- Email: [email protected]
A quantitative procedure for the dissociation of adult mammalian muscle into mononucleated cells
Printed in Sweden Copwighf @ 1976 by Academic Press. Inc. All righrs of reproduction in any form reserved
PRELIMINARY
NOTES
A quantitative procedure for the dissociation of adult mammalian muscle into mononucleated cells
plating efficiency and differentiation (determined by specific activity of creatine phosphokinase) for adult mammalian muscle.
ROSE YASIN, GISELA VAN BEERS, DITTA BULIEN and E. J. THOMPSON, Muscular Dys-
Materials
trophy Research Laboratories, London WC1 N 3BG, UK
The National
Hospital,
Summary. A new technique for quantitative analysis of dissociated adult skeletal muscle is described. Using adult hamster biceps we obtained cell yields of l-4 x 1W viable cells/g muscle, 4-10% plating efficiency (% cells attached at 20 h in vitro) and spontaneously contracting myotubes at day 7 which exhibited creatine phosphokinase specific activities of 0.4 pmoles/min/ mg protein at 25°C. This technique can be applied to other adult tissue.
Successful growth of adult tissue in culture is acknowledged to be more difficult than that of embryonic or neonatal tissues. As recently stated by Holtzer et al. [l], the technique conventionally used for culturing adult skeletal muscle (by explanting tissue pieces) is unsatisfactory because it is not possible to perform quantitative experiments. This problem would be eliminated if cultures could be established, starting with single cells which eventually differentiated into myotubes. The release of myogenic cells from adult rat skeletal muscle has been reported by Bischoff [2]; however, he was unable to count the number of cells released per gram of muscle or to determine the plating efficiency. Furthermore, no data was given to demonstrate the reproducibility of his technique. We now report a reproducible and quantitative technique for the production of fully differentiated muscle cultures derived from mononucleated cells released from adult hamster skeletal muscle. To our knowledge, this is the first report of quantitative data on cell yield,
and Methods
The biceps muscle from 612 week old male hamsters (DSN, The Coombehurst Breeding Establishment, Baughurst, Hants) was used for this study. General techniques for dissociation and cell growth have been described [3]. 200400 mg of muscle was freshly excised, placed in a beaker and incubated with g-12 ml of phosphate-buffered saline (PBS), pH 7.2 containing 0.1% collagenase plus 0.2 % trypsin (see table 1) in a slowly shaking water bath at 37°C. The muscle was teased with needles twice during this incubation. After 15 mitt, the supematant was decanted into an equal volume of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco-Biocult, without pyruvate) supplemented with 4 mM glutamine and 10% Foetal Calf Serum (FCS, Gibco-Biocult) to neutralize the enzymes. This neutralized solution contained numerous small muscle pieces, some of which were dispersed by triturating- gently 10 times with a 2.5 ml wide bore pipette (2 mm bore diameter). The remaining large debris, which was not dispersed by this procedure, was removed by filtration through 3 layers of 50 pm nylon mesh (R. Cadisch & Sons, London) after which the cells were sedimented at 50 a for 10 min at room temperature. The cells were suspended in 0.5-l ml of erowth medium: DMEM containinn 4 mM erlutamine. TO% FCS and 2% detoxified Chick Embryo Extract [4] (CEE, Flow Laboratories). The cells were stored in a CO* incubator (37°C) until plated. The undigested muscle remaining in the beaker was dissociated three more times, as described above, using fresh enzyme solution each time. At the end of the 3rd and 4th dissociations the supematants were decanted, as described above, and final dissolution of the muscle was achieved by triturating the remaining muscle in the following manner: 5 ml of neutralized supernatant was added to the undigested muscle fragments and they were slowly triturated with a wide bore 10 ml pipette (1 mm bore). The supernatant was decanted back into the original neutralized enzyme solution. After allowing the single cells to settle for 2 mitt, another 5 ml of neutralized supernatant was removed from the top portion of the solution and the trituration was repeated a second time with the supernatant similarly decanted. The neutralized enzyme solution was treated as described above. The cell suspensions were combined and the cells were counted in a Neubauer haemocytometer in the presence of 0.5% nigrosine. They were then plated on 35 mm Falcon dishes coated with rat tail collagen, in a total volume of 1.5 ml growth medium and were grown in a humidified atmosphere containing Exp Cell Res 102 (1976)
406
Preliminary notes
Table 1. Quantitative assessment of the dissociation, growth and differentiation of adult hamster muscle starting with single cells Expt no.
Yield-cells X lo”/g muscle
Cells plated (X 104/35mm dish)
Plating efficiency
CPK” (spec. act.)
1”
4.4
4.0
7.5
0.14 at 7 DIVd
:; 40
3.4 1.9 1.2
4.0 6.7 5.0
26.0 6.5 6.6
0.14 at 57 DIV 0.09 0.13 at 7 DIV
2:
1.8 1.9
8.2 5.2
13.0 5.8
0.13 at 7 DIV
;I: 9* lo*
0.9 1.9 1.2 2.0
4.8 4.2 10.5 12.0
5.6 ::i 5.9
0.17 0.15 at 78 DIV 0.12 at 7 DIV 0.12 at 7 DIV
’ b ’ d
0.1% collagenase Worthington CSL II (190 U/mg)+O.Z% Trypsin (Difco, 1: 250). 0.1% collagenase Sigma Type II (150 U/n&+0.2 % Trypsin (Difco, 1: 250). Creatine phosphokinase activity in pmoles creatinelminlmg protein at 25°C. Days in vitro.
1.5% CO*, 85% air. On the following day, the plates were washed twice with growth medium to remove the small debris, prior to determining the plating efficiency (i.e., the % of cells originally inoculated which had attached to the substratum after 20 h in vitro). Twenty to 40 fields were counted in each dish with an inverted microscope (Nikon) at x450 magnification (Field size, 0.2831 mm*). The medium was subsequently changed every second day. To harvest the cells, the medium was decanted and the plates were washed with cold PBS. The cells were scraped off the plates with a spatula coated with Teflon tape into 0.5 ml PBS, and sonicated for two 5-set periods. The CPK activity [5] was determined immediately. Protein was measured by the method of Lowry et al. [6].
Results The procedure outlined above yields mononucleated cells and is the result of a series Table 2. Time course of CPK and protein during myogenesis of adult hamster muscle cultures Days in vitro 3 (2) Protein, n-&plate CPK, pmoles creature/ min/plate CPK, spec. act.
4 (3)
5 (2)
7 (3)
0.048 0.21
0.33
0.35
0.002 0.013 0.041 0.067 0.047 0.065 0.13 0.19
No. of plates is given in parentheses. Results represent mean value. Exp CeNRes 102(1976)
of experiments comparing different methods of enzyme dissociation [7]. Table 1 lists the results obtained for ten different experiments. With this technique we obtained a yield of 1-4~ lo6 cells/g muscle wet weight, with 4-10% of the cells becoming attached 20 h after plating. On the basis of morphological differences [8], 4&50% of the cells attached at 36 h in culture appeared to be myoblasts. However, this method of measuring the myogenic cell population is rather dubious, since several workers have demonstrated that both the morphology and the number of cells which fuse into myotubes can be influenced by a variety of culture conditions, namely, a “fibroblast” can become part of a myotube [I, 91. A more objective procedure for measuring the myogenie population would be to estimate the specific activity of CPK in fully differentiated cultures, since this enzyme is conFig. 1. Phase contrast appearance of muscle cells in
culture. Barred line represents 10 pm. (a) Mononucleated cells liberated from adult hamster skeletal muscle 72 h after plating. Arrow indicates alignment of cells preparation for fusion; (6) appearance of myotubes after 7 days in culture.
Preliminary
notes
407
Exp CellRes 102 (1976)
408
Preliminary
notes
centrated in the myotubes. Table 2 demonstrates a CPK time course during the differentiation of the cultures. Fig. 1a illustrates the appearance of the single cells 72 h after plating. The cells with myogenic capacity began to fuse between the 3rd and 4th day (depending on the initial density) and by day 6-7 were fully differentiated into myotubes (fig. lb), which exhibited spontaneous contractions. The values obtained for the specific activity of CPK which reached a maximum at day 7, ranged between 0.13 to 0.17 pmoles creatine per min/mg protein at 25°C. Substituting 10% Human Serum (Type AB) for 20% FCS on day 4 increased the specific activities of CPK of such cultures to 0.40, thus comparing favourably with values reported by Yaffe’s laboratory [lo] of 0.67 pmoles/min/ mg protein (when recalculated to 2X), for primary muscle cell culture from newborn rat thigh muscle. Further data on CPK growth curves, total CPK and total protein/ plate will be discussed in a separate paper. This method for growing adult skeletal muscle in culture will be of value in comparing growth and differentiation of human muscle from normal and diseased material, particularly since the results of such comparisons reported in the past have been equivocal [II, 12, 131. We have recently successfully cultured normal and diseased adult human muscle with the same procedure and our preliminary results indicate the applicability of this technique to biopsy samples of as low as 150 mg tissue (manuscript in preparation). Depending on the condition of the human muscle, we have so far obtained 0.3 to 4~ log cells/g tissue wet weight with plating efficiencies ranging between l-10 %. This work was supported by the Muscular Dystrophy Group of Great Britain. Exp CellRes 102(1976)
References I. Holtzer, H, Jones, K W & Yaffe, D, J neurol sci 26 (1975) 1IS. 2. Bischoff, R, Anat ret 180(1974) 645. 3. Wilson, S H, Schrier, B K, Farber, J L, Thompson, E J, Rosenberg, R H, Blume, A J & Nirenberg, M W, J biol them 247 (1972) 3 159. 4. Skrbic, T R, Yasin, R, Van Beers, G, Bulien, D & Thompson, E J, Biochem sot trans 3 (1975) 4%. 5. Szasz, G, Busch, F W & Farahs, H B, Deut med Wochschr 9 (1970) 829. 6. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193(1951) 265. 7. Yasin, R, Skrbic, T R, Van Beers, G & Thompson, E J, Biochem sot trans 4 (1976) 53. 8. Morris, G E & Cole, R J, Exp cell res 75 (1972) 191. 9. Hauschka, S D, Research in muscle development and the muscle spindle (ed B Q Banker, R J Pryblyski, J P Van de Meuler & M Vickr) p. 53. Excerpta Medica, Amsterdam & London (1972). 10. Shainberg, A, Yagil, G & Yaffe, D, Dev biol 25 (1971) 1. II. Bateson, R G, Hindle, D &Warren, J, J neurol sci 15 (1972) 183. 12. Gallup, B, Strugalska-Cyanowska, H & Dubowitz, V, J neurol sci 17 (1972) 109. 13. Witkowski, J A & Dubowitz, V, J neurol sci 26 (1975) 203. Received March 2, 1976 Accepted May 25, 1976
DAPI fluorescence of plant chromosomes prestained with actinomycin D D. SCHWEIZER, Znstirure of Botany, University of Vienna, A-1030 Vienna, Austria Summary. Two plants, Ornithogalum caudatum and Scilla siberica, with contrasting types of heterochromatin, were employed to investigate the effect of prestaining with actinomycin D on the differential DAPI fluorescence in nuclei and chromosomes. Actinomycin D-pretreated preparations exhibited in both plants a significantly lower overall fluorescence intensitv than D-API controls. In 0. caudatum the fluorescence properties of DAPI-bright heterochromatin remained oualitatively unchanged, but in S. siberica chromosomes the differential fluorescence of DAPI-negative heterochromatin disappeared or occasionally was reversed. It is shown that the observations cannot be interpreted simply as a result of base specific drug-DNA interaction.
The DNA-binding compund 4’-f+diamidino-Zphenylindole (DAPI) [l] has proved a most valuable fluorochrome for the chro-
PRELIMINARY
NOTES
A quantitative procedure for the dissociation of adult mammalian muscle into mononucleated cells
plating efficiency and differentiation (determined by specific activity of creatine phosphokinase) for adult mammalian muscle.
ROSE YASIN, GISELA VAN BEERS, DITTA BULIEN and E. J. THOMPSON, Muscular Dys-
Materials
trophy Research Laboratories, London WC1 N 3BG, UK
The National
Hospital,
Summary. A new technique for quantitative analysis of dissociated adult skeletal muscle is described. Using adult hamster biceps we obtained cell yields of l-4 x 1W viable cells/g muscle, 4-10% plating efficiency (% cells attached at 20 h in vitro) and spontaneously contracting myotubes at day 7 which exhibited creatine phosphokinase specific activities of 0.4 pmoles/min/ mg protein at 25°C. This technique can be applied to other adult tissue.
Successful growth of adult tissue in culture is acknowledged to be more difficult than that of embryonic or neonatal tissues. As recently stated by Holtzer et al. [l], the technique conventionally used for culturing adult skeletal muscle (by explanting tissue pieces) is unsatisfactory because it is not possible to perform quantitative experiments. This problem would be eliminated if cultures could be established, starting with single cells which eventually differentiated into myotubes. The release of myogenic cells from adult rat skeletal muscle has been reported by Bischoff [2]; however, he was unable to count the number of cells released per gram of muscle or to determine the plating efficiency. Furthermore, no data was given to demonstrate the reproducibility of his technique. We now report a reproducible and quantitative technique for the production of fully differentiated muscle cultures derived from mononucleated cells released from adult hamster skeletal muscle. To our knowledge, this is the first report of quantitative data on cell yield,
and Methods
The biceps muscle from 612 week old male hamsters (DSN, The Coombehurst Breeding Establishment, Baughurst, Hants) was used for this study. General techniques for dissociation and cell growth have been described [3]. 200400 mg of muscle was freshly excised, placed in a beaker and incubated with g-12 ml of phosphate-buffered saline (PBS), pH 7.2 containing 0.1% collagenase plus 0.2 % trypsin (see table 1) in a slowly shaking water bath at 37°C. The muscle was teased with needles twice during this incubation. After 15 mitt, the supematant was decanted into an equal volume of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco-Biocult, without pyruvate) supplemented with 4 mM glutamine and 10% Foetal Calf Serum (FCS, Gibco-Biocult) to neutralize the enzymes. This neutralized solution contained numerous small muscle pieces, some of which were dispersed by triturating- gently 10 times with a 2.5 ml wide bore pipette (2 mm bore diameter). The remaining large debris, which was not dispersed by this procedure, was removed by filtration through 3 layers of 50 pm nylon mesh (R. Cadisch & Sons, London) after which the cells were sedimented at 50 a for 10 min at room temperature. The cells were suspended in 0.5-l ml of erowth medium: DMEM containinn 4 mM erlutamine. TO% FCS and 2% detoxified Chick Embryo Extract [4] (CEE, Flow Laboratories). The cells were stored in a CO* incubator (37°C) until plated. The undigested muscle remaining in the beaker was dissociated three more times, as described above, using fresh enzyme solution each time. At the end of the 3rd and 4th dissociations the supematants were decanted, as described above, and final dissolution of the muscle was achieved by triturating the remaining muscle in the following manner: 5 ml of neutralized supernatant was added to the undigested muscle fragments and they were slowly triturated with a wide bore 10 ml pipette (1 mm bore). The supernatant was decanted back into the original neutralized enzyme solution. After allowing the single cells to settle for 2 mitt, another 5 ml of neutralized supernatant was removed from the top portion of the solution and the trituration was repeated a second time with the supernatant similarly decanted. The neutralized enzyme solution was treated as described above. The cell suspensions were combined and the cells were counted in a Neubauer haemocytometer in the presence of 0.5% nigrosine. They were then plated on 35 mm Falcon dishes coated with rat tail collagen, in a total volume of 1.5 ml growth medium and were grown in a humidified atmosphere containing Exp Cell Res 102 (1976)
406
Preliminary notes
Table 1. Quantitative assessment of the dissociation, growth and differentiation of adult hamster muscle starting with single cells Expt no.
Yield-cells X lo”/g muscle
Cells plated (X 104/35mm dish)
Plating efficiency
CPK” (spec. act.)
1”
4.4
4.0
7.5
0.14 at 7 DIVd
:; 40
3.4 1.9 1.2
4.0 6.7 5.0
26.0 6.5 6.6
0.14 at 57 DIV 0.09 0.13 at 7 DIV
2:
1.8 1.9
8.2 5.2
13.0 5.8
0.13 at 7 DIV
;I: 9* lo*
0.9 1.9 1.2 2.0
4.8 4.2 10.5 12.0
5.6 ::i 5.9
0.17 0.15 at 78 DIV 0.12 at 7 DIV 0.12 at 7 DIV
’ b ’ d
0.1% collagenase Worthington CSL II (190 U/mg)+O.Z% Trypsin (Difco, 1: 250). 0.1% collagenase Sigma Type II (150 U/n&+0.2 % Trypsin (Difco, 1: 250). Creatine phosphokinase activity in pmoles creatinelminlmg protein at 25°C. Days in vitro.
1.5% CO*, 85% air. On the following day, the plates were washed twice with growth medium to remove the small debris, prior to determining the plating efficiency (i.e., the % of cells originally inoculated which had attached to the substratum after 20 h in vitro). Twenty to 40 fields were counted in each dish with an inverted microscope (Nikon) at x450 magnification (Field size, 0.2831 mm*). The medium was subsequently changed every second day. To harvest the cells, the medium was decanted and the plates were washed with cold PBS. The cells were scraped off the plates with a spatula coated with Teflon tape into 0.5 ml PBS, and sonicated for two 5-set periods. The CPK activity [5] was determined immediately. Protein was measured by the method of Lowry et al. [6].
Results The procedure outlined above yields mononucleated cells and is the result of a series Table 2. Time course of CPK and protein during myogenesis of adult hamster muscle cultures Days in vitro 3 (2) Protein, n-&plate CPK, pmoles creature/ min/plate CPK, spec. act.
4 (3)
5 (2)
7 (3)
0.048 0.21
0.33
0.35
0.002 0.013 0.041 0.067 0.047 0.065 0.13 0.19
No. of plates is given in parentheses. Results represent mean value. Exp CeNRes 102(1976)
of experiments comparing different methods of enzyme dissociation [7]. Table 1 lists the results obtained for ten different experiments. With this technique we obtained a yield of 1-4~ lo6 cells/g muscle wet weight, with 4-10% of the cells becoming attached 20 h after plating. On the basis of morphological differences [8], 4&50% of the cells attached at 36 h in culture appeared to be myoblasts. However, this method of measuring the myogenic cell population is rather dubious, since several workers have demonstrated that both the morphology and the number of cells which fuse into myotubes can be influenced by a variety of culture conditions, namely, a “fibroblast” can become part of a myotube [I, 91. A more objective procedure for measuring the myogenie population would be to estimate the specific activity of CPK in fully differentiated cultures, since this enzyme is conFig. 1. Phase contrast appearance of muscle cells in
culture. Barred line represents 10 pm. (a) Mononucleated cells liberated from adult hamster skeletal muscle 72 h after plating. Arrow indicates alignment of cells preparation for fusion; (6) appearance of myotubes after 7 days in culture.
Preliminary
notes
407
Exp CellRes 102 (1976)
408
Preliminary
notes
centrated in the myotubes. Table 2 demonstrates a CPK time course during the differentiation of the cultures. Fig. 1a illustrates the appearance of the single cells 72 h after plating. The cells with myogenic capacity began to fuse between the 3rd and 4th day (depending on the initial density) and by day 6-7 were fully differentiated into myotubes (fig. lb), which exhibited spontaneous contractions. The values obtained for the specific activity of CPK which reached a maximum at day 7, ranged between 0.13 to 0.17 pmoles creatine per min/mg protein at 25°C. Substituting 10% Human Serum (Type AB) for 20% FCS on day 4 increased the specific activities of CPK of such cultures to 0.40, thus comparing favourably with values reported by Yaffe’s laboratory [lo] of 0.67 pmoles/min/ mg protein (when recalculated to 2X), for primary muscle cell culture from newborn rat thigh muscle. Further data on CPK growth curves, total CPK and total protein/ plate will be discussed in a separate paper. This method for growing adult skeletal muscle in culture will be of value in comparing growth and differentiation of human muscle from normal and diseased material, particularly since the results of such comparisons reported in the past have been equivocal [II, 12, 131. We have recently successfully cultured normal and diseased adult human muscle with the same procedure and our preliminary results indicate the applicability of this technique to biopsy samples of as low as 150 mg tissue (manuscript in preparation). Depending on the condition of the human muscle, we have so far obtained 0.3 to 4~ log cells/g tissue wet weight with plating efficiencies ranging between l-10 %. This work was supported by the Muscular Dystrophy Group of Great Britain. Exp CellRes 102(1976)
References I. Holtzer, H, Jones, K W & Yaffe, D, J neurol sci 26 (1975) 1IS. 2. Bischoff, R, Anat ret 180(1974) 645. 3. Wilson, S H, Schrier, B K, Farber, J L, Thompson, E J, Rosenberg, R H, Blume, A J & Nirenberg, M W, J biol them 247 (1972) 3 159. 4. Skrbic, T R, Yasin, R, Van Beers, G, Bulien, D & Thompson, E J, Biochem sot trans 3 (1975) 4%. 5. Szasz, G, Busch, F W & Farahs, H B, Deut med Wochschr 9 (1970) 829. 6. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193(1951) 265. 7. Yasin, R, Skrbic, T R, Van Beers, G & Thompson, E J, Biochem sot trans 4 (1976) 53. 8. Morris, G E & Cole, R J, Exp cell res 75 (1972) 191. 9. Hauschka, S D, Research in muscle development and the muscle spindle (ed B Q Banker, R J Pryblyski, J P Van de Meuler & M Vickr) p. 53. Excerpta Medica, Amsterdam & London (1972). 10. Shainberg, A, Yagil, G & Yaffe, D, Dev biol 25 (1971) 1. II. Bateson, R G, Hindle, D &Warren, J, J neurol sci 15 (1972) 183. 12. Gallup, B, Strugalska-Cyanowska, H & Dubowitz, V, J neurol sci 17 (1972) 109. 13. Witkowski, J A & Dubowitz, V, J neurol sci 26 (1975) 203. Received March 2, 1976 Accepted May 25, 1976
DAPI fluorescence of plant chromosomes prestained with actinomycin D D. SCHWEIZER, Znstirure of Botany, University of Vienna, A-1030 Vienna, Austria Summary. Two plants, Ornithogalum caudatum and Scilla siberica, with contrasting types of heterochromatin, were employed to investigate the effect of prestaining with actinomycin D on the differential DAPI fluorescence in nuclei and chromosomes. Actinomycin D-pretreated preparations exhibited in both plants a significantly lower overall fluorescence intensitv than D-API controls. In 0. caudatum the fluorescence properties of DAPI-bright heterochromatin remained oualitatively unchanged, but in S. siberica chromosomes the differential fluorescence of DAPI-negative heterochromatin disappeared or occasionally was reversed. It is shown that the observations cannot be interpreted simply as a result of base specific drug-DNA interaction.
The DNA-binding compund 4’-f+diamidino-Zphenylindole (DAPI) [l] has proved a most valuable fluorochrome for the chro-