the assembly of the dispersed cells into clusters. Freshly dissociated cells displayed numerous elongated, randomly projecting filopodia. With the onset of cell reaggregation these filopodia bridged and connected distant cells becoming shorter as the cells came together and formed aggregates. In 24-h cell aggregates only short microvilli were seen, mostly on cell surfaces facing the periphery of the aggregate. Cells dissociated from retina tissue pre-treated with inhibitors of protein synthesis, or cells exposed to these inhibitors immediately after dissociation were mostly devoid of filopodial projections; such cells failed to re-aggregate histotypically. Thus, metabolic and biosynthetic processes are required for the changes in the cell periphery which result in formation or maintenance of filopodia, and which enable trypsin-dissociated cells to reform histotypic associations. Possible relationships between the formation of filopodia and histotypic reaggregation of cells is discussed.

Electrical activity in embryonic heart cell aggregates. Developmental aspects.

Action potential parameters were measured in beating heart cell aggregates which were formed from trypsin-dissociated cells of embryonic chick heats aged 2 1/2, 4 or 7 days. 1. In aggregates composed of cells from the whole heart there was an increase in the maximum diastolic potential, overshoot, maximum rate of rise of the action potential (V max), and action potential duration between days 2 1/2 and 7. 2. Action potential parameters from 4- or 7-day aggregates composed exclusively of atrial or ventricular cells were similar to those in whole heart aggregates of the same age with the exception of the action potential duration in which atrial less than whole heart less than ventricular. Between days 4 and 7 the increases in duration were approximately 14% in atrial, 35% in whole heart, and 50% in ventricular aggregates. Differences in action potential duration, within or between ages, were not due solely to differences in the rate of beating. 3. Action potentials in whole heart aggregates aged 2 1/2 days were insensivitive to TTX (10-5 g/ml) but abolished by D600 (1 MUG/ML). Conversely, at 7 days activity was suppressed by TTX (2 X 10-8 G/ML) WHILE D600 (1 mug/ml) shortened the action potential duration and reduced the overshoot without influencing V max. 4. Adrenaline (1 mug/ml) restored the action potential overshoot and duration in 7-day aggregates treated with D600. 5. Action potential development in embryonic heart cells appears to be characterized by the functional appearance of fast inward channels. The slow channel mechanism, previously utilized in action potential generation, may gradually assume its adult role of carrying inward current during the plateau phase. 6. In contrast to monolayer cultures, embryonic heart cells cultured in aggregate form seem to have membrane properties similar to those of intact tissue.

Studies on cellular adhesion-aggregation: Consideration of involvement of Concanavalin A receptors

Studies were made on the aggregation of trypsin-dissociated cells from chick embryos in the presence of Concanavalin A, a jackbean lectin, in its native tetrameric agglutinating form (n-ConA) and in a fragmented non-agglutinating form (t-ConA). The t-ConA was prepared by first trypsinizing native ConA and then subjecting the mixture to separation procedures to obtain and pool those fractions which failed completely to agglutinate cells. The presence of ConA in these fractions was demonstrated by equilibrium dialysis experiments using α-methyl- d-glycopyranoside which binds specifically to ConA. The agglutination of cells was conspicuously less in the presence of t-ConA + n-ConA than when n-ConA was added alone. The implication is that t-ConA competed with the untreated n-ConA for the same cell surface receptor sites and blocked them. The t-ConA at a final concentration of 200 μg/ml markedly inhibited the spontaneous aggregation of dissociated embryonic chick cells; the viability of these cells was unimpaired. This inhibitory effect was brought about by the t-ConA reacting with ConA sugar receptor sites. The results are correlated with current views on the possible way in which carbohydrates may be involved in cell-cell adhesion and a tentative explanation of how the t-ConA may be exerting its effect is put forward.

Effects of cytochalasins on the initial aggregation in vitro of embryonic chick cells.

ABSTRACT The initial aggregation of trypsin-dissociated cells from the skeletal muscle tissue of 9-day-old chick embryos in the presence of cytochalasins A and B was studied in order to discover the effects of these agents on contact and adhesion. Cytochalasin B (3 μg/ml) had a negligible effect on the rate of aggregation of cells over an 8-h period, but cytochalasin A at concentrations between 3 and 20 μg/ml markedly inhibited aggregation. Both agents altered the shape and size of aggregates and caused cells at their periphery to appear more spherical. The oxygen uptake of the treated cells was not noticeably different from that of the controls, despite the severe inhibition of isotopic carbon dioxide evolution. The effect of cytochalasin B on cell aggregation was reversible and although the cytochalasin A effect could not be abolished on return to medium free of A, the unaltered oxygen consumption was taken as an indication that permanent cellular injury did not occur. The effect of the cytochalasins on aggregate structure was interpreted on the basis of arrested cellular motility, but the singular inhibition by cytochalasin A of the rate of aggregation must await final confirmation of its site of action.

Identification of cardiac myocytes in vivo and in vitro by the presence of glycogen and myofibrils

On the basis of cell shape, refractility under phase optics, spontaneous beating and nucleolar number, two cell types have been distinguished in embryonic heart cell cultures: M cells (myoblastlike) and F cells (fibroblast-like). However, by different criteria, more than two cell types have been found in the heart in vivo. In the present study, heart cell types are redefined by properties which can be used for identifying cells both in vitro and in vivo. Trypsin-dissociated cells from 7-day chick hearts were cultured for 24 h. PAS staining indicated that all M (thick, refractile) cells contain glycogen (gly +), while most F (spread) cells do not (gly −). Under the electronmicroscope, only gly + cells contain myofibrils; most of these cells beat spontaneously in culture. Gly − cells do not beat. Gly + cells with myofibrils as well as gly − cells are found in the inoculum and in the heart in vivo. The ultrastructure of gly + cells in vitro and of muscle cells in the myocardium in vivo is similar. Therefore, it is concluded that gly + cells in culture are derived from the myocardium (muscle) of the heart. Gly − cells are apparently derived from the epicardium, the endocardium and the endothelial lining of blood vessels.

Fine structure of trypsin-dissociated cells of the rat anterior pituitary gland.

SummaryTrypsin-dissociated cells from the rat anterior pituitary appeared essentially intact and undamaged in the electron microscope. Suspensions contained five cell types which correspond closely to those seen in situ: thyrotroph (with cytoplasmic granules 1500 A approximate maximal diam), corticotroph (2000 A), gonadotroph (2000 A), somatotroph (4000 A), and mammotroph (7500 A).

Isolation of specific macromolecules required for adhesion of mouse tumour cells.

THE adhesive properties of cell surfaces which seem to determine many morphogenetic processes1 and which may be important in malignancy2 must reflect the biochemical characteristics of the cell surfaces. There have been attempts to isolate the macromolecules concerned by assaying for substances required for cell adhesion3, and one of us4 has described a promising experimental system for such studies. Trypsin-dissociated cells from an ascites-grown form of a mouse teratoma (teratocarcinoma, embryonal carcinoma)5,6 require L-glutamine in order to synthesize amino-sugars and to reaggregate in a solution of glucose and balanced salts, most probably because the amino-sugars are necessary for the formation of the complex carbohydrates involved in cell adhesion4. This communication describes experiments in which the amino-sugars are replaced by macromolecular substances in the ascites fluid in which the teratoma grew.

Studies on the mechanism underlying the inhibition by puromycin of cell aggregation in vitro.

ABSTRACT Cells dissociated with 0·25 % crude trypsin from the muscle tissue of 9-day-old chick embryos were employed to investigate the effect of puromycin on cellular metabolism. Parallel studies were also made, using the gyratory shaker, to confirm the effectiveness of puromycin in inhibiting cell aggregation and protein synthesis. Puromycin when introduced at a concentration of 10μg/ml into a suspension of cells in Eagle’s MEM did not completely inhibit cell aggregation. Small aggregates were formed in the first 4 h of the experiment. Protein synthesis of the rotated cells, as measured by the incorporation of L-[α -14C]leucine into proteins, was arrested by 91 · 7% within 15 min of introducing puromycin into a cell suspension. The antibiotic retained its inhibitory effect on protein synthesis for the 24-h period of rotation. Puromycin inhibited the cellular oxygen uptake and carbon dioxide evolution of the rotated cells by 40 % within 4 h of its introduction. However, treated cells were still respiring, though at a much reduced rate, at the end of the 24-h experimental period. The release of radioactive carbon dioxide by puromycin-treated cells was also inhibited by 40% at the 4-h stage but after 8 h no further14CO2 was evolved. The presence of the antibiotic markedly inhibited the uptake of glucose by trypsin-dissociated cells. The level of glycogen and lactate in cells suspended in Eagle’s MEM was reduced very considerably over a 24-h period. The presence of puromycin accelerated glycogen utilization over the first 6 h of rotation but at 24 h there was a difference of only 0 · 6 % between the glycogen content of treated cells and controls. At 24 h 11·3 % less lactate remained in the puromycin-treated cells than in the controls. The ATP/ADP ratio of trypsin-dissociated cells decreased from an initial value of 2 · 59 to 1 · 45 after rotation for 24 h. In the presence of puromycin the ATP/ADP ratio was 0 · 62 at 4 h and had further declined to 0 · 48 by 24 h. The effects of puromycin on the aggregation, protein synthesis and cellular metabolism of trypsin-dissociated cells are discussed in relation to cellular adhesive mechanisms.

Histotypic self-organization by trypsin-dissociated and EDTA-dissociated chick embryo cells

The phenomenon of aggregation of chick embryo cells in vitro has been investigated by the rotation-mediated cell-aggregation procedure. In serum-supplemented media trypsin-dissociated neural-retina cells and EDTA-dissociated cells produced aggregates which appeared very similar in size and form. Drugs which are known to interfere with macromolecular synthesis, and which prevent the formation of rotation-mediated aggregates by trypsinized cells appear to be less effective in inhibiting the aggregation of EDTA dissociated cells. The aggregation of EDTA-dissociated cells does not appear to be affected by periodate or by borate at concentrations that significantly inhibit the aggregation of trypsin-dissociated cells. The aggregation of both EDTA-dissociated and trypsin-dissociated retina and liver cells is greatly inhibited by 0.5 per cent glucosamine; however, numerous other aminosugars and other organic amines will cause a similar inhibition at concentrations of 0.5 per cent or less.

Neuron development from trypsin-dissociated cells of differentiated spinal cord of the chick embryo

Embryonic chick spinal cord in advanced stages of differentiation was dissociated into its constituent cells by trypsin digestion followed by mechanical separation. The dispersed cells, singly or in clusters, when seeded on or in blood plasma clots, developed typical nerve fibers up to several millimeters in length. This technique lends itself readily to studies of single neurons, as well as their reactions to controllable influences of the medium and of other cell types.