Conditions for monolayer cultures of chick skeletal muscle in a rich medium showed that non-muscle contaminants were only 20% of the population. Although the calcium content of this rich medium was 0·95 mM, multinucleate and long fibres were observed after 8 days in culture.

Either in rich or in restricted media, gelatin organized the pattern of cell and fibre development in the Petri plates. Cells grown outside the gelatin boundaries looked fibroblastshaped and many were vacuolated. Gelatin did not fully prevent the growth of fibroblast-like population.

Calcium in restricted media appeared to be very important for the acquisition of a definite elongated shape.

The possibility of the existence of myofibro-myoblasts was supported by the finding of multinucleated fibroblast-like cells during culture in restricted medium. Epigenetic factors, such as different media in a given culture or the origin of a serum batch utilized as a component of those media, affected the fate of the cultures and might also explain the myoblastic variance observed in this and other studies reported.

The capacity of phenotypic expression during the modulation change of muscle cells, in vitro, depends not only upon their genotypic origin but also on parameters such as the cell stage during this process, the type of nutrient media used and the interplay of both parameters; in vivo it depends upon specific cytological interactions.

The retention or change of characteristic properties of cell-multiplying populations has gained the attention of several groups of investigators during the past decade. This is a consequence of the development of experimental techniques that permit the various aspects of this phenomenon to be analysed at the cellular level (Königsberg, 1962; Reporter, Königsberg & Strehler, 1963; Königsberg, 1963; Coon, 1966; Cahn & Cahn, 1966; Coleman & Coleman, 1968; Richler & Yaffé, 1970).

Primary skeletal-muscle cultures in monolayer are usually initiated with suspensions of mononucleated cells which, after adhesive plating, appear to consist of two main classes -‘spindle-shaped’ and ‘fibroblast-like’ cells.

The photographs appearing in most publications of the muscle field, whether clonally or mass cultured, usually show more than one cell type at any of the stages studied (Königsberg, 1963; Yaffé& Feldman, 1965; Hauschka & Kö;nigsberg, 1966; De La Haba, Cooper & Elting, 1966; Yafié& Fuchs, 1967; Coleman & Coleman, 1968; Reporter, 1969; Cox & Simpson, 1970; Richler & Yafïé, 1970). By using clonal analysis the last authors have commented upon the capacity of various muscle cell lines to differentiate, but stressed that there are differences in the phenotype of the same cell line along passages.

Since the beginning of the clonal analysis (Königsberg, 1962) it was observed that there was a low efficiency (10%) of the colonies growing in a rich medium, which eventually produced well developed and fully differentiated muscle clones. From such findings, two important questions arise: firstly, what is the significance of the percentage of the differentiated clones; and secondly, what are the origin and stage(s) of the colonies that remain morphologically nondifferentiated? Thus in muscle tissue there is a claim that, both in vitro and in vivo, the same stem line of cells give rise to myoblasts and myotubes and on the other hand some presumptive myoblasts are generated and remain as ‘satellite cells’ for many years (Holtzer, 1970).

Modification of the muscle cell phenotype by differences in the composition of the culture media has not been considered extensively, but recently, in relation to the myotube formation from spindle-shaped cells, attention has been paid to the role that calcium plays in this phenomenon (Shainberg, Yagil & Yaffé, 1969; Ozawa & Ebert, 1970).

The present work reports, correlates and discusses what is known of the culture of muscle, either in rich or restricted media. The role of factors involved in the composition of those media is also analysed in terms of time-specificity during muscle development. With this type of analysis an explanation for the coexistence of differentiated and undifferentiated cells within the same culture could be provided.

Skeletal-muscle cultures

Rhode Island chick embryos of 11–12 days were employed routinely. Cells were obtained from the thigh muscle as described elsewhere (Ramírez & Alemán, 1972) and grown 9 days in the following media.

  1. Fresh Medium (FM) (Königsberg, 1963) containing 0·95 mM-CaCl2. Horse serum was from Myn Laboratories, México, D.F.; chick embryo extract was prepared in our laboratory.

  2. Eagle’s Minimum Essential Medium for suspension culture supplemented with non-essential amino acids (Sigma Co.) plus glutamine (MEM) and 10% Fetal Calf Serum (FCS) from Difco, containing 0·3 mM-CaCl2 (MEMS).

  3. National Cancer Institute solution (NCI) prepared in this laboratory contained 1·12 or 2·24mM-CaCl2 as properly indicated. It was supplemented with 10% FCS. Unless otherwise indicated, all media contained 100 i.u. of penicillin and 50μg of streptomycin per ml.

Coating of the slides and Petri plates was with gelatin.

Collagen was obtained from adult rat tails (Gallop, 1955). Stock solutions of collagen were tyndallized and kept frozen at –20°C; water dilute solutions were succesively filtered through Selas 01 and 02 candles for sterilization. Electron micrographs showed characteristic striations in collagen samples, but a randomly oriented material appeared in gelatin samples. With a 50 μI Hamilton microsyringe, narrow and broad strips of gelatin were gently aligned on slides washed previously with neutral detergent. On the other hand, after several assays with different doses, 35μg of gelatin were routinely applied to 10 cm diameter Falcon Petri dishes, diluted with 2·5 ml of double distilled water to completely cover the bottom of the plate. Petri dishes were gently rotated to homogenize the solution and left overnight at 45°C to evaporate the liquid.

Embryo extract

Nine-day-old chick embryos were decapitated, completely eviscerated and homogenized in Saline G solution (Königsberg, 1963) for 45 s in a Sorvall OmniMixer at 16000 rev/min, dispersed with 1·0 mg of hyaluronidase (Worthington) per 100 ml of the suspension for 1 h, centrifuged 20 min at 31000 g and the resultant supernatant spun down for 3 h in a Beckman SW 25·1 (or SW 25·3) rotor at 64000 g. The supernatant was sterilized by successive filtration through Selas 01 and 02 and kept frozen until used.

Gelatin stripped slides were seeded with hundreds of cells and covered with FM; the development of the culture was followed daily by direct observation under the microscope.

Stain and observation of cultures

The plates were rinsed with Saline G solution and fixed in 4% formaldehyde. Aniline blue orange and Delafield haematoxylin were used to stain the monolayers and covered with 5% polyvinylic alcohol. A Zeiss POL microscope was utilized for the microphotographs and a Honeywell 80S Repronar for the panoramic views.

During the early stages of development in FM there was a definite orientation of bipolar cells and myotubes were formed along their major axis which ran parallel, mainly at the edges of the strips of gelatin (Fig. 1 A). It is known, and we have confirmed, that myoblastic embryonic cells will grow in a similar manner in vivo (10–11 days); these are aligned along the connective tissue fibres (Fig. 1B). At a later stage, in vitro as well as in vivo, the ends of the cells approximate to each other and fuse to form myotubes. Remarkably, the strip guide in both cases is either connective tissue fibres or the gelatin lanes. Cells growing on the slides looked healthy and were bipolar within the boundaries of the strips, whereas the cells outside these limits were fibroblast-shaped, grew very poorly and many of them were vacuolated. It must be noticed that the alignment of cells in later stages of the culture, formed curls inside the strips of gelatin and they tended to remain straight along the border.

Fig. 1

Alignment and muscle fibre organization in gelatin lanes growing in vitro in FM (A) and connective tissue in vivo (B), ×250.

Fig. 1

Alignment and muscle fibre organization in gelatin lanes growing in vitro in FM (A) and connective tissue in vivo (B), ×250.

Another series of experiments in FM were performed in plastic Petri dishes that were previously covered with gelatin: the fibre pattern developed was a curled one, whose behaviour resembled that observed in the slides.

Although collagen or gelatin can organize the fibre pattern in vitro, it is known that during clonal analysis the critical time for cloning muscle cells in the presence of such protein is a very short one, namely the first 24 h of culture. However, it is also known that in mass cultures of fibroblasts the period for active synthesis of collagen for ‘conditioned medium’ in muscle development is the stationary phase; and it is the same period when contracting muscle fibres appear in primary cultures of chick embryo thighs (Königsberg & Hauschka, 1965).

Because of these facts, it was thought that in a muscle mass culture the number of myogenic cells would be improved if the gelatin could be maintained at a fixed concentration in a rapidly dividing population, which had been started with 106 cells per plate. On the other hand, this precaution would also ‘prevent’ excessive contamination from other cell types. Thus we scheduled medium changes which included the addition of 35 μg of gelatin per plate every time the medium was replaced. With cell densities of about 3 × 106 per Petri dish (10 cm in diameter), it was observed that around 24 h of culture the cells were bipolar and mononucleated (Fig. 2 A) and at 48 h myotube formation was clearly observed (Fig. 2B). Only 20% contaminant by non-characteristic cells appeared and these were interspersed mostly at the edges of the plate at the end of one week of the culture (Fig. 2C, D). This type of cell appeared even though the ‘differential adhesion’ technique was employed for cultivation of the muscle cells (Ozawa & Ebert, 1970).

Fig. 2

(A, B) Initiation of myotube formation from bipolar cells at 24 and 48 h of culture in FM; × 160 and × 63. (C, D) Monolayer muscle cultures grown in FM during 8 days; × 1·2 and × 12.

Fig. 2

(A, B) Initiation of myotube formation from bipolar cells at 24 and 48 h of culture in FM; × 160 and × 63. (C, D) Monolayer muscle cultures grown in FM during 8 days; × 1·2 and × 12.

Worthy of mention is the fact that Ca2+ concentration in FM measured by atomic absorption spectrometry was only 0·91 mM instead of the calculated 1·12 mM. Nevertheless, the average number of nuclei per fibre was higher than 100 (Fig. 3 A, B), although previous reports (Shainberg et al. 1969) have shown that 1·4mM-Ca2+ was the minimum for the onset of fusion and formation of multinucleated fibres in rat cultures. However, under conditions of low Ca2+ spontaneous contraction of the fibres was rather exiguous.

Fig. 3

A, B. Monolayer muscle cultures grown in FM during 8 days, × 160.

Fig. 4. Dusty aspect of muscle cells grown in FM containing double amount of CaCl2, after third day. ×63.

Fig. 5. A,B. Muscle cells grown in MEMS during 2 and 3 days; myotube formation and atypical syncytium, × 160.

Fig. 6. Atypical fibroblast-like cells containing more than one nuclei grown in MEMS during 4 days, × 250.

Fig. 3

A, B. Monolayer muscle cultures grown in FM during 8 days, × 160.

Fig. 4. Dusty aspect of muscle cells grown in FM containing double amount of CaCl2, after third day. ×63.

Fig. 5. A,B. Muscle cells grown in MEMS during 2 and 3 days; myotube formation and atypical syncytium, × 160.

Fig. 6. Atypical fibroblast-like cells containing more than one nuclei grown in MEMS during 4 days, × 250.

In some experiments Ca2+ was increased in FM to 2·24 mM. During the first 2 days of culture the growth of the cells was faster than at the lower Ca2+concentration. Furthermore myotubes were observed before 48 h of culture when 2·5 × 106 cells were plated by using the ‘differential adhesion’ technique. Nevertheless, despite the established routine of medium replacement every other day, the plates began to show a cloudy aspect after the third day in culture. Obviously, these were not optimal conditions for muscle culture, although fibres were wider and multinuclearity was found along the fibre (Fig. 4).

In order to test the components of FM and its capability to differentiate muscle cells, an experiment was performed employing FM with 2·24 HIM Ca2F, but horse serum was replaced by 10% FCS. The cells grew and differentiated poorly, but when medium was replaced by complete MEM containing 10% of FCS, the culture improved and myotubes began to appear in the plates (even though Ca2+ was 0·3 mw). This suggests that NCI in presence of FCS impairs the phenotypic expression of myotube formation.

The differentiation of embryonic muscle cells was tested in several media and the schedules used were as follows:

  • FM was renewed on days 2, 4 and 6.

  • FM was used on the first day and was replaced by MEMS containing 0·3 mM-Ca2+ on days 2, 4 and 6.

  • MEMS containing 0·3 mM-Ca2+ was renewed on days 2, 4 and 6.

  • NCI containing 7% FCS and T2 mM-Ca2+ (NCIS) was renewed on days 2, 4 and 6.

  • FM was used on the first day and was replaced by NCIS on days 2, 4 and 6.

During the first day of culture the MEMS and NCIS plates contained highly elongated bipolar cells and the myotube formation was initiated well in advance of the 50–60 h previously reported for a primary culture of rat skeletal muscle (Yaffé, 1971).

After the first replacement of media, plates growing in MEMS and those first grown in FM and replaced by MEMS showed the initiation of myotube formation with elongated mononucleated cells and eventually both showed a progressive development (Fig. 5 A). However, more myotubes appeared in the plates initiated with FM, although the cells were one-fifth shorter than those grown with MEMS alone. Remarkably, after 78 h in culture the plates containing MEMS showed very wide fibres with an axial ratio of approximately 5:1. It was also observed that their cytoplasm had at least 20 nuclei contained in three or four rows. On the other hand, the total length of such fibres was approximately four times that of an elongated myoblast (Fig. 5B). Disorganization within the syncytium was also observed. In comparison with the FM containing plates the population of MEMS plates was healthier but seemed exiguous.

Cells grown in MEMS, whether or not initiated in FM, developed into long-shaped myotubes that looked very healthy. Plates which were started with FM and subsequently fed with MEMS after the sixth day of culture had predominantly a well-developed population of fibres.

On the contrary, in FM there were abundant fibres but with very shortlooking myoblasts. After the renewal of the medium the cell pattern followed prior observations. Cells which were changed to or remained in FM, were more abundant and formed as much as 70% more fibres than cells growing in poorer media. In FM fibres became very wide.

Cultures grown in NCIS or in FM followed by NCIS were covered by a dusty medium and only isolated cells and few myotubes were observed.

From the above-mentioned results it seems apparent that a rich medium is needed for culture initiation in order to preserve the capability of myotubes to develop into fibres after a week, since the addition of gelatin every time the medium was changed was not enough to accomplish such effect.

In the first experiments evidence was obtained to support the role of gelatin in organization of the pattern of fibre growth. On the other hand, the replacement of medium containing gelatin scheduled every other day, permitted the muscle-type population to predominate over the ‘fibroblast-like’ cells within the same plate. The 80% proportion of the muscular population at the end of one week in culture must be considered in relation to the factors involved in such a culture. Thus conditioning of the surface of the plates prevents, delays or diminishes the probability that the non-muscle cell types will divide at the normal rate. This effect was reinforced in our cultures every time the medium was changed, since gelatin was added on each occasion in a rapidly growing population.

It is possible that gelatin in these culture experiments is playing a role in myogenesis, which is similar to that of collagen in connective tissue; that is, collagen may be important in the modulation of myogenesis in vivo.

With FM, collagen was utilized for clonal analysis of chick muscle and 43% of the colonies obtained were myogenic ones. Nevertheless, the clonal efficiency was not augmented whether or not more collagen was added to the plates (Königsberg & Hauschka, 1965).

In regard to the remaining fibroblast-like population, several points must be mentioned. Thus it is important to determine if these cells are true fibroblasts or are atypical myoblastic cells. We have observed trapezoidal cells from a primary muscle cell culture grown in MEMS contain three or four nuclei in their cytoplasm (Fig. 6), which supports the idea that cells other than spindle shaped are capable of forming a syncytium. Ca2+appears to be very important for the acquisition of a definite shape of the cells possibly by affecting their membrane (Ambrose, 1967). A Ca2+ concentration of only 0·9 mM in FM was enough to sustain fusion and formation of long multinucleated fibres although contractility was impaired. An increase in Ca2+ may be necessary to develop the non-typical myogenic cells to a spindle-shaped population. Thus at a Ca2+ content of 2·2 mM almost 100% of the population at the end of the first day was fusiform.

In regard to the Ca2+ concentration in a given medium, it should be mentioned that NCI as compared with MEM does not permit addition of Ca2+ above 1·3 mM, since precipitation occurred presumably because of the phosphate salts already present for buffering action.

Recent observations regarding the behaviour of spindle-or fibroblast-shaped cells in several environmental conditions have led to the hypothesis of myo-fibro-myoblastic cells (Lappano-Colleta, 1970).

It is possible that some cells of a given inoculum have to pass through proliferative mitosis before they acquire the capability for the so called ‘quantal mitosis’ (Holtzer, 1970). Our findings with cells cultured 24 h in MEMS and NCIS give support to this hypothesis since myotubes developed before 48 h. In earlier work which led to the discovery of the conditioned-medium containing collagen (Kdnigsberg, 1962), it was observed that with different cell densities of trypsinized muscle grown within the same medium, myotubes were formed at the same rate irrespective of the cell confluency shown by the culture. In other words, a high-density myoblastic population divides less rapidly, then a lower population can give rise to a permissive ‘quantal’ myoblastic cell. Similar findings with rat muscle have been reported recently (Yaffé, 1971). The interpretation was that at the onset of fusion there are myoblasts differing in their capacity to fuse and this is only dependent on the length of time the cells were maintained in vitro and was irrespective of their plating density. It is likely that, in our case, cells grown in MEMS and NCIS for 24 h could already contain some ‘mature’ cells to initiate myotube formation. Since the number of myotubes within a given plate is low at the beginning of the culture, it is likely that the distribution of such ‘permissive’ myoblasts is random in the plate, so the probability of meeting each other is low. However, we cannot exclude the possibility of local outbursts of myotubes.

The influence of a given serum batch must be pointed out. In our initial experiments with FM, cells and fibres were perfectly normal and accounted for the highest proportion of muscle cells type in a Petri plate ever obtained. On the other hand, a fresh batch of horse serum gave rise to short myoblasts grown initially or all the time with FM. Remarkably, FM initiated cultures which were subsequently transferred to MEMS began to change their myoblastic shape to highly elongated (4–5:1 axis ratio) mononucleated cells, thus favouring the hypothesis of myofibro-myoblastic cells. However, these cells were not as elongated as those cultivated in MEMS alone.

In regard to cells cultivated in MEMS there are some points to be discussed. Firstly, culture in these conditions always gave rise to a very elongated and bipolar cell population within the first 48 h, in contrast to cells growing in FM during the same time. Secondly, in MEMS plus gelatin after 78 h the width of the fibre, as well as the unusual disposition of the nuclei (three or four rows of nuclei in the same fibre) along its major axis, is a clear example of changes of phenotypic expression of cells under different environmental conditions. However, the length of the fibre in comparison with a mononucleated cell does not agree with the number and pattern of this nuclear distribution within the fibre. This finding suggests that there is an alternative explanation to that of the myoblast-ends fusion. One possibility is the multiplication of nuclei in the fibre without cytoplasmic participation; however, this possibility seems unlikely in view of the existing evidence in favour of fusion as the mechanism for syncytium formation in vitro (Königsberg, 1963) and in vivo (Mintz & Baker, 1967). One further alternative is lateral fusion of the highly elongated cells, even though two or three terminal cell fusions are in accordance with the length of the formed fibre.

In culture, once a strain cell achieves a definite stage it is not irreversibly obligated to remain in it (Cahn & Cahn, 1966; Richler & Yaffé, 1970). There is evidence to show the role played by certain epigenetic factors in the regulation of phenotype expression, such as intra-and inter-cellular metabolite interactions, cell contiguity, cell products of different strain(s), population densities (Königsberg, 1963; Grobstein, 1967), inducers or factors for strain recognition (Tiedemann, 1968; Humphreys, 1963). Since some of these factors can be changed in cell cultures, this provides a convenient method for the study of the mechanisms involved during changes in phenotypic expression.

It appears difficult to explain the periodical behaviour of muscle-cell lines cultivated in clones that failed to form fibres in spite of their proved capacity to produce them previously. They were also capable of regaining such an ability after various passages. Such findings were difficult to explain since it was shown that changes in ploidy do not affect the phenotypic expression of skeletal muscle cells (Richler & Yaffé, 1970). In the light of our present work we can speculate that different batches of horse serum could be responsible for such a myoblastic variance. The retention of the capacity of phenotypic expression of muscle cells appears to depend not only on their genotypic origin but on some other parameters: the stage of phenotypic expression during the modulation process, which includes specific cyto-and tissue-architecture; the type of nutrient media used, and the time-specificity in the action of a given medium to a given cell during development.

Nous decrivons des conditions pour faire des cultures de muscle squelettique de Poulet dans monocouches. Dans un milieu enrichie on n’a pas trouvé que 20% de contaminants par rapport á la population totale. Malgré la faible concentration de calcium dans ce milieu (0,95 HIM), après huit jours de culture nous avons observé des fibres longues et multinucléaires.

Aussi bien que dans le milieu enrichie, dans le milieu restreint, l’organisation du développement des cellules et des fibres a été guidée par la gélatine dans le boîtes de Petri. Les cellules qui ont poussé hors de la gélatine avaient formé fibroblastique et beaucoup d’entre elles ont été vacuolées. La gelatine n’a pas empeché complétement la croissance des cellules fibroblastiques.

Dans le milieu restreint, la calcium semble important pour l’acquisition d’une forme elongué définitive.

Le fait d’avoir trouvé cellules semblables au fibroblastes multinucléaires pendant la culture dans le milieu restreint, soutient la possible existence de myofibre-myoblastes.

Certains facteurs epigénetiques ont montré l’affectation des cultures. Nous avons observé par exemple, q’un milieu différent dans une culture donnée où la variation de la source d’obtention de la même espèce d’un serum utilisé dans dite milieu, ont montré l’affectation des cultures et ceci peut aussi expliquer la varietée de myoblastes observés au cours de ce travail et des autres publiés.

La capacité d’expresion phénotipique pendant la différentiation du muscle en culture dépend, non seulement de son origine génotipique, mais des parametres tels que: l’étape de différentiation des cellules, le sort du milieu de culture utilisé, ou tous les deux. In vivo cette capacité depende aussi des interactions entre cellules du même ou de type différente.

The authors wish to thank Dr C. S. Corker for correcting the manuscript and Dr E. A. Newsholme for helpful discussions.

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