DEVELOPMENTAL.

Retention

BIOLOGY

s&134-141(1976)

of Fully Differentiated Chick Embryonic MICHAEL

Department

of Physiology,

J.

University

Electrophysiological Heart Cells in Culture

MCLEAN

of

AND

Virginia

Accepted

School January

NICK

Properties

of

SPERELAKIS

of Medicine,

Charlottesville,

Virginia

22903

2,1976

Many cultured cells in spherical reaggregates (100-1000 Frn diameter), prepared using cells isolated from embryonic chick ventricles (16 days in ova), retain great sensitivity to tetrodotoxin (TTX; 0.1 @g/ml), rapidly rising action potentials (up to 200 V/set), high resting potentials (up to -90 mV), and they lack automaticity. The Na+ channels generating the action potential upstroke inactivate totally at about -50 mV. The chronaxie (hence, excitability), the ratio of P,,/P,, and the intracellular K+ concentration are about the same as in adult cells. Following blockade of the Na+ channels with TTX, norepinephrine produces slowly rising overshooting responses, indicating the presence of functional beta-adrenergic receptors. Thus, trypsin-dispersed myocardial cells can be made to retain highly differentiated membrane properties in vitro. INTRODUCTION

Many membrane electrical properties of chick myocardial cells have been shown to differentiate in the course of normal embryonic development (Sperelakis et al., 1975). For example, cells in young hearts (2-4 days in ouo) lack fast Na+ channels. Instead, the action potential is generated by kinetically slow Na+ channels which confer slow rates of rise (less than 20 VI set) and which are insensitive to tetrodotoxin (TTX), a blocker of fast Na+ channels (Narahashi et al., 1964). The dependence of the action potential on inward Na+ current in young and old hearts has been demonstrated (Sperelakis and Shigenobu, 1972). TTX sensitivity first appears on about Day 5, marking the initial appearance of fast Nat channels. During the period from about Days 5 to 8, TTX only partially reduces the rate of rise, reducing it to the level found in young embryonic cells. The residual Na+ influx must occur through slow Na+ channels still functional in TTX. The rate of rise increases progressively from Days 5 through 18, probably due to an increasing density of fast Na+ channels, reaching the adult level of about 150 V/set. After Day 8, TTX completely abolishes all excitability despite intense 134 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

stimulation, indicating that most slow Nat channels have become masked or have been removed. The resting potential also increases during development from about -40 mV on Day 2 to about -80 mV by Day 12. Furthermore, there is a decrease in the incidence of automaticity in ventricular cells as development proceeds. The last two factors are accounted for by a decrease in the ratio of P,,IP, from 0.2 on Day 3 to about 0.02 by Day 19 due to an increase in P,. 42K flux studies have corroborated that young hearts have a low PK (P, increases threefold between embryonic Days 7 to 19) (Carmeliet et al., 1975). However, when cells are dissociated from ventricles of old (16 day) embryonic hearts and cultured on glass or collagen substrates, the electrical activity often resembles that of cells in young embryonic heart, rather than that of the mature ventricle from which the cells were initially obtained (Sperelakis and Lehmkuhl, 1965). As judged by (a) loss of TTX sensitivity, (b) loss of fast Na+ channels and a regain of a high density of slow Na+ channels, (c) reduced resting potentials, and (d) high incidence of pacemaker potentials, the cultured cells rapidly reverted to the young embryonic state. The reversion can

MCLEAN

AND

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Differentiated

be partially prevented by culturing in media containing elevated K+ concentrations (McLean and Sperelakis, 1974). Cells grown in this manner maintain their sensitivity to TTX; however, the rates of rise are slow (20-30 Vlsec) and the resting potentials average only about -70 mV. Reports from other laboratories also have indicated that cultures of embryonic chick heart cells of younger age than used in the present studies can be produced which have not completely reverted, i.e., the cells retained partial TTX sensitivity (Lieberman et al., 1975a,b; DeHaan and Fozzard, 1975). For example, Lieberman et al. (1975b) observed moderately fast-rising ventricular-shaped action potentials, but blockade by TTX occurred only with TTX concentrations of 25 Fglml and higher. In the present report, we present evidence that fully mature (differentiated) electrophysiological properties, including TTXsensitive fast Na+ channels, can be maintained in vitro. METHODS

The ventricles of 16-day chick embryonic hearts were washed free of blood and dispersed into single cells by stirring in modified Tyrode’s solution (Ca2+- and Mg”+free) containing 0.05% trypsin (1:250; Nutritional Biochemicals Corp.) and 100 mg% glucose. The composition (in mM) of the normal Tyrode’s solution was: 144.0 Na+, 2.7 K+, 1.8 Ca’+, 1.0 Mg2+, 145.2 Cl-, 1.1 HZP04-, and 6.0 HCO,-. For some experiments, the composition of the dissociation medium was altered by increasing the concentration of K+ to 25 r&Z by isosmolar substitution for Na+, or by addition of 5 mM ATP. At 15-min intervals, the cloudy cell-containing supernatant was decanted into chilled culture medium, and fresh trypsinizing solution was added; this procedure was repeated four times. The composition of the culture medium was: 10% horse serum, 40% Puck’s N-16 or Medium 199 nutrient solution, 50% Hanks balanced salt solution, and penicillin and

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135

streptomycin (50 units/ml). The cells were diluted with culture medium to a concentration of about lo6 cells/ml, and 0.5 ml of this suspension was placed into a well (area of 1.75 cm? within a glass culture vessel. The well was formed by a glass ring sealed on a cellophane square (cut from dialysis tubing) with silicone vacuum grease. Culture medium (2 ml) was added to the area surrounding the well. Cells that were dissociated in 25 mM K+ were also cultured in elevated K+. Within 3-5 days, the cells reaggregated spontaneously into various groupings: spheres (0.1 to 1.0 mm in diameter), monolayers, or long cylindrical strands. The factors controlling the shape of the aggregation are not known. All data reported here were from the spherical reaggregates, although similar results were obtained from the monolayers and strand preparations. Most of the spheres were lightly attached to the cellophane. The aggregates were cultured for 3-14 days prior to experimentation. For testing, the aggregates were transferred by Pasteur pipet to an uncirculated bath (at 37°C) containing either fresh culture medium or Tyrode’s solution with a [K+l, of 2.7 m&f; there was no difference in the results obtained in the two solutions. Cells grown in medium containing elevated K+ were washed several times with normal Tyrode’s solution ([K+], of 2.7 n-&J prior to commencing experimentation. Aggregates which did not beat spontaneously contracted in response to external electrical field stimulation (rectangular pulses, 0.3-4 msec in duration, applied via platinum plate electrodes positioned 1 cm apart). The air over the tissue bath was humidified to minimize evaporation. The pH of the bathing solution (not bubbled with 5% CO,) was adjusted to 7.3 and did not change during the course of the experiments. Intracellular micrelectrode penetrations were made using standard methods (Sperelakis, 1967; McLean and Sperelakis, 1974). Maximal rates of rise were estimated from photographs of tracings

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taken at. fast sweep speed. A bridge technique was used to apply polarizing current pulses through the voltage-recording microelectrode; the validity of this technique has been previously discussed (Sperelakis, 1969; Sperelakis, 1972). Arguments criticizing the bridge technique have also been advanced (Lieberman et al., 1975b). RESULTS

Intuit

Noncultured

Hearts

Intact 16-day chick embryonic hearts (ventricles) had resting potentials of about -80,mV and action potentials with maximu.m rates of rise of about 150 VJsec (Fig. 1A). The a.otion potentials were completely abolished by TTX (Fig. lB), and isoproter:I’*

/. IN1At.T

l&DA” r

CONTROL

VOLUME

50, 1976

enol rapidly elicited slowly rising, but overshooting, electrical responses (Fig. 1C). Cultured

Cell Reaggregates

Nonpacemaker cells. Automaticity was not observed in many cells in the spherical reaggregates prepared from Is-day ventricles. Many of these nonpacemaker cells had high resting potentials (up to -90 mV), and the upstroke velocity of the action potentials was very fast, about 175 VI set (Fig. 1D). The action potentials were completely abolished by TTX (0.05 pg/ml) even though intense stimulation was applied (Fig. 1E). Thus, the resting potential, maximal rate of rise of the action potential, and TTX sensitivity of these cells in the reaggregates are virtually +TTX

+I50PREL

6 --------

--v/SEC

:

I

SPHERICAL

\

E _-_-----

REAGGREGATE

FIG. 1. Comparison of tetrodotoxin (TTX) sensitivity of cells in intact 16-day-old chick embryonic ventricle with cultured spherical reaggregates of cells isolated from 16-day embryonic hearts. (Al-(C) Recordings from one cell in an intact heart. Control action potentials had fast rates of rise (+V,,,; 150 V/set) (A); after TTX (1 @g/ml), all excitability was abolished despite tenfold increase in stimulus intensity (B). Isoproterenol (isoprel; lo-” M) produced a slowly rising action potential in the TTX-blocked heart CC). CD)(F) Recordings from one nonpacemaker cell in a spherical reaggregate. Control action potential with +Vimar of 175 V/set (D) was abolished by TTX (0.05 pg/ml) (E). Isoproterenol induced a slow response (F). (G)-(H) Recordings from a pacemaker cell in a reaggregate. Control action potential with an intermediate +v,,, of 70 V/set and pacemaker potential (V,) (G). TTX (2 kg/ml) suppressed automaticity, but stimulation elicited an action potential with +Vimax reduced to 10 V/set (H). Time and voltage calibrations in panel H apply throughout. Field stimulation applied in all panels except G.

MCLEAN

AND

SPERELAKIS

Differentiated

identical to those of the intact heart. Cells with equally fast rates of rise and TTX sensitivity have also been observed in monolayer-s and strands. The incidence of such “fast” cells in preparations dispersed in the presence of ATP was 25% and that in preparations dispersed and cultured in 25 mM K+ was 50%. When both elevated K+ and ATP were used, the incidence was 80%. The other nonpacemaker cells in the reaggregates had slow (~25 Vlsec) or intermediate (30-85 Visec) rates of rise; many of these cells were completely sensitive to TTX, although some were only partially sensitive or insensitive. In the presence of TTX (0.1 pgiml), addition of isoproterenol allowed slowly rising overshooting electrical responses to be elicited (by field stimulation) within 1 min (Fig. lF), and visible contractions reappeared concomitantly. The threshold for the slow response was considerably greater than that required to elicit the normal action potential. The slow response was completely blocked by Mg2+ (1 mM), D-600 (0.1 pg/ml), or propranalol (lo-” M). The catecholamine-induced slow response is similar to that produced in old intact hearts (Fig. 1C). Pacemaker cells. Cells exhibiting pacemaker activity were also found in the reaggregates. The incidence of pacemaker cells was higher when the cells were separated in low KC concentrations and in the absence of ATP. Many pacemaker cells (about 50%) had high maximal diastolic potentials (about -80 mV) and had action potentials with intermediate rates of rise (60-80 V/set) (Fig. 1G). When TTX was added, even in high concentrations, the rate of rise was only diminished from 70 to 10 V/set (Fig. 1H); accompanying contractions persisted also. Automaticity of these cells was suppressed by TTX, and action potentials often had to be elicited by stimulation (Fig. 1H). Such cells appear to have partially reverted. The other pacemaker cells had slow rates of rise and were insensitive to TTX. The fact that these

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137

cells possess automaticity suggests a decrease in P, toward the level found in young hearts. DeHaan et al. (1975) have observed similar partially TTX-sensitive pacemaker cells in spherical reaggregates formed by the rotation method of Moscona. Some additional properties of the nonpacemaker cells. When the maximal rate of rise (+$‘,,,) of the “fast” cells was plotted against the membrane potential (E,n), the channels generating the action potential upstroke inactivated completely at -50 mV (Fig. 2A1, as do adult fast channels. In these experiments, the resting potential was varied by elevation of [K’],, by isosmotic substitution for Na’. The plot of E,,, against the log of external K’ concentration (IK’I,,) has a linear region with a slope of 60 mV/decade which extrapolates to an internal K+ concentration ([K+J) of about 130 n&! (Fig. 2B). ]K+],, was elevated by isosmolar substitution for Na’. The experimental data closely fit a theoretical curve calculated from the Goldman constant-field equation for aP,,IP, ratio of 0.02. The form of the equation used for the calculations was E,

= -61mV

log

[K’li + J’dJ’KWa+lJ [K+l,, + PNaIPd[Na+lo) ’

assuming P,, to be negligible. Strength-duration curves gave chronaxies ranging from 0.4-0.7 msec (Fig. 2C). Since Shimizu and Tasaki (1966) showed that chronaxie of developing chick heart decreased from 4 msec on Day 2 to 1 msec by Day 16, the fast cells in spherical reaggregates match the excitability of old embryonic and adult chicken hearts. Polarizing current pulses were applied through a dc bridge circuit which allowed simultaneous recording of current and transmembrane potential. Voltageicurrent plots were determined for several cells in the same aggregate as well as for cells in different aggregates. The plots were linear (at least up to ?25 mV) with a slope giving an input resistance (rin) of

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PN,/ PK = 0.02

CNo10 = ISOrnM Cr,i CNa];=

CN.l,+CKl,=

=I30 20 153

AI InA)

FIG. 2. Summary of some electrophysiological properties of “fast” nonpacemaker cells in spherical reaggreates. (A) Plot of +vimax versus membrane potential (E,) from aggregates in four separate cultures (different symbols) with complete inactivation of the fast Nat channels at about -50 mV. Resting potential varied by elevation of [K+&. Each set of symbols represents a different series of experiments on different cultures; each point represents the mean of four to seven values obtained from different reaggregates within one culture. (B) Plot ofE, versus the log of external K+ concentration ([K’l,) (equimolar substitution of K’ for Na+) extrapolates to an internal [K+l, of 130 mM. The slope of the points in high lK+l, is about 60 mV/ decade; the dashed line gives the K’ diffusion potential (E,) calculated from the Nernst equation. The dotted line was calculated from the Goldman constant-field equation for aP,,/P, ratio of 0.02 and for the ion concentrations given in the figure. (Cl Strength-duration curve obtained from one impaled cell gave a chronaxie of 0.5 msec. (D) Voltage/current relation gave an average input resistance of about 5 MR; data obtained both from cells in several different aggregates and from several measurements in each aggregate. These aggregates varied in size between 150 to 300 pm.

about 5 Ma (Fig. 2D). This value is comparable to that previously obtained by Sperelakis and Shigenobu (1972) for old intact embryonic hearts using the bridge technique. The aggregates studied had diameters ranging between 150 and 300 Frn, and there was no apparent dependence of input resistance on aggregate size. Assuming the surface area of a single cell within the aggregate to be 80 x 10-fi cm’ (cell dimen-

sions assumed to be 16 pm in diameter and 150 pm long), membrane resistivity calculates by this method to be 400 a-cm2. DISCUSSION

Cells in spherical reaggregates of trypsin-dispersed embryonic ventricular cells can be made to demonstrate essentially adultlike electrophysiological properties, including (a) high resting potentials, (b)

MCLEAN

AND

SPERELAKIS

Differentiated

high [K+li, (c) lack of automaticity, (d) rapidly rising action potentials, (e) complete TTX sensitivity, (0 receptors for inotropic agents such as isoproterenol, and (g) brief chronaxie (high excitability). While no single factor can be identified as essential for the maintenance of differentiated properties, we feel that several factors may interact to prevent extensive reversion, including the composition of the dissociation medium and the manner of reaggregation of the cells in vitro.. The present experiments suggest that the cell dispersal step is important in determining the membrane properties of the cultured cells. We cannot give mechanisms for these effects at present; however, one possibility is that high concentrations of K+ and ATP in the dissociation medium reduce their loss during cell separation. Seraydarian et al. (1972) showed that ATP content of the cultured cells was lower than that of control hearts. Sperelakis (1972) gave evidence based on electrical measurements that LK’I, was decreased in cells cultured by standard methods, a change which was prevented by the present methods. Although such procedures could conceivably result in the selection of a population of cells which retain differentiated membrane properties, this is more or less immaterial because we want a selected population of highly differentiated cells to study, regardless of how they are obtained. Because reaggregates of 2.5-day-old cells (prepared by the present methods, i.e., with elevated K+ and ATP in the dissociation medium) retained young electrophysiological properties (unpublished observations), these procedures probably do not induce de nouo synthesis of fast Na+ channels, but rather may protect against their loss. However, our experiments do not allow unequivocal determination as to whether high K+ and ATP prevent the loss of fast Na+ channels or stimulate the resynthesis of channels. Sachs et al. (1973) previously suggested that protein synthesis was important for

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139

regaining TTX sensitivity in aggregates prepared from ‘I-day hearts which had been rendered insensitive by trypsinization. However, their reaggregates showed pacemaker activity and intermediate rates of rise similar to that shown here in Fig. lG-H, and their cells were not completely sensitive to TTX (DeHaan et al., 1975). Our own experiments (McLean and Sperelakis, 19741, including the present results, indicate that trypsin does not irreversibly destroy TTX sensitivity. “Fast” cells (i.e., possessing fast rates of rise and complete TTX sensitivity) generated slowly rising, plateaulike electrical responses when catecholamines were added after blockade of fast Na+ channels. Shigenobu and Sperelakis (1972) have shown that the slow response which occurs in intact embryonic chick hearts is dependent on both extracellular Na+ and Ca’+, and can be blocked by Ca’+ antagonists. This slow response is also dependent upon cellular ATP levels (Schneider and Sperelakis, 19751. In all respects, the present results resemble the slow responses produced in adult cardiac cells. Thus, functional receptors for catecholamines are retained in the cultured cells described here. The membrane resistivity calculated by the present method is considerably lower than that reported by Lieberman et al. (1975a) for strand reaggregates and by DeHaan and Fozzard (1975) for spherical reaggregates. DeHaan and Fozzard used the total surface area of all the cells (within the reaggregate) to obtain the high value from their calculation, rather than that for a single cell within the aggregate as used here. They based their assumption on the fact that they found good electrical coupling between many cells. For a given measured input resistance, the larger the surface area of the total membrane through which the applied current flows, the larger is the calculated membrane resistivity. If the cells are electrically connected by low-resistance pathways, then the effective surface area is larger, and the

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calculated membrane resistivity is correspondingly larger. Lieberman et al. (1975a) also used the total surface area of the cells for their calculations. In contrast, when our reaggregates were impaled with two microelectrodes, we did not observe electrical coupling between most cell pairs; this indicates that the cells in our aggregates do not behave as one giant isopotential cell (unpublished observations). This justified our use of the surface area of a single cell for calculations of R,. Obviously, this difference in method of calculation would account for the much larger resistivities that the others reported. The availability of cultured cells which closely resemble cells in the intact myocardium provides a preparation which should facilitate a number of types of experiments which cannot be performed accurately on the intact heart. As one example, reaggregates consisting nearly entirely of myocardial cells can be analyzed for various metabolites. Furthermore, the presence of functional receptors in the membranes of the cultured cells make this preparation useful in studying pharmacological properties of myocardial cells in the absence of neuronal influences. The ability to stimulate various stages of development in vitro may also lead to the discovery of the factors which regulate myocardial differentiation. Note added in proof. We have now prepared spherical reaggregate cultures of trypsin-dispersed old embryonic chick heart cells by the gyrotatory method of Moscona, without using elevated K+ or ATP in the dispersal medium or culture medium, and have obtained a large proportion of cells with highly differentiated membrane electrical properties. This research was supported by a grant from the U.S. Public Health Service, National Institutes of Health (No. 5SOlRR05431-13). M.J.M. is a Predoctoral Trainee supported by an NIH Training Grant (HL-05815). An abstract of these results has appeared in Fed. Proc. 34, 391 (1975). REFERENCES CARMELIET, E. E., HORRES, and VEREECKE, J. S. (1975).

C. R., LIEBERMAN, Potassium

permeabil-

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ity in the embryonic chick heart: Changes with age, external K, and valinomycin. Zn “Developmental and Physiological Correlates of Cardiac Cells” (M. Lieberman and T. Sano, eds.), pp. 103116. Raven Press, New York. DEHAAN, R. L., and FOZZARD, H. A. (1975). Membrane response to current pulses in spheroidal aggregates of embryonic heart cells. J. Gen. Physiol. 65, 207-222. DEHAAN, R. L., MCDONALD, T. F., and SACHS, H. G. (19751. Development of tetrodotoxin sensitivity of embryonic chick heart cells in vivo and in vitro. In “Developmental and Physiological Correlates of Cardiac Cells” (M. Lieberman and T. Sano, eds.1, pp. 155-168. Raven Press, New York. LIEBERMAN, M., SAWANOBORI, T., KOOTSEY, J. M., and JOHNSON, E. A. (1975al. A synthetic strand of cardiac muscle. Its passive electrical properties. J. Gen. Physiol. 65, 527-550. LIEBERMAN, M., SAWANOBORI, T., SHIGETO, N., and JOHNSON, E. A. (197513). Physiological implications of heart muscle in tissue culture. In “Developmental and Physiological Correlates of Cardiac Cells” (M. Lieberman and T. Sano, eds.1, pp. 139-154. Raven Press, New York. MCLEAN, M. J., and SPERELAKIS, N. (1974). Rapid loss of sensitivity to tetrodotoxin by chick ventricular myocardial cells after separation from the heart. Exp. Cell Res. 86, 351-364. NARAHASHI, T., MOORE, J. N., and SCOTT, W. R. (1964). Tetrodotoxin blocking of sodium conductance increase in lobster giant axons. J. Gen. Physiol. 47, 965-974. SACHS, H. G., MCDONALD, T. F., and DEHAAN, R. L. (1973). Tetrodotoxin sensitivity of cultured embryonic heart cells depends on cell interactions. J. Cell Biol. 56, 255-258. SCHNEIDER, J. A., and SPERELAKIS, N. (1975). Slow Ca++ and Na+ responses induced by isoproterenol and methylxanthines in isolated perfused guinea pig hearts exposed to elevated K’. J. Molec. Cell. Cardiol. 7, 249-273. SERAYDARIAN, M. W., ARTAZA, L., and ABBOTT, B. C. (1972). The effect of adenosine on cardiac cells in culture. J. Molec. Cell. Cardiol. 4, 477-484. SHIGENOBU, K., and SPERELAKIS, N. (19721. Ca++ current channels induced by catecholamines in chick embryonic hearts whose fast Na’ channels are blocked by tetrodotoxin or elevated K+. Circ. Res. 31, 932-952. SHIMIZU, Y., and TASAKI, K. (19661. Electrical excitability of developing cardiac muscle in chick embryos. Tohoku J. Exp. Med. 88, 49-55. SPERELAKIS, N. (19671. Electrophysiology of cultured chick heart cells. In “Electrophysiology and Ultrastructure of the Heart” (T. Sano, V. Mizuhira, and K. Matsuda, eds.1, pp. 81-108. Bunkodo Co., Tokyo. SPERELAKIS, N. (1969). Lack of electrical coupling between contiguous myocardial cells in vertebrate

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hearts. In “Comparative Physiology of the Heart: C,urrent Trends” (F. V. McCann, ed.), pp. 136-165. Birkhauser Verlag, Basel. SPERELAKIS, N. (19721. The electrical properties of embryonic heart cells. In “Electrical Phenomena in the Heart” (W. C. De Mello, ed.), pp. 1-61. Academic Press, New York. SPERELAKIS, N., and LEHMKUHL, D. (1965). Insensitivity of cultured chick heart cells to autonomic agents and tetrodotoxin. Amer. J. Physiol. 209, 693-698.

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SPERELAKIS, N.,and SHIGENOBU, K. (1972).Changes in the membrane properties of chick embryonic hearts during development. J. Gen. Physiol. 60, 430-453. SPERELAKIS,N.,SHIGENOBU, K.,and McLEAN,M.J. (1975). Membrane cation channels - Changes in developing hearts, in cell culture, and in organ culture. In “Developmental and Physiological Correlates of Cardiac Cells” (M. Lieberman and T. Sano, eds.), pp. 204-234. Raven Press, New York.

Retention of fully differentiated electrophysiological properties of chick embryonic heart cells in culture.

DEVELOPMENTAL. Retention BIOLOGY s&134-141(1976) of Fully Differentiated Chick Embryonic MICHAEL Department of Physiology, J. University Elec...
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