Photoelectric monitoring of single beating heart cells in culture

This paper characterizes a photoelectric recording system which monitors individual beating heart cells in vitro. A culture procedure which supports continued beating for up to three months without confluent overgrowth or aggregation is described.     

Growth of dissociated beating human heart cells in tissue culture

Fetal human heart cells have been dissociated by trypsinization and successfully grown in tissue culture on two occassions. The characteristics of the growth and mitotic activity were quite similar to those previously found for rodent heart cells. The maximum beating rates seen in the cultures from each specimen were 33 and 75 per minute.     

Further studies on preservation of the beating rhythm of myocardial cells in culture

1. 1. Single myocardial cells from fetal mouse heart beat spontaneously in monolayer culture. In standard medium they maintained a constant beating rate for at least 5 h. After the beating rate of individual cells had been accelerated for a short time by electrical stimulation, the original beating rate could be immediately restored by interrupting the stimulation. Quiescent myocardial cells from neonatal mouse atrium could be induced to beat by electrical stimulation and most of them ceased to beat again immediately by interrupting the stimulation.

2. 2. After the spontaneous beating of individual myocardial cells had been stopped or slowed down for a short time by incubation in medium of low temperature or high potassium or low calcium concentration, the original beating rate could be restored by replacing the cells in the original, normal medium.

3. 3. After the spontaneous beating of individual myocardial cells had been stopped by adding a metabolic inhibitor, such as 2,4-dinitrophenol or 2-deoxyglucose, the original beating rate could be restored by replacing the cells in the original, normal medium.

4. 4. Both single myocardial cells and cell clusters showed arrhythmia, including flutter and fibrillation, in medium of low potassium or high calcium concentration. After a short period of arrhythmia, the original beating rate could be restored by replacing the cells in the original, normal medium. The arrhythmia of cell clusters produced in either low potassium or high calcium medium was also corrected immediately by addition of quinidine sulfate.

     

In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix

We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24-48 h and can be maintained in culture for 5-6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.     

Closed loop television tracking of beating heart cells in vitro

The motion of beating heart cells in vitro has been used as a sensitive indicator of the presence of drugs. The combination of closed loop television video tracking and latex microspheres placed on the cells as markers results in an improved measurement of cell motion. Latex microspheres, 5.2 micrometers, are allowed to settle onto beating myocardial heart cells in vitro, where they move in concert with the cell motion. These microspheres, when viewed by televised transmission microscopy with normal optics, present high contrast circular targets that are excellent for closed loop television video tracking techniques. With video tracking, an analog voltage is generated proportional to the horizontal center of intensity of the target being tracked. This signal represents the location of a video demodulator gate that is maintained centered over the video target by feedback control.     

Development of heart cells in culture: studies using an affinity purified antibody to a myosin light chain

Cultured neonatal rat heart cells can be used to study the factors that regulate cardiac contractility and myocyte development in vitro. An antibody to the 26,000 dalton light chain of myosin (MLC1), has been produced and purified on a Sepharose 4B affinity column prepared with rat heart myosin. When primary cultures of myocytes are studied by indirect immunofluorescence using this antibody a predictable pattern of myofibrillar structure is observed to develop over 72 h. This myosin cytoskeleton is highly organized and the myosin fibrils exhibit cross striations. The antibody does not stain non-muscle heart cells and there is no evidence for myocyte division in culture. The qualitative immunofluorescent pattern of myosin organization is the same in both spontaneously beating and in non-contracting cells.     

In vitro cultivation and behavior of aortic endothelium cells in a low serum culture medium

Endothelial cells isolated from calf aorta were used in the subculture No. 4 to 10 for experiments to establish standardized and well reproducible conditions of cultivation. The cells can be cultivated in the commercial medium Eagle-MEM with following supplements: 0.1 g L-glutamine, 5.0 g peptone, 0.5 g serum albumin, and 10 ml (= 1%) bovine serum per 1000 ml medium (MEMPAS). With the aid of immunofluorescence technique the cell type specific marker Factor VIII antigen was shown to be localized especially in the perinuclear region of the cells. The cells were characterized with regard to their growth behaviour. Both, the MEMPAS and the Eagle-MEM with 10 per cent serum increases the cell number in the first 4 days of the exponential growth to the same values. The use of MEMPAS in connection with a strict cultivation regime from the deep frozen cell suspension to culture in scintillation vials guaranteed well reproducible conditions of cultivation. In 14 non-selected experiments distributed over a longer period of time it was found that with regard to the values of the cell number on the respective day the cultures can be divided into two groups, which differ with statistical significance. In further experiments it was possible to confirm this result. Medium, conditioned by endothelial cells (K-MEMPAS) increases the cell number and the growth rate. From these results it was concluded that endothelial cells of vessels are able to produce growth factors with self-stimulating effects. At this time the endothelial cell line is stored in deep frozen state up to the 25th subculture. The endothelial cells cultivated in the described standardized conditions are useful for screening of cell type specific factors with angiogenic activity.     

In vitro bioassay of erythropoietic activity in serum using mouse spleen cells. The effect of heat inactivation on serum erythropoietin

Untreated human serum is known to be toxic to in vitro assays for erythropoietin, including the mouse spleen cell assay system (MSCA). This phenomenon had previously been shown to be mediated by complement-dependent IgM heteroantibodies and can be overcome by heating the serum at 56 degrees C for 30 minutes. Using the MSCA, we have found that the toxic effect of serum could also be removed by treatment with a precipitating antibody against the C3c component of complement. The effects of the two methods of complement inactivation on the measurement of stimulatory activity in serum have been compared. For normal serum, the results after heat inactivation and antibody treatment were similar. In contrast, serum from a patient with aplastic anemia gave a result equivalent to 327 mU erythropoietin/ml after heat treatment, but after antibody treatment equivalent to 1,520 mU erythropoietin/ml. Gel permeation chromatography of unheated, heated, and antibody-treated sera showed that heating markedly reduced the activity of the erythropoietin peak. Seventy percent of the activity of partially purified urinary erythropoietin was lost during heating in the presence of normal serum. In addition, heating caused the appearance of high molecular weight compounds that are stimulatory in the MSCA. The level of this activity appeared to be directly related to the stimulatory activity of the unheated serum.     

Intrinsic factors influencing the maintenance of contractile embryonic heart cells in vitro. 3. The effect of inoculum level

Seven-day embryonic heart cells are tested for their ability to condition their own medium by comparing cell responses at various inoculum levels. The data show that contractile activity, spreading on glass, and survival increase as inoculum level rises. The data also show that the cell death rate is inversely proportional to the rate at which cell spreading and contractile activity increase.     

In vitro cytokinin binding to a particulate fraction of tobacco cells

At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco (Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known.Abbreviations BA N ⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - K_d equilibrium dissociation constant - R_t total concentration of binding sites In partial fulfillment of the requirements for the Ph.D. degree in the Department of Botany and Plant Pathology, Michigan State University     

In vitro studies on information transfer in cells from allotype-suppressed rabbits.

Data are presented which show that cells from allotypically suppressed rabbits resist in vitro "rescue" attempts in which informational ribonucleic acid (i-RNA) coding for the suppressed allotypic marker is used. It is shown that peritoneal exudate cells from such thoroughly suppressed animals contain cells that yield i-RNA coding for the suppressed marker, and that central lymphatic tissue possesses cells that produce Ig of the suppressed type.     

In vitro effect of rabbit anti sea star lymphocyte serum on axial organ cells

The axial organ (AO-cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated or not into two populations: adherent (B-like) and non adherent (T-like) to nylon wool. Rabbit anti sea star lymphocyte serum induces the proliferation of axial organ cells. The T-like antiserum stimulates the T-like cells exclusively; the whole axial organ cell antiserum only stimulates the whole axial organ cell population.