Creatine kinase isoenzymes in cultured human muscle cells. II. A study of carrier females for Duchenne muscular dystrophy by needle and open biopsy.

Creatine kinase (CK) isoenzymes, total and specific CK activities and protein concentrations were measured in the cultured cells from muscle biopsies of 18 carriers of Duchenne muscular dystrophy (DMD) and the results compared with those in the cultures of patients with DMD and other neuromuscular disorders. The proportion of MB and BB isoenzyme in the carriers was similar to that in boys with DMD and in patients with other myogenic disorders, and significantly different from neurogenic patients. CK isoenzyme analysis appears to be a more sensitive index of in vitro differentiation than estimation of CK activities. Cell differentiation is reduced and the incidence of cell death increased in cultures derived from needle rather than open biopsies.

An abnormality of cell behaviour in human dystrophic muscle cultures: a time-lapse study.

The initiation of monolayer mass cell cultures from adult human biopsies has revealed a striking abnormality in the growth and development pattern of muscle cultures from Duchenne-type dystrophy. This abnormality in cell behaviour was seen as early as 4 days in culture, well before myotube formation or confluence, and consisted of areas where cells clustered together in a multilayered mass rather than showing the typical monolayer distribution normally observed. To gain some insight into the mechanism of cell cluster development, we have examined such a culture by time-lapse cinematography and also the cell behaviour of other control cultures. The results of this study show that the clusters enlarged primarily by cell division and, to a lesser extent, by the acquisition of neighbouring cells. Furthermore, none of the single cells surrounding the clusters exhibited contact inhibition of movement. This behaviour was not observed in the other cultures examined. These findings could be indicative of an abnormality in the cell surface or cell-locomotory machinery of dystrophic cells.

A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells.

A quantitative and reproducible technique for establishing primary surface cultures from normal and diseased human muscle is described. Successful cultures were prepared from both fresh muscle and that stored up to 96 hr at 4 degrees C. The CPK activity of the muscle cells ranged between 0.5-3.0 micronmoles creatine per min per mg protein at 30 degrees C, thus indicating a high degree of differentiation. Spontaneous contractions were observed in 4 out of the 22 cultures established. Nerve cells were not required to achieve this level of differentiated function. No gross differences in plating efficiency, rate of myotube formation or CPK specific activity were found for the diseased muscle cells cultured so far. However, a 5--10-fold higher cell yield was obtained from muscles of patients with an inflammatory myopathy. The advantages of this technique for carrying out comparative studies on normal and dystrophic muscle cells are discussed.