Beating hamster heart cells in tissue culture

Hamsfer 4. 5. 6.

herrrf

613

tissue culture

0’. F., DAVIS, E. V., GLOVER, R. L. and R.4KE, G. \I’., .J. Immunol. 79, 428 (1957). MILLONIG, G., J. Appl. P&s. 32, 1637 (1961). OSSER>XAN, E. F., RIFKIND, R. A., TSKATSUKI, F. and L.~WLOR, D. P., Ann. X.1’. Acad.

MCLIJIANS,

Sci. 113, 627 (1964’). 7. PERSONS,

D.

Biophys.

F.,

BENDER,

M. A.,

DARDEN,

E. B.,

PRATT,

G. T. and

LINDSLEY:

D. L.,

.I.

Hioehem. Cgtol. 9, 369 (1961).

8. PETTENGILL, 0. S. and SOREXSON, G. D., J. Cell Viol. 23, 724 (1964). In vitro 2, 128 (1966). 10. PETTENGILL, 0. S., SORE~SOS, G. D. and ELLIOTT, hI. L., Arch. P&h. 82, 483 (1966). 11. POTTER, M. and FAHEY, .J. L., .J. iVat Cancer Inst. 24, 1153 (1960). 12. POTTER, M., FAHEY, .J. L. and PILGRIM, I. A., Proc. Sot. Ezpfl Biol. Med. 94, 327 (1957). 13. R.4ssos, A. S., O’CONOR, G. T., BARON, S., \YHANG, J. J. and LE~ALLAIS, 1’. Y., Jnf. J.

9. ~-

Cancer 1, 89 (1966). 11. 13.

S~II~TINI, D. D., BENESCH, I<. and BARNETT, R. J., J. Cell Biol. 17, 19 SORESSON, G. D. and PETTESGILL, 0. S., J. Cell Hiol. 31, 1llA (1966).

BEATING

HAMSTER

HEART

W. DEW. ANDRUS*

CELLS

IN TISSUE

(1963).

CULTURE]

and F. F. STRASSER

Pasadena Foundation for Medical Research, Pasadena, CaZi$ 91101, U.S.A.

Received January 6, 1967

SmGm mammalian

heart cells were first cultured by Harary and Farley [I, 21 from trypsinized newborn rat hearts. For a study of malignant transformation by polyoma virus Porwit-Bobr et al. [7], prepared non-beating cultures from hamster myocardium disaggregated with trypsin. Mark and Strasser [6] have described modifications of Harary and Farley’s methods that allow preparation and observation of rat heart cultures with a high proportion of beating cells. In spite of the occasional use of hamster heart in culture as explants [4, 81, successful culture of single beating hamster heart cells has apparently not previously been reported. The present paper deals with procedures that provide cultures containing beating myocardial cells from the golden hamster (Mesocricetus auratus) and briefly describes some features of these cells in culture. Materials and Methods.-Ventricles from l- to 4-day-old hamsters were cut into 3 or 4 pieces and trypsinized as described by Mark and Strasser [6] except that as 15 to 20 min trypsinizations were used and the first two many as seven successive discarded. Approximately 1.25 x lo5 cells/ml medium (modified Eagle’s MEM) were investigation was supported in part by Public Health Service Fellowship No. l-FS-HEthe National Heart Institute, by the U.S. Army Medical Research and Development Command, Department of the Army, under Grant No. DA-MD-49-193-66-G183, and by U.S.P.H.S. Research Grant No. NB-03113 from the National Institute of Neurological Diseases and Blindness. 2 Permanent address: Zoology Department, Pomona College, Claremont, Caiif. 91711, U.S.A. 1 This

10, 203-01 from

Experimental

Cell Research 47

614

W. DeW. Andrus and F. F. Strasser

seeded into conventional 2 ml Rose chambers. One or two days later the chamber was dismantled and a sheet of perforated cellophane (Microbiological Associates, Washington, D.C.) was laid directly over the cells that had become attached to the bottom coverslip and the chamber reassembled, but this time with a gas-permeable polystyrene coverslip as the top of the chamber (see [6]). Results.-Mark, Hackney and Strasser [5] have observed a differential effect of cellophane on rat endothelioid cells (see their Fig. 3), but with hamster hearts the effect is much more pronounced. In hamster cultures if the cellophane overlay is not used, a distinctive type of non-beating cell, assumed to be endothelial in origin, will overgrow the myocardial cells and cause a rapid decline in the number of beating cell groups such that beating usually stops in 6 to IO days. With the cellophane present, however, the endothelioid cell overgrowth is retarded (though not completely blocked) and cultures will show contractions in vigorous muscle groups for several weeks. Fig. 1 emphasizes the effect of cellophane on the growth habit of hamster ventricle cells in uifro, showing in Fig. la the typical radiating appearance of muscle cell groups under cellophane; in Fig. 1 b the crowded appearance of endothelioid and typically non-beating muscle cells within a pore, and in Fig. 1 c the type of cell sheet that develops in the absence of cellophane. Fig. 2 shows cytological details (a) in muscle and (b) endothelioid cells. Z bands in the myofibrils of the muscle cell and the more filamentous mitochondria and prominent golgi zone of the endothelioid cells, apparent also in the rat [3], are readily seen. Hamster myocardial cells in culture differ from those of the rat, particularly in the relationship between adjacent beating muscle groups. Mark and Strasser [6] demonstrated that while two separate rat myocardial cells may beat at differing rates, soon after they become joined together, whether by direct muscle-to-muscle cell contact or through contact of each beating cell with another non-beating (endothelioid) cell, they will be beating in synchrony. By this contact synchrony process large portions of the cell sheet soon contract at the same rate. Fig. 3 shows that conditions for such contact synchrony are not the same in hamster heart cultures. By Figs. 3 b and 3 c, although both muscle cells appear to be in contact with the endothelioid cell background, they still beat at differing rates, and it is only when the two muscles connect Fig. l.--Cultured hamster heart cells (8 days) phase contrast; a. cells cultured with a cellophane overlay. Part of a 0.65 mm diameter pore (P). A prominent muscle group (M), linked with smaller muscle clusters, shown against a background of endothelioid cells (E); b, detail of a cellophane pore crowded with endothelioid cells; c, culture without a cellophane overlay showing early overgrowth by endothelioid cells. Fig. 2.-Cultured hamster heart cells. High power phase contrast; a, typical muscle elements displaying myofibrils (MF) with z bands; b, endothelioid cells showing filamentous mitochondria (MT) clustered around probable golgi zone (G). Fig. 3.-Stages in the development of contact synchrony between adjacent hamster heart muscle cells followed with still photography from day 3 through day 6 of a culture. The edge of a cellophane pore is visible at the lower margin of each photograph. a, at 3 days 2 individual muscle cells which beat at different rates; b, at 5 days the apparent contact of these muscle cells with the advancing sheet of endothelioid cells did not lead to beat synchrony; c, at 6 days the two cells, still apparently in contact with the endothelioid sheet are not synchronized; d, 1 hr later than c the cells are firmly in contact with each other (see arrow) and beating in synchrony. Experimental

Cell Research 47

(i 15

Hrtmster heart tissue culture

directly with each other (1 hr later in Fig. 3d) that they begin to beat synchronously (we have also followed the phenomenon with natural speed and time-lapse cinematographic records). What significance this difference in ease of synchrony may have for the intact animal is not clear. We wish to thank

Dr Frederick

H. Kasten

for the photographs

in Fig. 2.

Experimental

Cell Research 47

616

J. Michl and J. Svobodovci

REFERENCES 1. 2. 3. 4. 5.

HARARY, I. and FARLEY, B., Science 131, 1674 (1960). -Exptl Cell Res. 29, 451 (1963). KASTEN, F. H., BOVIS, R. and MARK, G. E., J. Cell Biol. 27, 122A (1965). LYMAN, C. P. and BLINKS, D., J. Cell Comp. Physiol. 54, 53 (1959). MARK, G. E., HACKNEY, J. D. and STRASSER, F. F., in Proceedings of the Gordon Research Conference on Factors Influencing Mvocardial Contractility, Aug. 22-26. In Dress. 6. MARK, G. E. and STRASSER, F. F., Expi Ce% Res. 44, 217 (1966): 7. PORWIT-BOBR, A., CHLAP, Z., ROKITOWA, Z. and JASZCZ, W., Acta Med. Polona 4, 209 (1963). 8. kHYIDTMANN, iw., 2. ges. exptl Med. 45, 714 (1925).

RNA

TURNOVER

AND

HUMAN

THE

CELLS

J. MICHL

GROWTH

POTENTIAL

OF

IN CULTURE

and J. SVOBODOVA

Tissue Culture Laboratory, Institute of Physiology, Czechoslovak Academy of Sciences, Research Institute for the Medical Radioisotopes, Prague, Czechoslovakia

Use of

Received January 16, 1967

IN

previous experiments it was found that the levels of 32P soluble intermediates in cells are in a positive correlation with the growth rate of cells; significant differences were found between HeLa cells and human diploid cells [6]. For this reason we studied in further experiments the relationship between the acid soluble pool and metabolic state of RNA-probably the most important macromolecular cell component which can be in an equilibrium with soluble compounds (11. In the experiments HeLa cells and human diploid cells, derived from embryonic lungs, strain LEP 14, were used [3]. The cells were cultivated in 1200 ml Roux flasks in a synthetic medium [4] supplemented with 10 per cent of inactivated calf serum. After the incubation with Na,H32P0, (0.1 &/ml) or 3H-uridine (1.0 ,&/ml) the cells were washed two-fold with a fresh medium and scraped off from the glass with a rubber policeman. The cells were sedimented at 140 g and the packed cell volume was recorded.

The cells were

then

extracted

with

cold

8 per cent

trichloroacetic

acid

and after the centrifugation the radioactivity of aliquots of the supernatant was measured. In the experiments with 3H-uridine the cells were further extracted with 80 per cent ethanol, 96 per cent ethanol, with a mixture ethanol-chloroform (3: 1) and with

ether;

the remaining

Chicago) and the radioactivity Tracerlab [5]. Experimental

Cell Research 47

sediment

was solubilized

in NCSTM

was measured with the liquid

solubilizer

scintillation

(Nuclear

counter