Collagenase- and trypsin-dissociated heart cells: A comparative ultrastructural study

Journal

of Molecular

andCellular

Cardiology

(1976)

8,747-757

Collagenaseand Trypsin-Dissociated A Comparative Ultrastructural MIREILLE Department

MASSON-PEVET,

8 October

Cells :

J.

DE

BRUIJNE

and Department for Electron Amsterdam, the .Netherlands

Microscopy,

H. J. JONGSMA

of Physiology, University of Amsterdam Faculty of the Free University, (Received

Heart Study AND

1974, and accepted 24 February

Medical

1976)

M. MASSON-P~VECT, H. J. JONGSMA AND J. DE BRIJIJNE. Collagenaseand Trypsin-Dissociated Heart Cells: A Comparative Ultrastructural Study. Journal of Motecuiar and Cellular Cardiology (1976) 8,747-757. Two enzyme preparations were used to dissociate new-born rat hearts: crude trypsin and crude collagenase. An ultrastructural study of the cardiac cells has been made at two stages: just after their isolation and centrifugation and again after they had been plated and grown into monolayers. A comparison was made with the ultrastructure of Z-day-old rat ventricular cells fixed iz situ. Immediately after centrifugation, i.e. fixed in the pellet, trypsinized heart cells were severely damaged whereas no evident injury was observed in collagenase-dissociated cells. Once cultured in monolayers, the recovery time of these cells was very different, depending on the enzyme employed: one week after using trypsin, and 2 days after using colfagenase. Therefore, we conclude that collagenase is clearly superior to trypsin as a means to dissociate heart cells, and that collagenase-treated cells represent a much better model of the intact heart, especially when used for experiments within the first week of cultivation. KEY

WORDS:

Rat heart;

Trypsin;

Collagenase;

Ultrastructure;

Tissue culture.

1. Introduction

Cultured heart cell preparations are used increasingly by physiologists to gain insight into the active electrical properties of heart cells, which is understandable if one considers the difficulties encountered in intact heart preparations [see 191. In order to extrapolate the results obtained with in vitro preparations to the intact heart, one must be sure that the results are not dependent upon the properties of the cultured cells, and thus partly on the technique used to dissociatethe hearts. In this study, two enzyme preparations were used to dissociate newborn rat heart cells, crude trypsin and crude collagenase.Trypsin has been used becauseit is the most widely used dissociating agent for heart tissues (chick embryo [5, 7, 8, 11, 17, 18, 30, 40, 45, 491, mouseembryo [Zl], newborn rat [6, 14, 21, 23, 24, 26, 27, 29, 32, 441) and collagenasebecauseit has been reported to dissociateembryonic chick hearts [4, 15, 421 and adult rat hearts [2, 12, 281. In this paper we report a comparison of the ultrastructure of isolated heart cells obtained just after centrifugation, and again after they had been plated and cultured in monolayer-s.Comparison is made with the ultrastructure of ‘L-day-old rat ventricular cells fixed in situ.

748

M.

MASSON-PiVET,

H. J. JONGSMA

2. Material

and

AND

J.

DE

BRUIJNE

Methods

Tissue culture technique Ten to 12 hearts of 2-day-old Wistar rats were excised under sterile conditions. After removal of blood vessels, the hearts were cut in small pieces and incubated in the dissociation medium. Trypsin dissociation The cells were cultured as described by Jongsma [,?I]. fragments of heart muscle were incubated at 37°C with 20 ml of a 0.05% (NBC; 1:300) solution in pucks saline A [31], which is Ca and Mg free. min the supernatant was decanted and discarded to remove cellular debris blood cells. The procedure was repeated 3 times for 20 min each time with aliquot of trypsin solution.

In brief, trypsin After 20 and red a fresh

Collagenase dissociation Fragments of hearts were incubated for 2 x 30 min in Pucks saline A [31] pH 7.2 to 7.4 (1.5 ml medium per heart) containing 450 units/ml of crude collagenase (Worthington Biochemical Corp.; CLS fraction type I), 0.001% DNase (Worthington Biochemical Corp. ; DP fraction 1400 units/mg) to digest the slimy material formed during the dissociation process [46], 0.01 mM Ca2+ and 0.6 mM Mgs+. Bicarbonate buffer was replaced by 20 mM HEPES buffer. After the first incubation period of 30 min, the tissue fragments were mechanically dispersed by passing them several times through a large-bore pipette. After both trypsin and collagenase dissociation, the resulting cell suspensions were cooled in ice for 5 min, and centrifuged for 5 min at 250 g. The pellets were washed with Pucks saline A [31]. The final cell suspensions were pooled, counted with an haemocytometer and plated in plastic dishes (Falcon plastics; 0 5.0 cm). Growth medium was added to obtain a final cell concentration of 5.105 cells/ml. The dishes were incubated at 37°C in an atmosphere of air and CO2 with a relative humidity of 80 to 90%. pH in the culture dishes ranged between 7.2 and 7.4. The medium was changed every 48 h. The growth medium consisted of Hams FlO medium [13] containing 10% fetal calf serum and 10% horse serum. Tryptic activity of crude trypsin and of crude collagenase has been determined according to Bergmeyer [I].

Preparation for electron microscopic examination The cultured cells were fixed and embedded directly in the plastic dishes at different times after plating, from 20 h to 10 days. They were rinsed in Hanks salt solution prior to fixation, fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 10 min at room temperature, and postfixed in phosphate buffer-osmium

PLATE 1. Portions of ventricular cells of a P-day-old rat heart fixed in situ. The myofilaments are organized in myofibrils (myo). Glycogen granules (gl) are abundant. These cells are well differentiated. mit mitochondria; N nucleus. x 13 500. PLATE 2. Cross section of a T tubule (T) in a ventricular cell of a two day old rat heart fixed ir2 situ. Two cisternae of the SR are closely apposed to this tubule forming a triad or interior coupling (arrow heads). gl glycogen granules; mit mitochondria. x 25 000. PLATE 3. Intercalated disc (ID) between two ventricular cells of a Z-day-old rat heart fixed in situ. This disc is of a “step” type, and fasciae adherentes (fa), desmosomes (d) and nexus (n) are present in it. x 25 000. PLATE 4. Heart cell fixed in the pellet after trypsin dissociation. The nucleus (Nj lies against the sarcolemma, mitochondria (mit) are aggregated around the myofilaments (myo) which form a tangled mass in the clear cytoplasma. Cytoplasmic vacuoles (V) are quite numerous. Free mitochondria from broken cells are observed on the left bottom. x 13 500. PLATE 5. Heart cells after 2 days in culture following trypsin dissociation. The volume occupied by the myofilaments (myo) is low. Extensive rough endoplasmic reticulum (rer) and Goigi complex (g), numerous polyribosomes (pr) are present in the sarcopiasma. Mitochondria (mit) are small with few cristae. 2 Z line material. X 3 1 500. PLATE 6. Heart cell after 3 days in culture following trypsin dissociation. Fibrillar material (f) is formed at the periphery, electron dense Z line material (Z) being associated with these filaments. mit mitochondria; pr polyribosomes. x 27 000. PLATE 7. Heart cell after 3 days in culture following trypsin dissociation. Filaments (f) and Z substance (2) have migrated to the center of the cell. A new sarcomere (S) has been formed. Polyribosomes (pr) in a helical array are often associated with new filaments (arrow heads). x 21 000. PLATE 8. Heart cell after 7 days in culture following trypsin dissociation. An interconnected branching pattern of myofibrils is observed. Tubules of the sarcoplasmic reticulum (ST) in longitudinal section are present between the myofibrils. Z Z line. x 30 800. PLATE 9. Heart cell after 3 days in culture following trypsin dissociation. Tubules of the sarcoplasmic reticulum in cross section (arrow heads) are illustrated here at the level of the Z line (Z). mit mitochondria. x 28 500. PLATE 10. Heart cell after 7 days in culture following trypsin dissociation. The tubules in cross section (arrow heads) at the level of the Z line (Z) are not filled with horseradish peroxidase which has been used to mark the extracellular space. Unstained section. x 28 000. PLATE 11. Heart cell after 7 days in culture following trypsin dissociation. Most of the myofilaments are organized in myofibrils (myo) running parallel to each other. Nevertheless, at the top of the picture, some myoliiaments can be seen free in the sarcoplasm, surrounded by numerous polyribosomes. mit mitochondria; pr polyribosomes. x 12 700. PLATE 12. Heart cell after 20 h in culture following trypsin dissociation. A prominent basal lamina (bl) with a wavelike appearance is covering the plasma membrane (pi). x 43 300. PLATE 13. Heart cell after 7 days in culture following trypsin dissociation. The basal lamina (bl) looks like that found around heart ceils fixed in sit@. pi plasma membrane. x 33 500. PLATE 14. Two heart cells after 4 days in culture following trypsin dissociation, separated with an early intercalated disc (ID) following a rather straight course. Fasciae adherentes (fa) desmosomes (d) and nexus (n) can be seen. x 39 000. PLATE 15. The wavelike appearance of an intercalated disc (ID) between two 8 days old cultured heart cells after trypsin dissociation is illustrated on this micrograph. x 29 500. PLATE 16. Heart cell fixed in the pellet after coliagenase dissociation. Myofibrils (myo) are well organized, but follow an angled course especially at the 2 line level (Z) (arrow heads). Mitochondria (mit) are present between the myofibrils. Vacuoles (V) are found in the sarcoplasma. Giycogen granules (gl) are observed between the myofibrils and at the periphery of the cell. x 24 000. PLATE 17. Heart cells after 40 h in culture following collagenase dissociation, cut parallel to the base of the dish. An interconnected branching pattern of myofibrils is observed. Nexus (n), desmosome (d) and fasciae adherens (fa) are present in the wavy intercalated disc. N nucleus; mit mitochondria; g Golgi complex; L lipid droplet. x 22 500. PLATE 18. Heart cells after 43 h in culture following collagenase dissociation, cut perpendicularly to the base of the dish. Myofibrils (myo) in cross and longitudinal section are present side by side. Tubules in cross section are observed at the level of the 2 lines (Z) ( arrow heads). mit mitochondria. x 34000. PLATE 19. Heart cells after 3 days in culture following collagenase dissociation. Peroxidase which marks the extracellular space does not penetrate into the cells. The myofibrils (myo) are running parallel to each other. Unstained section. N nucleus; mit mitochondria. x 12 500. [facing page 7481

-‘t :’ . <.

; ,6

.‘ .mmit i‘

PLATES 1-3

J‘

PLATE 4

PLATE 5

4. .. _-’. .

7

PLATES 6 and 7

..z ,o,

,,

f 4 , i. t

PLATE 16

PLATE I8

ULTRASTRUCTURE

OF CULTURED

MYOBLASTS

749

tetroxide [36] for 15 min at 4°C. Dehydration was made in graded ethanols. Transition to Epon embedding medium was accomplished via l/l 100% ethanol to Epon. Pure Epon was left to polymerise for 48 h at 60°C in Falcon dishes. The pellets were fixed in the same way, directly in the centrifugation tubes after a careful rinse in Pucks saline A [31]. In order to obtain a better contrast, some monolayers were incubated in 0.5% uranyl magnesium acetate in 0.9% NaCl for 15 min prior to dehydration. In some experiments, horseradish peroxidase was used to trace the extracellular space. The cells were then incubated in 0.25% horseradish peroxidase in Hanks salt solution for 15 min at 37°C. After fixation in glutaraldehyde, they were quickly rinsed in 0.2 M cacodylate buffer pH 7.4 and incubated in 0.1 M cacodylate buffer containing O.l1o/o diaminobenzidine and 0.01 o/o hydrogen peroxyde for 1 h at room temperature. After incubation, the monolayers were washed in buffer, post-fixed in phosphate buffered-osmium tetroxide for 15 min and embedded in Epon. After polymerisation of the Epon the dishes were sawed into blocks. The cells were sectioned, either perpendicular or parallel to the base of the dish on a Reichert ultra microtome with glass knives. Sections were mounted on copper grids with parlodion film, stained with lead citrate or double stained with uranyl acetate and lead citrate and examined with a Philips 301 electron microscope. Two-day-old rat hearts were fixed in situ as follows: the hearts were perfused with a physiological salt solution [34], and fixed similarly with 1.5% glutaraldehyde in 0.09 M phosphate buffer pH 7.4 for 10 min. The pieces were postfixed by immersion in 1 o/o 0~04 in the same buffer for I h, dehydrated in graded ethanols and embedded in Epon.

3. Results Ultrastructure

of 2-day-old rat ventricular cells jked in situ

A brief review of the ultrastructure of 2-day-old ventricular cardiac cells is given here first so as to compare it with the ultrastructure of heart cells in culture, and thus to study the influence of enzymatic digestion on these cells. We will not go into detail as newborn rat heart cells, at a first approximation, have an organization similar to that described by other investigators for adult mammalian heart cells [lo, 20, 38, 43,471. The nucleus was fusiform, situated near the center of the cell, and oriented along its long axis. Near the poles of the nucleus, the cytoplasm contained a Golgi complex, mitochondria, some cisternae of rough endoplasmic reticulum and glycogen granules. Myofibrils showed the same organization, and seemed to have the same distribution as in adult rat heart cells (Plate 1). Glycogen granules were very

750

M. MASSON-PIhET,

H.

J. JONGSMA

AND

J. DE

BRUIJNE

abundant : they were found particularly between the myofilaments at the level of the I bands, and around the mitochondria (Plate 1). The sarcoplasmic reticulum formed interconnected networks that were frequently more intricate in the region of the Z lines. When the cardiac cells were separated from each other by a large intercellular space, the plasma membrane was covered by a basal lamina. A transverse tubular system or T system was present, but quantitatively less abundant than in adult ventricular cells. The sarcoplasmic reticulum formed couplings with the sarcolemma (peripheral couplings), and with the membranes of the T tubules (interior couplings: diads or triads) (Plate 2). The cells were interconnected by step-like intercalated discs presenting the three types of junctional specialization found between adult ventricular cells : fasciae adherentes, maculae adherentes and nexuses (Plate 3).

Ultrastructure

of 2-day-old

rat heart cells directly after trypsiniration,

and after plating

The isolated heart cells obtained after trypsinization and centrifugation and fixed in the pellet were rounded with numerous rather thin finger-like extensions. They apparently were severely damaged by the dissociation procedure (Plate 4). The nucleus, which was found in the center of the cell or against the sarcolemma, was often distorted with finger-like extensions. The mitochondria were aggregated in the cell center or along the margin at the cell membrane. Their ultrastructure generally showed no signs of damage. The myofibrils were completely disrupted and the myofilaments formed a tangled mass in which thin and thick filaments could hardly be recognized. The Z line material had nearly completely disappeared. Numerous vacuoles were present. Free organelles, and especially mitochondria were commonly observed extracellularly around the heart cells fixed in the pellet, attesting to the fact that plasma membranes had been broken during the dissociation (Plate 4). Once they were plated, the isolated cells attached themselves to the bottom of the dish and start contracting after 15 to 20 h of incubation. After 2 days in culture, they had divided and grown enough to form a monolayer which contracted synchronously [22]. Twenty hours after plating, the volume occupied by myofibrils in these cardiac cells was much smaller than before the dissociation (Plate 5) and their degree of organization was often very low. These young muscle cells in culture had a clear cytoplasm filled with an extensive rough endoplasmic reticulum, many free polyribosomes, which were mostly arranged in clusters or in a helical array, rather small mitochondria, which mostly had sparse cristae, and a large Golgi complex (Plate 5). The nucleus was big and contained one or more nucleoli, the perinuclear cistern was wide and nuclear pores abundant. The formation of new filaments was commonly observed (Plate 6). The first

ULTRASTRUCTURE

OF CULTURED

MYOBLASTS

751

fibrillar material seen consisted of entangled filaments about 50 nm in diameter, which were located mainly at the periphery of the cell just under the sarcolemma (Plate 6). Always associated with these filaments were electron dense areas with irregular contours (Plate 6), which presumably contained Z line material. After some time, both Z line material and filaments left the sarcolemma and migrated towards the center of the cell (Plate 7). The long axis of the filaments became oriented parallel to the cell membrane. At this stage the filaments seemed to be inserted into the Z material which had proliferated in elongated bands with no defined shape. Later on these bands of Z material were divided into smaller fragments into which filaments were always inserted. In this way sarcomeres were formed in the cytoplasma (Plate 7). When the cells had been between 4 and 7 days in culture after trypsinization, myofibrils were present, but not all of them were running in the same direction: as in embryonic hearts, an interconnected branching pattern of myofibrils could be observed in longitudinally sectioned cells (Plate 8). When sarcomeres were present, sarcoplasmic reticulum (SR) tubules formed a lacelike network in close apposition to the surface of the myofibrils (Plate 8). In longitudinal sections, tubules in cross section were commonly found at the Z line level (Plate 9). These tubules could not be filled with horseradish peroxidase which was used to mark the extracellular space (Plate 10). This suggests that they do not communicate with the extracellular space and that they belong to the SR. Peripheral couplings were often observed (Plate 10). After 7 days of culturing, the well organized myoblasts were characterized by myofilaments arranged in parallel myofibrils (Plate 11). However, even at this time some free myofilaments were found in the cytoplasm with free polyribosomes in helical arrays at their surface (Plate 11). The number and size of the mitochondria were larger. The mitochondrial matrix was dense and the cristae often had an angulated appearance. The rough endoplasmic reticulum, the number of polyribosomes and the Golgi complex were less extensive. A basal lamina was not always observed around the young cultured cells. When present, it often was thick with a wavelike appearance (Plate 12). Its thickness may reach over 100 nm. As the cells in vitro became older, the basal lamina became thinner and appeared more similar to that seen around newborn rat heart cells. Its thickness was then about 40 nm, and it sometimes appeared subdivided into an electron-dense outer zone and an electron-lucent zone adjacent to the plasma membrane (Plate 13)) like that around ventricular heart cells. Intercalated discs between cultured heart cells were present in a rudimentary form as early as 1 day after plating. These early discs were short and followed a rather straight course (Plate 14). Already at this time, fasciae adherentes, desmosomes and nexuses were present (Plate 14). After some days in culture, the parallel membranes within the discs were thrown into finger-like projections and had a wavelike appearance (Plate 15).

752

M. MASON-PhET,

Ultmstructure

of Bday-old

H. J. JONGSMA

AND

J. DE

BRUIJNE

rat heart cells directly after collagenase dissociation, and after plating

The isolated heart cells were either rounded with some thin finger-like projections or elongated. The nucleus was situated in the cell center. Well organized myofibrils were present but were compressed and angular, especially at the Z line level (Plate 16). Mitochondria were present between the myofibrils, and vacuoles were found in the cytoplasm. Many glycogen granules were seen just under the sarcolemma, at the periphery of the cells and in the projections. Some mitochondria were present in the pellet between the cells, but far less than after trypsin dissociation. The effects of trypsin and collagenase on heart cells in the pellet are summarized in Table 1. TABLE

1

Ultrastructure of isolated heart cells in the pellet After trypsin dissociation After collagenase dissociation .____-.Round with finger-like Round with some extensions extensions or elongated

Cell shape Nucleus Mitochondria Myofibrils

Often

distorted

Disorganized,

aggregated Z line

material

Free organelles

Oval

Mostly in the pellet

myofilaments

into a mass

disappeared

Numerous

with

smooth

outline

Dispersed between myofibrils

Aggregated

Well organized but angled course

Present within Very few

with

myofibrils

During the first hours of culturing, the cells attached themselves to the bottom of the Petri dishes and spread out. They started beating about 10 h after plating and they beated synchronously after 24 h in culture. The myofibrils, which were angled in newly isolated cells, were stretched out parallel to the plane of the bottom of the dish (Plate 17), although not all of them were running in the same direction. Thus, when the cells were cut perpendicularly to the dish base, myofibrils in cross and longitudinal section were observed side by side (Plate 18). An interconnected branching pattern of myofibrils was commonly observed during the first day of culturing, and to a lesser degree during the second (Plate 17). Two days after plating, the myofibrils were running parallel to each other (Plate 19). At the Z line level tubules in cross section were often observed (Plate 18). Horseradish peroxidase used to mark the extracellular space did not fill these tubules (Plate 19), and it is probable that they belong to the SR. Mitochondria and rough endoplasmic reticulum did not show apparent differences to those in hearts fixed in situ, but the Golgi apparatus was larger and the number of free polyribosomes higher (Plate 17). No difference was observed regarding the basal lamina between cells obtained by trypsin and collagenase dissociation. Intercalated discs with a wavelike appear-

ULTRASTRUCTURE

OF CULTURED

MYOBLASTS

ante were present as early as 30 h after plating. Nexuses, desmosomes adherentes were present in these discs (Plate 17).

753 and fasciae

Remarks about collagenase technique We tried to dissociate young rat hearts with highly purified collagenase, but very few cells were isolated. It is clear that the dispersing power of crude collagenase depends, at least partly, on impurities. As trypsin was the most abundant impurity present, we measured tryptic activity of crude collagenase. It was found to range from 3 to loo/, of tryptic activity of crude trypsin we used. We tried to dissociate hearts using 0.005% crude trypsin as dissociating enzyme solution, this amount of trypsin being the maximum present in the crude collagenase we used. Only a few cells were isolated, too few to culture.

4. Discussion As we have shown, 2-day-old rat ventricular cells were well differentiated, and in many aspects comparable to adult ventricular cells. Fixed just after their isolation, these cells showed many differences, depending on the enzyme used to dissociate the heart muscle. After trypsinization, the structural damages were dramatic. Similar results have already been published by Kasten [25] on neonatal rat heart cells, and by Fischman and Moscona [II] on 6-day-old embryonic chick and 14-day-old embryonic mouse ventricular heart cells. These last authors also showed that myofibrillar damage could not be ascribed to the incubation of heart fragments in a calciumand magnesium-free salt solution as is necessary with trypsin dissociation. Centrifugation employed to sediment the cells can also be excluded as the cause for the observed structural changes, since cells in the pellet after collagenase dissociation show far fewer structural changes. Thus we can definitely state that trypsin is responsible for the myofibrillar damage observed. Wollenberger [49] reported that trypsinization of embryonic chick hearts disrupts the myofibrillar structures of the cells, presumably because the enzyme penetrates into the cells and digests myosin and actin [35]. Using fluorecent and electron-microscopical autoradiography techniques to study the localisation of trypsin in cultured mammalian cells, Hodges et al. [16] observed an intracellular penetration of trypsin as well as its presence at the cellular surface. The intracellular penetration was very marked at 37°C and enzyme activity could be demonstrated in cultured cells for up to about 48 h. During the first days after plating, trypsinized heart cells were actively synthesizing proteins, as shown by the typical appearance of the nucleus and the nucleolus as

754

M.

MASSON-PiVET,

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J.

JONCSMA

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J.

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BRr-IJNE

well as by the presence of multiple polyribosomes amidst slender newly formed filaments. The pattern of development of the myofilaments was identical to that observed in embryonic rat ventricular myoblasts [33] and has been described in cultured ventricular cardiac cells [29J. Only one week after plating the cells had reached an ultrastructural level of organization comparable to that of 2-day-old rat ventricular cells in situ. In this period of time, new filaments were synthesized, internal cell structures rearranged according to the new geometry of the cells, and intercalated discs between the cells formed. After use of collagenase, the recovery time is much shorter: 2 days after plating, the cells were already comparable to those of P-day-old rats in situ. But in this case, even directly after centrifugation, the cells were in much better condition. The internal cell structures were not damaged by the enzyme preparation and especially, myofibrils were not disrupted. These results are in agreement with those of other authors who used collagenase to dissociate embryonic chick hearts [4,42]. We found that the dispersing power of highly purified collagenase to dissociate heart cells was nearly negligible. This has already been mentioned by Kono [28], and is understandable because collagen fibers are not present around every heart cell. On the other hand, O.OO5o/o crude trypsin used to dissociate cardiac muscle permited the isolation of very few cells. We know that this amount of trypsin is the maximum present in the crude collagenase we used. It means that the good dispersing effect we get with crude collagenase is due to the enzyme combination of collagenase-trypsin, trypsin being present only in a very low amount. The influence of some other impurities such as caseinase on the dispersing power of crude collagenase has not been investigated, and cannot be completely excluded. No damage comparable with that observed after use of trypsin was found in cells dissociated with crude collagenase. The concentration of trypsin present as an impurity probably was too low for such effects to become evident. Trypsin is known to provoke ultrastructural alterations of the cell surface [3, 9, 471, releasing glycoproteins and sugars from the cell membrane [37, 411 and has been shown to adsorb to cell surfaces, and to prevent the formation of glycoprotein material [39]. The enzyme persists there, in an active form, for as long as 24 h after application [39]. Although our findings pertaining to the basal lamina are not conclusive, it may be stated that the contaminants of crude collagenase, mainly trypsin, might be responsible for loosening intercellular material other than collagen and thus cell surface material, although the concentration of this enzyme is too low to damage the cell organelles. This would explain the poor results obtained with pure collagenase. Work is now going on in our laboratory to establish to what extent the membrane is damaged by different enzyme preparations. When we use cultured myocardial cells as a simplified model for the heart, the maintenance of a “normal” organization of the cell is most important. It appears on the basis of our results that collagenase may be considered clearly superior to trypsin as a means of dissociating heart cells, if one considers the ultrastructure of

ULTRASTRUCTURE

OF CULTURED MYOBLASTS

newly dissociated cells, and of cells from the monolayers week of culturing.

observed

755 during

the first

Acknowledgements

The authors wish to thank Prof. Dr L. N. Bouman, Dr Elisabeth C. M. Hoefsmit and Prof Dr J. James for their helpful discussions, and C. C. Hollander-Schoonlingen, J. C. Demmers and J. 0. Boorsma for technical assistance. This investigation was partly supported by the Netherlands foundation for pure research (ZWO).

REFERENCES 1.

BERGMEYER, H. U. In Methods

ofEnzymatic Analysis, Vol. 1. pp. 515-516. New York: Chemie, Weinheim/Academic Press Inc. (1975). 2. BERRY, M. N., FRIEND, D. S. & SCHEUER, J. Morphology and metabolism of intact muscle cells isolated from adult rat heart. Circulation Research26,67%687 (1970). 3. BOERM, B., LUNDI, P. M., NORBY, K. & SCHELI, U. Ultrastructure of a mouse sarcoma in its solid, enzymatically dissociated and ascitic form. International Journal of Cancer 3, 283-290 ( 1968). 4. CAVANAUGH, D. J., BERNDT, W. 0. & SMITH, T. E. Dissociation of heart cells by collagenase. Nature 200,261-262 (1963). 5. CAVANAUGH, M. W. Pulsation, migration and division in dissociated chick heart cells in vitro.Journal of Experimental .Zoolosy 128,573-585 (1955). 6. CEDERGREN, B. & HARARY, I. In vitro studies on single beating rat heart cells. VII. Ultrastructure of the beating cell layer. 3oumal of Ultrastructure Research 2, 443-454 (1964). 7. DE HAAN, R. L. & GO~LIEB, S. H. The electrical activity of embryonic chick heart cells isolated in tissue culture singly or in connected cell sheets. Journal of General PhysioloD 52,643-665 (1968). 8. DE HAAN, R. L. & HIRAKOW, R. Synchronization of pulsation rates in isolated cardiac myocytes. Experimental Cell Research 70,2 14220 (1972). 9. EDWARDS, G. A. & FOGH, J. Micromorphologic changes in human amnion cells during trypsinization. Cancer Research 19,608-611 (1959). 10. FAWCETT, D. W. & McNwrr, N. S. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle.Journal of Cell Biology 42, l-45 (1969). 11. FISCHMAN, D. A. & MOSCONA, A. A. Reconstitution of heart tissue from suspensions of embryonic myocardial cells: ultrastructural studies on dispersed and reaggregated cells. In Cardiac Hyfiertrophy. N. R. Alpert, Ed. pp. 125-139. New York and London: Academic Press (197 1). 12. GLICK, M. R., BURNS, A. H. & REDDY, W. J. Dispersion and isolation of beating cells from adult rat heart. Analytical Biochemistry 61,32242 (1974). 13. I%M, R. G. An improved nutrient solution for diploid Chinese hamster and human cell lines. Experimental CellResearch 29,515-526 (1963). 14. HARARY, I. & FARLEY, B. In vitro studies on single beating rat heart cells. I. Growth and organization. Experimental Cell Research 29,451-465 (1963). 15. HILFER, S. R. & BROWN, J. M. Collagenase. Experimental Cell Research 65, 246-249 (1971). Verlag

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25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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