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P R E P A R A T I O N OF A D U L T M A M M A L I A N S K E L E T A L M U S C L E C U L T U R E S U S I N G P A P A I N D I G E S T I O N

Submitted by

JULIE MORGAN and LOUIS COHEN

Department of Medicine University of Chicago Chicago, Illinois 60637

I. INTRODUCTION

A method is described by which adult mammalian skeletal muscle can be enzymatically separated into viable individual muscle cell pieces. One does not separate whole muscle cells, because many of them are very long and usually many millimeters to centimeters in length. As the maximum length of piece obtained in the procedure is about 400 ~m, it is most unusual to separate a single cell. In addition, it is unusual to get "small aggregates of cells" because again they would only be pieces of cells. The use of the term "pieces" should not convey the idea that whole muscle cells can be separated. This can be done with the papain, but only with very gentle separation using micro- manipulator tools. These long muscle cell pieces do not regenerate very well in any case, and thus are not useful for establishing cultures with a high percentage of myoblasts. These cell pieces are used to observe skeletal muscle regeneration in vitro {1,2}. The use of explants is not satisfactory because the early events of degeneration and regeneration are obscured. Crude papain was con- sidered for enzymatic separation because, in addition to the digestion of intercellular proteins, the presence of some nucleases prevent the formation of glutinous material which interferes with the separation of the individual muscle cell pieces. We have found the method described below to be ef- fective for separating skeletal muscle of many mammalian species. I t has also been useful in the preparation of cultures from cardiac muscle and aorta samples.

II. MATERIALS

Glassware All glassware is sterilized by autoclaving at

15 lbs pressure for 30 min Glass bottles: 100-ml serum bottles,

GIBCO, 1 and 500-ml and 1000-ml sterile water bottles, Travenol 2

Pasteur pipets, cotton-plugged, 53A-inch length, Chase, 3 packaged for sterilization with six to a 20-cc syringe packet, Chief- tain No. 25526-010 AHS 4

Glass centrifuge tubes, 10-ml, capped with aluminum foil or stainless-steel closures with fingers, No. 16 Bellco ~

Plastic ware All disposable plastic ware is obtained

sterile Culture flasks, 30-ml, No. 3012 Falcon 6 or

No. 25100 Corning 7

1 Grand Island Biological Co., Grand Island, NY. 2 Travenol Laboratories, Deerfield, IL. 3 Chase Instruments Corp., Lindenhurst, NY. 4 American Hospital Supply, Evanston, IL. s Bellco Glass, Inc., Vineland, NJ. 6 Falcon Plastics, Oxnard, CA. 7 Corning Glass Works, Corning, NY. 8 Linbro Scientific Co., Inc., New Haven, CT. 9 Becton, Dickinson and Co., Rutherford, NJ.

~o Baltimore Biological Laboratories, CockeysviUe, MD.

Petri dishes, 60-mm diameter, No. 30026 Multi-dishes, 24-well, No. FB-16-24-TC Lin-

bro s Pipets, graduated 10-ml, No. 75516 Plastic syringes, 1-ml, 5-ml, 10-ml, Plas-

tipak, B-D 9 Solutions and media

Horse serum 1 (or BBL 1~ Fetal bovine serum 1'1~ Human serum: 40 ml blood is collected from

a volunteer (always female in our case) af- ter an overnight fast and allowed to clot. The blood is centrifuged at 200 to 250 X g for 10 min and the serum pipetted into a sterile tube. The serum is mixed by gentle inversion of the tube and then dispensed in 1-ml portions into small sterile glass tubes which are capped or sealed with parafilm. The serum is stored frozen until needed. I t is neither sterilized in any way nor heat-inactivated. No contamination of the cultures has occurred using this procedure.

Eagle's Minimum Essential Medium (MEM) with Earle's salts, bulk or individ- ual packets of powder, No. F-11,1 diluted with sterile water and sterilized by filtra- tion through a 0.45-~m Millipore filter.

Sterile water for irrigation, for preparing

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MEM and modified BSS ~ Papain, Crude Powder, type II, Sigma ~ Cysteine hydrochloride, No. C-562 Fisher ~ Antibiotic solution: penicillin-streptomycin

mixture, 20 ml (10,000 units per ml pen- icillin; 10,000/~g per ml streptomycin) TM

Sodium bicarbonate, 8.4%, Abbott ~ The complete medium to be used contains:

400 ml Eagle's MEM {with Earle's salts); 100 ml fetal bovine serum; 12 ml of 8.4% sodium bicarbonate solution; 2.5 ml anti- biotic solution {final concentration of 50 units penicillin, 50 ~g streptomycin per mD.

The complete medium with increased con- centrations of antibiotics: same as above complete medium, but containing 200 units penicillin and 200 ~g streptomycin per ml

Modified balanced salt solution (BSS), made up as a 10X strength stock solution con- taining: NaC1 68.0 g, KC1 4.0 g, glucose 10.0 g, sodium bicarbonate 22.0 g, and phenol red 0.05 g made up to 1 1 with ster- ile water and sterilized by filtration through a 0.45-~m Millipore filter. For use, 100 ml of the 10X stock solution is di- luted to 1 1 with sterile water. This dilute solution then contains: NaC1 6.8 g, KC1 0.4 g, glucose 1.0 g, sodium bicarbonate 2.2 g, and phenol red 0.005 g in 1 l, with an osmolality of 300 mOSM.

Papain solution (3) contains: 0.10 g papain and 0.02 g cysteine hydrochloride in 100 ml dilute 1X BSS prepared and warmed to 37~ just prior to use. The amount of papain and cysteine hydrocloride is halved for most species other than dog.

Other equipment Hypodermic needles, 20- or 21-gauge by 1.5-

inch, Yale, Luer-Lok Hub ~ Scissors, straight operating, regular, length

4�89 No. SU-1700 Mueller ~4 Surgical blades, No. 10, SU-1370 and No.

21, SU-1375 ~4 surgical handles, ASR push-button release, SU-1360 (for No. 10 blades) and SU-1361 (for No. 21 blades) TM

Forceps, Brophy tissue, No. MO-500 ~" Parafilm, American Can TM

Filter unit and 0.45-~m pore size filter, No. HAWPO4700, Millipore ~

" S igma Chemical Co., St. Louis , MO. '* F i sher Scientific Co., Fa i r Lawn, NJ . ~ A b b o t t Labora tor ies , Nor t h Chicago, IL. '" V. Mueller, Chicago, IL. ,s Amer i can Can Co., Neenah, WI. ~' Millipore Corp., Bedford, MA. ~ H o t p a c k Corp., Phi ladelphia, PA. ~ In te rna t iona l E q u i p m e n t Co., N e e d h a m He igh t s , MA. ~ Carl Zeiss Inc., New York, NY.

CO2 incubator, set at 5% CO2, Model 351814 Hotpack ~

Centrifuge, Model HN International ~8 Inverted phase contrast microscope with

camera attachment, Zeiss ~ Kodak Plus X or Kodachrome II film

III . P R O C E D U R E

A. Establishment of culture from fresh tissue

1. Collect small pieces of skeletal muscle {5mm 3 or less} by sterile techniques, using a scalpel with a disposable blade, and im- merse at 37~ in Eagle's MEM {with high antibiotic concentration} for at least 30 min to destroy external contamination. Samples can remain at room temperature or 37~ without further treatment for 0.5 to 14 hr without apparent effect on viabil- ity, but they are generally placed in the refrigerator (4~ if they cannot be pro- E

d th d z cesse e same ay. ~ ~ o

2. Transfer the muscle to a 60-mm plastic ~ ~ | Petri dish containing about 10 ml papain ~ ~ .-~ solution (3). It has not been found neces- ~ "~ sary to filter the dilute 1X BSS after the ~ ~ ~ d

~ t o _ m addition of the papain and cysteine. No ~ ~ ~ contamination has occurred from this ~ ~ = sourc

3. Mince the tissue into small (1 mm 3) pieces ~ "' with scissors. When mincing larger sam- ._o .~ r

ples, pull away and discard adhering con- ~ V- nective tissue. <

4. Transfer the minced muscle plus papain solution to a 30-ml plastic culture flask and place in a water bath at 37~ Agi- tate once or twice during a 10-rain period.

5. Allow the cell pieces to settle for 1 min, and decant the cell suspension into a cen- trifuge tube containing 1 ml horse serum for every 9 ml suspension. Add the horse serum to stop the enzyme action and to protect the cells during the subsequent centrifugation step.

6. Add fresh papain solution to the remain- ~.J ing tissue pieces in the flask for further 10-min incubation.

a 7. Perform six or more such consecutive pa-

pain treatments. The number varies de- ~ - - Z pending on the size of the original sample ~ z d

and the degree of agitation given during ~ ~ z each treatment. ,,, > ~,

m z -~ 8. Centrifuge the cell suspensions at o a -o

{D 100 • g fo r 10min. ~-z o I - - -

o e 9. Resuspend the resulting cell pellets in Ea- z o_

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gle's MEM (with low antibiotic concen- tration). The volume in which each pellet is suspended can be varied as necessary. At this stage count the number of cells or muscle cell pieces per ml (using a hema- cytometer). We do not use any stain for the counting procedures. In the majority of samples of human muscle, obtained by muscle biopsy, only 1 ml of medium, con- taining 500 to 750 muscle cell pieces, is used.

10. Place the suspensions 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 in diameter by 18-mm deep. Cultures can also be estab- lished in 60-mm plastic Petri dishes in 4 ml Eagle's medium or any other suitable container.

11. Maintain cultures in a water-saturated at- mosphere at 37~ in an incubator sup- plied with 5% CO2 in air.

12. Change the medium for the first time af- ter 1 week.

13. Transfer the supernatant fluid, plus any cell pieces still suspended, to the second row of six wells in the Linbro plate or to new Petri dishes.

14. Place fresh Eagle's medium (low antibio- tic) in the original culture chambers. This procedure avoids loss of cell fragments and allows the morphology of the earliest attached cells to be examined without interference from floating debris.

15. Make similar transfers as cells attach in the second row of wells. By the time three transfers have been made (21 days) the growth of the cells in the initial row is of- ten such that a confluent monolayer is formed.

16. To obtain striated human myotubes, re- place (this is essential in most case) the fe- tal bovine serum with human serum. Use nonsterilized-non heat inactivated serum which is freshly prepared from whole clotted blood and stored in 1-ml aliquots and frozen until needed.

B. Establishment of subcultures

1. To establish subcultures, remove the cul- ture medium from the confluent cultures.

2. Briefly rinse each well with 0.5 ml dilute 1 >< BSS.

3. Place 1 ml of a solution containing 0.05% papain and 0.01% cysteine hydrochloride in dilute BSS into each well.

4. When most of the cells become rounded, loosen the remainder by vigorous pipet- ring of the papain solution.

5. Transfer the entire 1 ml papain plus sus- pended cells to a sterile plastic Petri dish containing 4 ml Eagle's medium prepared as described above. All cell types, includ- ing those myotubes which do not frag- ment during the papain treatment, attach again rapidly. After subculture, fibro- blasts begin to spread out in the first 1 to 2 min, and myoblasts a n d myotubes within 10 to 15 min.

6. Follow events of all stages of culture (sep- aration of muscle into cell fragments, cell attachment, cell division, cell fusion, myotube development, and the appear- ance of myofibrils) with phase contrast microscopy, using a Zeiss inverted micro- scope with a 35-mm photographic attach- ment on Kodak Plus X or Kodachrome II film.

IV. DISCUSSION

Papain is a mixture of proteases from Carica papaya, the papaya plant (3). I t is used as a meat tenderizer because of its ability to di- gest intercellular proteins. I t contains en- zymes which are similar to pepsin but which can act in acid, neutral or alkaline medium. In the procedure described here, it rapidly separ- ates the long muscle fibers with very little agitation.

In dog and monkey samples, at least 90% of the muscle cell pieces (of all sizes) eventually become attached to the substratum and give rise to cells, the number of which is propor- tional to the original size of the muscle. I t is difficult to talk about viability of muscle cell pieces, except that those pieces that have a frayed or flared appearance at the broken ends will probably not produce new cells. The number of mononucleated cells produced from any given muscle cell piece appears to be directly related to its length and probably the number of nuclei present. The situation is fur- ther complicated by the fact that many cell pieces with frayed ends will become sealed at points distant from the ends and then pro- duce mononucleated cells. In all the species we have studied, except dog, the muscle cell pieces that settle produce mononucleated cells in 5 to 9 days. Dog muscle produces "buds," also in 5 to 9 days, which fall from floating pieces as well as from those which have settled. The buds may form either myo- blasts or fibroblasts. As we have had very few (1 human and 2 rabbit) cultures in which there

465

has been no growth at all, we continue all cul- tures until all the necessary observations or analyses have been made.

The best and fastest development of myo- tubes in all species is seen in those suspen- sions in which the muscle cell pieces are sub- jected to 50 min of papain treatment. Occa- sionally, in human cultures, however, large muscle cell pieces produced by shorter per- iods of papain treatment do not show any changes or proliferation of cells and remain floating in the medium for many weeks with- out any apparent change. Fibroblasts also survive welt. Their relative number can be re- duced in a given culture by performing sev- eral brief papain treatments and subsequent transfers of the cell suspension at the approp- riate time, as described earlier. The method can be used on muscle samples as small as 2 mm ~ and will yield muscle cell pieces suffi- cient to establish three or four cultures. This procedure has proved effective in culturing rabbit psoas, adult mammalian (human, dog, pig, sheep and rabbit) cardiac muscle, and rabbit aorta. Pig skeletal muscle has been grown but the myotubes 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 cul- ture conditions, which are suitable for canine and primate species, but are inadequate for the pig.

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 sus- pensions to remove the papain solution. The

rapidity with which the papain solution loses its activity may also be a problem in some cases. Similar loss of activity at room tem- perature is seen with other proteolytic en- zymes, such as trypsin. We have found that a piece of muscle can be separated successfully with six papain treatments in about 1.5 hr.

The limitations of this method relate to the fact that the fibroblasts also survive. The ideal enzyme to separate muscle is one which would successfully separate viable muscle cells and at the same time destroy the fibro- blast population.

This procedure has many potential uses. I t is possible to observe the muscle cell pieces con- tinuously during treatment in papain, im- mediately after separation or injury and through many weeks of the subsequent growth period. The papain solution has also been used to separate individual muscle cells from rabbit psoas using microneedles. By this method, cultures can be established from very small muscle pieces.

V. REFERENCES

1. Morgan, J., and L. Cohen. 1974. Use of papain in the preparation of adult mammal- ian skeletal muscle for tissue culture. In Vitro 10: 188-195.

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

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

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