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

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 growth from biopsy material from boys with Duchenne’s muscular 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 Ca²⁺-and Mg²⁺-free balanced salt solution and transferred in the papain solution to a flask, in which they are incubated at 37°C for 10 min with occasional 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 subcultured and collected for indefinite frozen storage in dimethylsulfoxide.