use of papain in the preparation of adult mammalian skeletal muscle for tissue culture

IN VITRO Vol. 10, Nos. 3 & 4, 1974

USE OF PAPAIN IN THE PREPARATION OF ADULT MAMMALIAN SKELETAL MUSCLE FOR TISSUE CULTURE 1

JULIET MORGAN: AnD LOUIS COHEN

Department of Medicine, University of Chicago, Chicago, Illinois 60637

SUMMARY

This report describes in detail a method of enzymatic separation of adult mammalian muscle using papain. The procedure has proved valuable in the preparation of suspensions of muscle cell pieces from normal human skeletal muscle obtained from patients of all ages, from 3 months to 79 years. Muscle cultures have been successfully grown from biopsy material from boys with Duchenne's musc~flar dystrophy and from their mothers. The procedure was initially established with adult canine skeletal muscle and has also been used for monkey muscle.

Small pieces of skeletal muscle are chopped in a solution of 0.05% papain and 0.01% cysteine hydrochloride in Ca 2.- and Mg~+-free balanced salt solution and transferred in the papain solution to a flask, in which they are incubated at 37~ for 10 min with oc- casional agitation. The resulting cell suspension is collected and the remaining pieces are treated with further portions of fresh papain until only connective tissue remains. The cell pellets obtained by centrifugation are resuspended in Eagle's minimum essential medium (supplemented with 20% fetal calf serum) and transferred to culture chambers. The muscle can be observed at all times, during the separation procedure and subsequently in culture. The events occurring during skeletal muscle regeneration can be followed. Using the same papain preparation, myoblasts and myotubes may be subcul- tured and collected for indefinite frozen storage in dimethylsulfoxide.

INTRODUCTION

Most observations on the regeneration of adult mammalian skeletal muscle in vitro have been made using small pieces of tissue or explants (1-5). Because the use of explants ob- scures the early cellular changes which take place, we investigated several enzyme systems for their ability to produce viable cell cultures. The enzyme trypsin (6) has been used successfully for embryonic skeletal muscle, but does not produce viable muscle eel[ suspensions from adult muscle. An effective method is described here, using papain, by which we have been able

1 This work was supported by a grant-in-ald from the American Heart Association with funds con- tributed in part by the Chicago Heart Association, the Pharmaceutical Manufacturers Association, and National Institutes of Health Research Grant NS 10385 from the Institute of Neurological Diseases and Stroke.

2 Address reprint requests to: Dr. Juliet Morgan, Department of Medicine, University of Chicago, Box 401,959 E. 59th Street, Chicago, Illinois 60637.

to establish cultures of skeletal muscle from several mammalian species, including dog, monkey, and man.

~V~ETttODS

Small pieces of skeletal muscle (5 mm 3 or less) were collected by sterile techniques and im- mersed at 37~ in Eagle's minimum essentiaI medium (MEM) containing 20~/o fetal calf serum, 0.17% sodium bicarbonate, 200 tLg of streptomycin, and 200 units of penicillin per ml. These samples could remain without further treatment for 0.5 to 14 hr without apparent effect on their viability, as measured by subsequent culture.

The muscle was transferred to a small, sterile plastic Petri dish containing 10 ml of a solution of 0.05% papain (Sigma Chemical Company; crude powder, type II) and 0.01% cysteine hydrochloride (7) in sterile Ca ~*- and Mg2*-free balanced salt solution, prepared and warmed to 37~ immediately before use. I t has not been

188

PAPAIN IN SKELETAL MUSCLE CULTURE 189

found necessary to filter the balanced salt solution after the addition of the papain and cysteine. No contamination occurred from this source. The tissue was chopped into small (1 mm ~) pieces with scissors. With larger samples, adhering connective tissue was pulled away and dis- carded during the mincing, but with most small samples the entire piece of tissue was treated. The muscle plus papain solution was transferred to a 35-ml sterile plastic flask, which was placed in a water bath at 37~ and agitated once or twice, during a 10-min period. The cell pieces were allowed to settle for 1 min, and the cell suspension was decanted into a sterile centrifuge tube containing 1 ml of horse serum for every 9 ml of suspension. Fresh papain solution was added to the tissue pieces in the flask for a further 10-min incubation. Six such consecutive papain treatments were given, but the number could be varied depending on the size of the original sample, the cell density required in each culture chamber, and the degree of agitation given during each treatment.

The horse serum was added because it has been found (8, 9) to increase viability and, when trypsin was used, to stop the action of the enzyme (9). However, the action of the papain solution was found to diminish very quickly over the 10-min period, and for this reason several short treatments are preferable to one or two longer ones. The papain solution itself is stable for only 2 to 3 hr at room temperature.

The cell suspensions were centrifuged at 100 • g for 10 rain. The subsequent cell growth was satisfactory even when all of the suspensions were retained until after the sixth treatment, and centrifuged at the same time, providing the horse serum was added. The resulting cell pellets were resuspended in M E M containing 20% fetal calf serum, 0.17% sodium bicarbonate, 50 ~g of streptomycin, and 50 units of penicillin per ml. The volume in which each pellet was suspended was varied, depending on the size of the original muscle sample and the culture chamber used. The number of muscle cell pieces (MCP) per ml was counted in a hemocytometer at this stage. Because the majori ty of the MCP in the earlier treatments contained up to 12 nuclei, counts indicated only the number of MCP and not the number of nuclei present or the number of cells which would be produced. In the majori ty of samples of human muscle, obtained by muscle

biopsy, only 1 m[ of medium, containing 500 to 750 MCP, was used and the suspensions were placed in the first row of wells in a Linbro culture plate. These plates contain 24 wells (four rows, six wells per row), each with a total volume of 3 ml and measuring 15 mm • 18 mm. Cultures were also established in 60-min Falcon plastic Petri dishes in 4 ml of Eagle's medium, with similar results. Cultures were maintained in a water-saturated atmosphere at 37~ in an in- cubator supplied with 5% CO~ in air.

The medium was first changed after 1 week. The supernatant fluid plus any cell pieces still suspended was transferred to the second row of six wells in the Linbro plate or to new Petri dishes. Fresh Eagle's medium was placed in the original culture chambers. This procedure avoided loss of cell fragments and allowed the morphology of the earliest attached cells to be examined without interference from floating de- bris. Similar transfers were made as cells were seen to at tach to the second row of wells. By the time three transfers had been made (21 days), the growth of the cells in the initial row was often such that a confluent layer had formed.

Subcultures were established in Petri dishes by the following method. The culture medium from the confluent cultures in the Linbro plate was removed. Each well was briefly rinsed with 0.5 ml of papain solution. Thereafter, 1 ml of papain solution was placed in each well. When most of the cells became rounded, the remainder were loosened by vigorous pipetting of the papain solution. The entire 1 ml of papain plus suspended cells was transferred to a sterile plastic Petri dish containing 4 ml of Eagle's medium prepared as described above. All cell types, including those myotubes which did not fragment during the papain treatment, attached again very rapidly. Fibroblasts began to spread out in the first 1 to 2 min and myoblasts and myotubes in 10 to 15 min after subculture. The procedure avoids a centrifugation stage and has been found especially useful when only a few cells are to be transferred.

Representative cultures from each sample containing a high proportion of myoblasts and myotubes were collected and deep-frozen. Petri dishes containing a confluent or nearly confluent layer of cells were treated with papain solution as described above for subcultures. The restflting cell suspension was transferred to a sterile

190 MORGAN AND COHEN

centrifuge tube containing 1 ml of horse serum. After a slow (100 • g) 10-min centrifugation, the cell pellet was resuspended in 0.65 ml of Eagle's MEM (with no added serum, sodium bicarbonate, or antibiotics), 0.20 ml of fetal calf serum, and 0.15 ml of dimethylsulfoxide (DMSO). The contents of the tube were well mixed and allowed to stand at room temperature until the color of the phenol red in the medium indicated that the pH was 7.2 to 7.4. At this time, the cell suspension was transferred to a small, sterile-labeled glass vial. The vials were placed immediately into a Revco deepfreeze unit at --86~

To reestablish the cell cultures following re- moval from the freezer, the vials were placed in warm (37~ water and thawed as quickly as possible. The 1 ml of cell suspension was transferred to a Petri dish containing 4 ml of Eagle's medium made up as described above. Cells began to attach to the plastic in the first 10 min after thawing occurred. Cells preserved in this manner continued to divide, fused into myotubes, and became mature striated muscle in the same way as cells which have not been frozen. Under favor- able conditions, 40 to 60% of the cells were viable after thawing. Cell division was observed after 5 to 6 days. Similar satisfactory results with frozen material have been obtained (3) using 10% DMSO in TC 199 plus human AB serum (s:2).

To obtain complete maturation of human myotubes to striated muscle, it was essential in most cases to replace the fetal calf serum with human serum. The serum was stored in 1- ml aliquots and frozen until needed. I t was not sterilized in any way nor heat-inactivated. No contamination of the cultures occurred using this procedure. Although many different sera have

been tested, dog myotubes did not become striated under the present culture conditions. Monkey myotubes, on the other hand, rapidly became striated in the Eagle's medium with 20% fetal calf serum.

Events at all stages of culture (separation of muscle into cell fragments, cell attachment, cell division, cell fusion, myotube development, and the appearance of myofibrils) were followed with phase contrast microscopy using a Zeiss inverted microscope with a 35-mm photographic attachment on Kodak Plus X or Kodachrome I I film.

]:~ESULTS

Initial e]:]ects o/papain treatment. During the initial chopping of the muscle in papain solution, the effect of the enzyme became apparent after only 2 or 3 min. At this time, the muscle fibers could be separated by pulling with forceps. Mi- croscopic examination revealed that muscle cells first became broken by transverse cracks into short lengths, 100 to 200 t~m long. The length of the MCP produced was often uniform, even though they were still held in a connective tissue network.

After a 20-min treatment in papain (Fig. la) , the average size of the MCP was 120 • 60 t~m. Each was approximately rectangular in outline and contained about 6 to 12 nuclei. With in- creasing time of papain exposure, the MCP were reduced in size so that by 50 min (Fig. lb) , the average size was 60 • 25 tLm. Each of the smallest fragments contained only one or two nuclei and was generally spindle-shaped in outline. However, because the supernatant fluid was not completely decanted after each treatment, a few large MCP were always carried over to the next tube.

With this procedure, in which muscle is treated

FIG. 1. Appearance of muscle cell pieces separated from dog skeletal muscle after 20 mi~ (a) and 50 min (b) of treatment in 0.05% papain solution. Phase contrast; • 166

FAPAIN IN SKELETAL MUSCLE CULTURE 191

for varying time periods from 10 to 60 min, the MCP produced exhibit a wide range of size. Each MCP, however, probably contains the number of nuclei that would be present in a piece of muscle cell of the same size. I t is not possible to determine what percentage of the nuclei are contained in satellite cells at the time of papain- ization. Satellite cell nuclei are said to comprise 5% of the nuclei (10) present in normal unin- jured adult human skeletal muscle. No values are available for dog or monkey muscle. The number of nuclei present in each MCP, especially the larger MCP, can therefore not all be designated "satellite cell" nuclei.

Appearance of myoblasts. The term "myoblast" is used in this report to refer to a mono- nucleated cell, which under the appropriate culture conditions will fuse with other such cells to form a multinueleatcd myotube. Myoblasts first began to develop from the MCP on day 4 or 5, and were usually seen first in the cultures established from MCP treated for 40 or 50 rain with papain. The method of myoblast formation was not the same in all three species. In canine cultures (11), larger MCP containing up to about 12 nuclei, produced cells by the development of buds or protrusions on the surface of

the MCP. The buds increased in size until they were 13 to 15 ~m in diameter. Each bud was attached to the parent MCP by a stalk. A nucleus passed from within the MCP into the bud, and the stalk between the bud and MCP de- creased in size until the bud was pinched off. The bud floated away from the MCP and dropped to the surface of the culture dish. A cell spread out to form a typical myoblast which could subsequently be observed to fuse with other such cells into myotubes. Smaller canine MCP settled and transformed directly into myoblasts. This occurred more quickly (5 to 6 days) than the appearance of buds (6 to 9 days). On one occasion, an MCP (120 tLm long) containing 11 nuclei elongated with loss of striations to form a myotubc also containing 11 nuclei. The loss of striations in all MCP allowed the number of nuclei to be determined in many instances.

In the dog cultures (Fig. 2a), the myoblasts were distinguishable from the fibroblasts by the characteristic appearance of the nucleus, which contained a single, large, almost square nucleolus. In monkey myoblasts (Fig. 2b), the nucleus contained either one or two more circular nucleoli of variable size. Human myoblasts (Fig. 2c) contained nuclei with one large or a large

FIO. 2. Appearance of myoblasts in culture: a, dog myoblast fusing with a myotube, the nucleus contains a single large, almost square nucleolus; b, three monkey myoblasts aligning themselves prior to fusion, the nuclei contain one or two circular nucleoli of variable size; c, a human myoblast just prior to fusion, showing a nucleus containing a single nucleolus; other myoblasts (not illustrated) may also contain one or more smaller nucleolar- like bodies. Phase contrast; a and b, • 550; c • 1,200.


and one or more smaller, nucleolus-like bodies. The perinuclear envelope of myoblast nuclei of all three species was relatively more conspicuous than in the fibroblast. Fibroblasts of all three species were similar. The nuclei contained mul- tiple small, faint nucleolus-like bodies and the perinuclear envelope was not distinct.

In both human (12) and monkey (13) cultures, it was difficult to distinguish myoblasts from fibroblasts. In these two species, cells developed from the larger MCP in the following way. MCP became attached to the substratum and after 5 to 7 days, the striations began to disappear, and nuclei often became visible. At this stage, a cytoplasmic extension appeared

from the MCP and spread out at the surface of the culture chamber. A nucleus passed from within the MCP into the emerging cytoplasm and subsequently a separate cell moved away from the MCP. The number of cells to emerge from a given MCP appeared to depend on its size and presumably on the number of nuclei present. Smaller MCP (30 • 15 tLm or less) changed directly into single cells, as in the canine cultures.

Myotube ]ormation. Following myoblast formation, in the early stages of the culture, the cells became aligned in rows, end to end. Usually three to five, but sometimes up to 20 cells, arranged themselves in this way. If cell fusion

Fro. 3. The morphology of myotubes and myotube nuclei which develop in culture from myoblasts of a and b, dog; c and d, monkey; and e and ], human. In each species, the lower photograph shows an enlarged view of the nuclei present in the myotube. Phase contrast; a, c, and e, x 190; b, d, and ], • 800.


occurred, a multinucleated cell or myotube was formed. Fusion did not always take place between cells so aligned. Fusion between myoblasts, between myoblasts and myotubes, and between myotubes, took place usually only at the ends of the cells or the leading edge of the myotube. In all three species, the gross morphology (Fig. 3, a, c, and e) of the myotubes appeared similar, except for their respective nucleolus-like bodies (Fig. 3, b, d, and 1). The largest myotube assembly was seen in monkey cultures, in which nearly the whole of a 60-mm plastic Petri dish was covered by an interconnected network of giant myotubes. The possibility that this extensive fusion was caused by viral contamination is being investigated in a series of ultrastructural studies on the untreated muscle and the cultures concerned.

Maturation of myotubes to striated contractile muscle cells. The completion of development of the myotubes into contractile striated muscle cells took place only in monkey and human cultures. Cross striations appeared in mon-

key myotubes (Fig. 4a) without any change in the culture medium and in the presence of fetal calf serum. Striations rarely occurred in human cultures in Eagle's medium with 20% fetal calf serum, but frequently (Fig. 4b) appeared in cultures with well developed myotubes, 36 to 48 hr after the substitution of 10% human serum for the fetal calf serum.

Whatever factor in the human serum was nee- essary for the development of cross striations, it maintained the cells in this form for only a few days. Additional fresh serum was required after 4 to 5 days, to conserve or renew the cross striations. Not all of the myotubes became striated in any one culture chamber. Spontaneous contractions could be induced in striated areas by abrupt tapping of the edge of the microscope stage.

~)ISCUSSION

Trypsin has been used with success to prepare cell suspensions from many tissues of many ani- mal species. I t has been used to separate em-

FIG. 4. Striations seen in myotubes of a, monkey; and b, human skeletal muscle cultures. Phase contrast ; a, X 1,300 ; b, • 1,200.


bryonic chick (14) and rat muscle (15). When used to treat adult muscle, trypsin causes the production of a mucinous material, probably in large part DNA, which makes the muscle pieces adhere and prevents the separation of MCP.

Papain is a mixture of proteases from Carica papaya, the papaya plant, which is used as a meat-tenderizer because of its ability to digest intercellular proteins. It contains enzymes which are similar to pepsin but which act in acid, neutral, or alkaline medium. In the procedure described here, it rapidly separates the long muscle fibers with very little agitation and in a short time interval. The viability of the nuclei in the MCP appeared to be high. The best and fastest development of myotubes in all species was seen in those suspensions in which the MCP had been subjected to a 50-rain papain treatment. In many instances, myoblasts developed from nuclei surrounded by only a small rim of cytoplasm. It is likely that a good proportion of the myonuclei present in the initial muscle sample survive the separation procedure to develop into myoblasts. The actual percentage which do ultimately develop into myoblasts will not be known until the myonuclei are labeled with a radioactive isotope before papain treatment and subsequently assessed by counting or autoradiography techniques. Both in the dog and monkey samples, at least 90% of the MCP, of all sizes eventually become attached to the substratum, and give rise to cells, the number of which was proportional to the original size of the MCP. Occasionally, in human cultures, however, MCP produced by shorter periods of papain treatment did not produce cells, but re- mained floating in the medium for many weeks without any apparent change. Fibroblasts also survive well, but their relative number can be reduced in a given culture by performing several brief papain treatments and subsequent transfers of the MCP at the appropriate time, as described earlier.

This appears to be the first report of the suc- cessful culture of adult canine skeletal muscle and monkey skeletal muscle. Human skeletal muscle was first grown in 1946 (1) and many reports have been published since that time. The work up to 1969 was reviewed by Ross and Hudgson (2) and since then, several other ex- cellent papers have appeared (reviewed in 3). All of these authors have used explants to estab-

fish the cultures and human serum in the initial culture medium. The procedure described has been most useful for dog, monkey, and human samples. Pig and rabbit skeletal muscle have been grown, but the myotubules that form in these cultures are small, contain no more than six to eight nuclei, and are difficult to locate. This may be explained by the rapid growth of fibroblasts, which mechanically interfere with myoblast fusion, or by the culture conditions, which are suitable for canine and primate species, but are inadequate for the rabbit and pig. This procedure has proved effective in culturing human fetal skeletal and heart muscle, and adult mammalian (human, dog, pig,. sheep) cardiac muscle. In addition, the method can be used on muscle samples as small as 2 mm 3 and will yield MCP sufficient to establish three or four cultures. The loss of activity of the papain over a 2- to 3-hr period at room temperature has proved to be an added advantage when transferring only a few cells to another culture chamber, as there is no need to centrifuge the cell suspensions to remove the papain solution.

The limitations of the method mainly concern the fact that the fibroblasts are also viable. An ideal enzyme to separate muscle would selec- tively deter the growth of fibroblasts. Papain digests mainly intercellular proteins, but other enzymes such as nucleases are probably present. It does not seem to be injurious to the intact cell or nucleus. Much larger samples than those used for this study, when treated with trypsin, yielded no viable cells. The rapidity with which the papain solution loses its activity may also be a problem in some cases, but as an average time to separate a piece of muscle with six papain treatments is about 1.5 hr, it has not been any disadvantage in our hands. Similar loss of activity at room temperature is seen with other proteolytic enzymes, such as trypsin.

The procedure has many potential uses. It is possible to observe the muscle cell pieces con- tinuously during treatment in papain, immediately after separation or injury and through many weeks of the subsequent growth period. Adult canine skeletal muscle (11) was used to establish the method. It was then extended to normal adult human skeletal muscle (12), muscle from boys with Duchenne's muscular dystrophy (DMD) (16), muscle from the female


carriers of DMD (17), and finally to an examination of the growth differences of muscle from corn off-fed monkeys and from those on a high cholesterol diet (13).

By this method, combined with ultrastructural studies, it should be possible to answer many of the unsettled basic questions regarding skeletal muscle regeneration, such as: Do the myonuclei contribute to the newly formed myoblast popula- tion? Is the satellite cell just an intermediate cell stage in the regeneration of skeletal muscle ?

Finally, the testing of new therapies in patients is a long, costly effort. The culture meth- ods described here may eventually prove useful for the assessment of the myopathic or thera- peutic effects of new compounds on each stage in the regeneration of skeletal muscle.

REFERENCES

1. Pogogeff, I. A., and M. It. Murray. 1946. Form and behavior of adult mammalian skeletal muscle in vitro. Anat. Itec. 95 : 321-335.

2. Ross, K. F. A., and P. Hudgson. 1969. Tissue culture in muscle disease. In: J. N. Walton (Ed.), Disorders oJ Voluntary Muscle. 2nd ed. Little, Brown and Company, Boston, pp. 319-361.

3. Bishop, A., B. Gallup, Y. Skeate, and V. Dubowitz. 1971. Morphological studies on normal and diseased human muscle in culture. J. Neurol. Sci. 13 : 333-350.

4. Pearce, G. W. 1963. Tissue culture in the study of muscular dystrophy. In: G. H. Bourne, and M. N. Golarz (Eds.), Muscular Dys- trophy in Man and Animals. l=[afner Pub- lishing Co., Inc., New York, pp. 178-191.

5. Mendell, J. It., It. I. Itoelofs, and W. K. Engel. 1972. Ultrastructural development of ex- planted human skeletal muscle in tissue culture. J. Neuropathol. Exp. Neurol. 31: 433- 446.

6. Moscona, A. A. 1952. Cell suspensions from organ rudiments of chick embryos. Exp. Cell Res. 3 : 535-539.

7. Paul, J. 1965. Cell and Tissue Culture. E. and S. Livingstone Ltd., London, p. 199.

8. Morgan, J. On the transformation of lympho- cytes in vitro. M.Sc. thesis, Macquarie Uni- versity, Sydney, 1968.

9. Bateson, R. G., D. Hindle, and J. Warren. 1972. Growth pattern in vitro of normal and diseased adult human skeletal muscle. J. Neurol. Sci. 15 : 183-191.

10. Fischman, D. A., H. Y. Meltzer, and It. W. Poppei. 1973. The ultrastructure of human skeletal muscle: variations from archetypal morphology. In: C. M. Pearson, and F. K. Mostofi (Eds.), The Striated Muscle. Wil- liams & Wilkins Co., Baltimore, pp. 58--76.

11. Morgan, J., and L. Cohen. 1974. In vitro culture of adult canine skeletal muscle. Proc. Soc. Exp. Biol. Med., submitted for publica- tion.

12. Morgan, J., and L. Cohen. 1974. Regeneration of normal adult human skeletal muscle in tissue culture. In Vitro 9: 360-361.

13. Morgan, J., and L. Cohen. 1974. The regeneration of adult monkey skeletal muscle in vitro and the effect of prior diet. Fed. Proc. 33 : 654.

14. O'Neill, M. C., and F. E. Stockdale. 1972. A kinetic analysis of myogenesis in vitro. J. Cell Biol. 52 : 52-65.

15. Yaffe, D., and M. Feldman. 1964. The effect of actinomycin D on heart and thigh muscle cells grown in vitro. Dev. Biol. 9 : 347-366.

16. Morgan, J., It. E. Cohen, and L. Cohen. 1973. Differences in normal and dystrophic human skeletal muscle cells revealed by papain treatment and growth in tissue culture. Clin. Ires. 21 : 862.

17. Morgan, J., L. Cohen, and M. Malerich. 1974. Characteristics of skeletal muscle from carriers of Duchenne's muscular dystrophy after papain treatment and in tissue culture. Neurology 24 : 352.

use of papain in the preparation of adult mammalian skeletal muscle for tissue culture

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