Myogenesis on microcarrier cultures

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1983

727

MYOGENBSISON MICROCARRIERCULTURES A. Shainberg*, A. Isac, S. Reuveny ‘, A. Mizrahi ’ and A. Shahar l Life Sciences Department, Bar-Ilan University, Ramat-Can 52100, and Institute for Biological Research, P.O.B. 19, 70450 lIsrae1 Ness-Ziona, Israel.

ABSI’RACT The capacity of embryonic chick myoblasts to grow in vitro on DEAEcellulose microcarriers (MC) has been investigated 6ioscall.y and The cells attached to the MC, replicated and fused morphologically. These myotubes synthesized muscleto form elongated myotubes. such as creatine kinase (CK) and acetylcholine specific proteins, Some of the receptors (AChR), and they contracted spontaneously. development of advantages of this technique are: a> Tridimensional myotubes on MC with orientation of fibers parallel to each other; b) Wscle cells can be cultured on MC for long periods (months); c> Easy harvesting of samples at any time during cultivation; d) DEAE-cellulose MC are commercially available, inexpensive and easy to handle.

INTRODUCT ION Mononucleated myoblasts obtained from chick embryonic skeletal muscle, proliferate and subsequently fuse to produce multinucleated myotubes in monolayer (ML) cultures (Konigsberg, 1963; Shainberg et al., 19711. Steps in the differentiation of myoblasts in vitro are similar to those of muscle tissue in situ. However, whxeskeletal myofibers developing in vivo orient parallel to each other in the direction of efficientphysiologic function, myofibers in vitro in ML cultures develop in a nonoriented random branching pattern cannot be (Turner et al., 1983). Moreover, myotubes in ML cultures maintained for a long period since they peel off the dish following 2-3 weeks of cultivation, due to their intensive contractions. In addition there are difficulties in collecting samples of cells from ML cultures throughout cultivation. We tried to overcome these disadvantages by growing muscle cells on a tridimensional support made of DBAE-cellulose MC (Reuveny et al., 1982; Shahar et al., 1983). In this report we describe morphological and biochemical differentiation of chick myoblasts grown on these

MC.

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MATERIALSAND METHODS Microcarriers: DE-53, microgranular cylindrical DEAE-cellulose anion exchanger [Whatman, England) having an exchange capacity of 2 milliequivalents per gram dry material, were used as a microcarrier for cultivation of muscle cells. They were equilibrated with phosphate buffered saline (PBS), pH 7.4 and autoclaved in hatches of 15 g in lOOm1 PBS. Cells and growth medium: Myoblasts were obtained by mechanical dissociation from the thigh muscle of II-12-day-old embryos of white Most fibroblasts were eliminated by preplating. Leghorn chickens. Cultures were grown in high glucose Dulbecco’s modified Eagle medium (DMFM), supplemented with 10% horse serum and 2% chick embryo extract, in a humid 90% air, 10% CO2 atmosphere. 1. ML cultures were carried out on collagen-coated 30mmdiameter Chick cells were plated at a denplastic dishes (Nunc, Denmark). medium was rein 1.5 ml medium. Nutrient sity of 5x105 cells placed twice a week. 2. MC cultures Prior to addition of cells, MC were washed twice in the medium was 7 in nutrient medium. The final MC concentration mg/ml, in 30 mmplastic dishes without substrate coating. Plating of About 50% of cells on MC was in the same density as for monolayer. the medium was replaced twice a week in the suspended MC cultures. Processing for transmission electron microscopy (TEN) and scanning electron microscopy (SEM1: The cell -MC conglomerates were transferred to Eopendorf’s tubes in which they were washed in PBS and fixed in 2.5%-glutaraldehyde, post-fixed in 1% osmium tetroxide and dehydrated in increments of ethanol. For TEM, embedding was made in Epon. A much harder mixture of Epon than that routinely used for monolayer cultures was required because of the coarseness of the DE-53 MC. For SEM, critical point drying of pellets was made according to Jones and Gillett (1973) in Polaron apparatus, using liquid CO2. Conglomerates were,then spread on a double Scotch tape and vacuumcoated with 200 A of gold-palladium (Polaron sputtering unit). Observations were made in a JEOL 100s TEM and JSM 35C scanning electron microscope at 25 KV. Assay for AChR; The level of AChR was measured by exposing the cells to 6x10-8M 1251-a-BuTX (So-150 Ci/mmol) for 1 hr in DMEM according to Birnbaum et al. (1980). CK activity; Cultures were rinsed in PBS and then homogenized in sodium phosphate buffer (0.1 M, pH 7.01, containing 0.5% Triton Xwas determined on the homogenate at 30°C as previ100. CK activity In brief, the ATP formed ously described (Shainberg et al., 1971). phosphorylates by interaction of ADP with creatine phosphate, The produced glucose-6glucose in the presence of hexokinase. phosphate reduces NADP to NADPH, which is determined by recording absorption changes at 340 nm. of nucleic acids and protein Determination of the rate medium consynthesi s : Cultures were incubated for 1 hr in nutrient

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taining methyl-3H-thymidine (46 Ci/mmol, Amersham) and then washed homogenized and several times with PBS. The cells were collected, precipitated with an equal volume of cold 20% trichloroacetic acid. The precipitate was dissolved in lml 0.5N NaOH, 0.5ml portions were counted in a liquid scintillation counter (Kontron, Switzerland) 10-20~1 portions were taken for protein determination (Lowry). The same procedure was also used for measurement of 3H-uridine (41 Ci/mmol), or 3H-leucine (52 Ci/mmol) incorporation. RESULTS The MC in the plating dishes remained in suspension as single isolated particles in the nutrient medium. Within 24 hr following the addition of myoblasts, most of the cells attached to the MC and aggregated to form cell-MC-conglomerates. These conglomerates were composed of several MC and kept floating in the medium during the of cultivation. The cells on MC-conglomerates entire period multiplied and actively synthesized protein and nucleic acids, although at a lower rate than ML cultures (Table I>. TABLE I. RNA, DNA and protein monolayer muscle cultures

Monolayers

(days 1

(dpm/mg protein)

8 2 8 3 H-Leucine

in

Age

2

3 H-Thymidine

synthesis

microcarrier

and

Microcarriers (dpm/mg protein)

29,814 t 1,256 6,069 -+ 306

16,658 -+ 1,052 162 2,179 +

121,458 -+ 20,101 622 19,450 t

80,411 -+ 5,164 13,694 -+ 692

2

12,255 +

586

8

14,979 -+

945

7,302 -+ 6,562 -+

622 448

In SEM (Fig. 1AB) dividing of myoblasts were observed in conglomerates during the first 2-4 days. Thereafter cells fused to form myotubes, mainly towards the end of the first week in culture. Unlike ML cultures, in which myotube networks were oriented randomly, the myotubes on MC were oriented parallel to each other forming bundles in the same spatial direction.

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tiscle fibers which were small and flat during the first week of cultivation on MC became thicker and cylindrical thereafter. Their surface appeared covered by an abundance of spherical microprojections and microvilli in addition along the myotubes there were equally spaced elongated microvilli approaching the adjacent myotubes. Large swellings noticable in the cytoplasm are probably indicative of nuclear location (Fig. 1B). In TEM (Fig. lC, D), myotubes showed a fully developed sarcomerit organization. They exhibited vesicular nuclei (with a few lobulations) which contained euchromatin with often one prominent Among the cytoplasmatic organelles the most striking nucleolus. bundles of myofibrils with a well evident cross were : structured a large number of mitochondria, part of them oriented striation; several Golgi apparatus cisternae and a typialong the myof ibrils; cal smooth endoplasmic reticulum with a honeycomb-like structure. Myotubes in both ML and MC began to contract spontaneously on the 5th day. Since all the conglomerates moved as one unit, it was sometimes possible to observe spontaneous contractions even without the aid of a microscope. As soon as myoblasts differentiated morphologically, there was also a biochemical differentiation which was manifested by an increase in the activity of CK and the amount of AChR. A comparison of the development of these two proteins in MC and ML is shown in per plate in ML cultures was higher Fig. 2. Although CK activity than that of MC cultures, it became almost the same with respect to The amount of AChR was signifispecific activity (per protein). cantly higher in MC cultures. DISCUSSION DEAE-cellulose particles were shown to provide a tridimensional anchorage for cultured chick myoblasts; these myoblasts replicate their DNA and fuse to form typical myotubes. IGscle cells colonize the MC in culture and form large conglomerates which remained floata fully ing in the nutrient medium. Each conglomerate represents differentiated muscle entity composed of bundles of striated contractile myotubes oriented along the MC. In this way the spatial orientation of the myotubes on MC resembles muscle orientation in samples of one or more conglomerates can l% vivo. In addition, aected (without interfering with the rest of the culture) at any time during cultivation for TIP4, SF%! or biochemical analysis. myotubes in 2-3 week-old ML cultures contract very Furthermore, strongly and thus tend to peel off the dish; subsequently, they myotubes grown on MC establish more In contrast, degenerate. lie firm contacts with the substrate and also among themselves. adherence of the cells to the MC and the flexibility of the floating conglomerates probably increase the survival of the myotubes and enable cultivation on MC for a longer period.

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FIG. 1. SIN (A,B) and TEN (C,D) micrographs of MC muscle cultures. nrt of a conglomerate 3 days in culture with already fused myotubes; B - Adjacent cylindrical myotubes in a 14-day-old culture. (Note spherical microprojections, microvilli and a large swelling indicative of nuclear location.); C,D - A part of a sarcomeric organization, showing a honeycomb-like agranular endoplasmic reticulum (arrow) CC), and nucleus CD). C = x10,800; D = x9,000.

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1983

aI 2 -z - 1000 it 22 ..-5 = .L

500

oy

I

0

I

I

I

I

5

10

15

20

TIME

5 TIME

EL-----5 0

(days)

15

15

20

TIME (days)

(days)

10

10

I

I

I

1

5

10

15

20

TIME (days1

FIG. 2: Comparison of CK and AChR in ML and MC muscle cultures. The activities of CK and the levels of AChR were measured at different times during the myogenesis in ML (O--O>, or MC (O---O) muscle cultures. Each point is an average of 2-3 replicates.

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1983

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Biochemical differentiation of skeletal muscle in culture is characterized by biosynthesis of contractile proteins, cytoplasmic membrane components (Fambrough, 1979; Buckenzymes, and specific In studying the developmental sequence ingham, 1977; Yaffe, 1969). of these characteristics it was suggested that the synthesis of discrete membrane components is regulated independently of cytomuscle differentiation (Patterson and plasmic proteins during Prives, 1973). Therefore, our biochemical analysis was done on both cytoplasmic enzyme CK and the membrane protein AChR. Although measurement of CK activity has been used for some time as an index of muscle differentiation in cell cultures (Turner et al., 1976; Shainberg et al., 1971; Coleman and Coleman, 19681, its usefulness is limited because a large increase in CK activity was found even when cell fusion was inhibited (Morris and Cole, 1979; Shainberg and Brik, 1978; Merlie and Gros, 1976; Turner et al., 1976). Thus, we used AChR as an additional marker for muscle differentiation. We found the development of these two proteins to be similar in MI and in MC cultures. In myoblasts there is a basal level of these proteins in both MC and ML. As soon as myoblasts fuse to form the elongated myotubes, a concomitant increase occurs in both of these proteins. Finally, the synthesis of these proteins reaches a plateau and there is no further increase. Since the half-life of the receptors is about 20 hr, it is suggested that the plateau is achieved not because the cell approached a state where it is not active in protein synthesis, but because the rate of protein synthesis and protein degradation are similar. The findings that the level of AChR in MC-muscle cultures is higher than in corresponding ML cultures could indicate that the spontaneous contractions of myotubes grown in ML are more frequent in comparison to those grown on MC. Indeed, we have previously shown that the levels of AChR are decreased by electrical stimulation and increased by agents which inhibit contractile activity (Shainberg and Burstein, 1976). In conclusion, several of the following features favor MC muscle cultures as a more appropriate model for the study of muscular differentiation than ML cultures: a> Maintenance for longer periods in culture ; b) Spatial orientation of myotubes closer to the in vivo conditions; c> Easy harvesting of samples for biochemical analysis and morphological evaluation at any time during cultivation; d) Growing of muscle tissue to a higher amount and at a better efficiency ; e> Finally, cell-MC conglomerates can be used as differentiated muscular entities to be transplanted into a damaged or a sick muscle. This possibility is now under investigation. REFERENCES Birnbaum, M. , Reis, M.A. and Shainberg, A. (1980) Role of calcium in the regulation of acetylcholine receptor synthesis in cultured muscle cells . Pflugers Archiv 385, 37-43.

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1983

Buckin ham, M.E. (1977) International Review of Biochemistry (ed. J. PiXll F vol. 15, p. 269, University Park Press, Baltimore. Coleman: J.R. and Coleman, A.W. (1968) Muscle differentiation and macromolecular synthesis. Journal of Cellular Physiology 72, Suppl. 1, 19-34. Fambrough, D.M. (1979) Control of acetylcholine receptors in skeletal muscle. Physiological Review 2, 176-216. Jones, G.E. and Gillett, R.A. (1973) A simple method of preparing a cell suspension for scanning electron microscopy. Experientia 31, 1244-1246. Konigsberg, I.R. (1963) Clonal analysis of myogenesis. Science 140, 1273-1284. Merlie, J.P. and Gros, F. (1976) In vitro myogenesis. Expression of muscle specific function in the absence of cell fusion. Fxper i ‘mental Cell Research 97, 406-412. Morris, G.E. and Cole, J.J. (1979) Calcium and the control of muscle specific creatine kinase accumulation during skeletal muscle differentiation in vitro. Developmental Biology 69, 146-158. Paterson, B. and Prives, J. (1973) Appearance of acetylcholine recept or, in differentiating cultures of embryonic chick breast muscle. Journal of Cell Biology 2, 241-245. Reuveny , S. , Silberstein, L., Shahar, A., Freeman, E. and Mizrahi, A. (1982) DE-52 and DE-53 cellulose microcarriers. I. Growth of primary and established anchorage-dependent cells. In Vitro 2, 92-98. Shahar, A., Reuveny, S., Amir, A., Kotler, M. and Mizrahi, A. (1983) Synaptogenesis and myelination in dissociated cerebral microcarrier-cell cultures. Journal of Neuroscience Research (in press). Shainberg , A. , Yagil, G. and Yaffe, D. (1971) Alterations of enzymatic activities muscle differentiation in vitro. during Developmental Biology 25, l-29. Shainberg, A. and Burstez, M. (1976) Decrease of acetylcholine receptor synthesis in muscle cultures by electric stimulation. Nature 264, 368-369. Shainberg, A. and Brik, H. (1978) The appearance of acetylcholine receptors triggered by fusion of myoblasts in vitro. FEBS Letters 88, 327-331. Turner, D.C., Gmur, R., Siegrist, M., Burckhardt, E. and Eppenberger, H.M. (1976) Differentiation in cultures derived from embryonic I. Muscle-specific enzyme changes before fusion chi cken muscle. in EGTA-synchronized cultures. Developmental Biology 9, 258-283. Turner, D.C., Lawton, F., Dollenmeier, P., Ehrismann, R. and Chiquet, M. (1983) Guidance of myogenic cell migration by oriented Developmental Biology 95, 497-504. deposits of fibronectin. Yaffe, D. (1969) Cellular aspects of muscle differentiation in vitro . Current Topics in Developmental Biology 4, 37-77.

Received:

27th May 1983.

Accepted:

10th June 1983.