Culturing of diagnostic muscle biopsies as spheroid-like structures: a pilot study of morphology and viability (original) (raw)
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Preparation of Isolated Human Muscle Fibers: A Technical Report
In Vitro Cellular & Developmental Biology - Animal, 2002
The aim of this study was to develop a technique to culture satellite cells from isolated intact fast or slow human muscle fibers. Previous studies have been carried out on small rodent muscles where the fibers run from tendon to tendon, but this is the first description of the modification of this technique for much larger human muscles. We have demonstrated that the human muscle fibers are in fact segmental, and we have also shown that it is possible to obtain very pure satellite cell cultures. We discuss the importance of this technique as a source of highly purified muscle cell cultures, which can be used for further studies on satellite cell behavior.
Relative contribution of different classes of myogenic cells to muscle fiber formation in culture
Experimental Cell Research, 1973
Mitotically active cells were labelled in the explant with SH-thymidine for 3 h and then chased with an excess of cold precursor for 12 h before plating in culture. In these conditions no further incorporation of radioactivity occurs in culture. The participation of labelled cells in the fiber formation was followed by autoradiography. The data reported show that the formation of muscle fibers in culture occurs preferentially by fusion of myogenic cells that are actively duplicating their DNA in the primary explant. The participation of cells already differentiated and unable to divide mitotically in the explant appears to be less relevant.
Biotechnology and bioprocessing, 2021
Background: Skeletal muscles are attached to bone and are responsible for the axial and appendicular movement of the skeleton and for maintenance of body position and posture. Objectives: The present review aimed to high light on embryonic development of skeletal muscles, histological and ultrastructure, innervation, contraction and relaxation, causes, pathophysiology, and treatment of volumetric muscle injury. The heterogeneity of the muscle fibers is the base of the flexibility which allows the same muscle to be used for various tasks from continuous low-intensity activity, to repeated submaximal contractions, and to fast and strong maximal contractions. The formation of skeletal muscle begins during the fourth week of embryonic development as specialized mesodermal cells, termed myoblasts. As growth of the muscle fibers continues, aggregation into bundles occurs, and by birth, myoblast activity has ceased. Satellite cells (SCs), have single nuclei and act as regenerative cells. Satellite cells are the resident stem cells of skeletal muscle; they are considered to be self-renewing and serve to generate a population of differentiation-competent myoblasts that will participate as needed in muscle growth, repair, and regeneration. Based on various structural and functional characteristics, skeletal muscle fibres are classified into three types: Type I fibres, Type II-B fibres, and type II-A fibres. Skeletal muscle fibres vary in colour depending on their content of myoglobin. Each myofibril exhibits a repeating pattern of cross-striations which is a product of the highly ordered arrangement of the contractile proteins within it. The parallel myofibrils are arranged with their cross-striations in the register, giving rise to the regular striations seen with light microscopy in longitudinal sections of skeletal muscle. Each skeletal muscle receives at least two types of nerve fibers: motor and sensory. Striated muscles and myotendinous junctions contain sensory receptors that are encapsulated proprioceptors. The process of contraction, usually triggered by neural impulses, obeys the all-ornone law. During muscle contraction, the thin filaments slide past the thick filaments, as proposed by Huxley's sliding filament theory. In response to a muscle injury, SCs are activated and start to proliferate; at this stage, they are often referred to as either myogenic precursor cells (MPC) or myoblasts. In vitro, evidence has been presented that satellite cells can be pushed towards the adipogenic and osteogenic lineages, but contamination of such cultures from non-myogenic cells is sometimes hard to dismiss as the underlying cause of this observed multipotency. There are, however, other populations of progenitors isolated from skeletal muscle, including endothelial cells and muscle-derived stem cells (MDSCs), blood-vessel-associated mesoangioblasts, muscle side-population cells, CD133+ve cells, myoendothelial cells, and pericytes. Volumetric muscle loss (VML) is defined as the traumatic or surgical loss of skeletal muscle with resultant functional impairment. It represents a challenging clinical problem for both military and civilian medicine. VML results in severe cosmetic deformities and debilitating functional loss. In response to damage, skeletal muscle goes through a well-defined series of events including; degeneration (1 to 3days), inflammation, and regeneration (3 to 4 weeks), fibrosis, and extracellular matrix remodeling (3 to 6 months).. Mammalian skeletal muscle has an impressive ability to regenerate itself in response to injury. During muscle tissue repair following damage, the degree of damage and the interactions between muscle and the infiltrating inflammatory cells appear to affect the successful outcome of the muscle repair process. The transplantation of stem cells into aberrant or injured tissue has long been a central goal of regenerative medicine and tissue engineering.
The regenerative response of single mature muscle fibers isolated in vitro
Developmental Biology, 1975
Segments of individual differentiated muscle fibers, ranging from l-10 mm in length, were dissected from the pectoral muscle of juvenile Japanese quail and cultured for periods ranging up to 2-3 weeks under conditions known to promote the proliferation and eventual differentiation of embryonic myoblasts. Approximately 22% of such fibers give rise to a colony of bipolar cells which expands in area and cell number. Sometime during the second or third week the process of myoblast fusion is initiated and a network of long cross-striated multinuclear cells is formed. In the vast majority of fibers the colony arises from only one highly localized site along the fiber suggesting that proliferative competence is restricted to extremely few fiber nuclei. This same conclusion is suggested, as well, by the observation that fiber degeneration, in terms of the loss of intact nuclei, occurs rapidly and is completed within 24 hr after explantation (and perhaps as soon as 4-8 hr). Those nuclei which survive (usually one per fiber) are found to be contained within separate bipolar cells closely applied to the fiber. An examination of the fine structure of these surviving cells shows them to be separate, mononucleated cells contained within the basement lamina of the degenerating fiber. These cells are identical, on the basis of their ultrastructure as well as their location, to satellite cells associated with muscle fibers in the source tissue.
Skeletal muscle. A review of its development in vivo and in vitro
Physical therapy, 1982
Physical therapists are well-aware of the form and function of mature skeletal muscle. The steps that muscle goes through to become that way, however, are generally less familiar. This article reviews some highlights of myogenesis as revealed by in vivo and in vitro studies. It also describes how tissue culture may help elucidate the mechanisms of genetic myopathies.
Biology of the Cell, 1997
The present study was performed to determine the influence on human satellite cell yield, proliferation, and differentiation rates of: 1) sex and age of donors; 2) site of the muscle biopsy; and 3) delay before processing of the muscle biopsy sample. We used a standardized primary muscle cell culture procedure on 206 normal muscle samples obtained from different muscle groups of patients aged from 20 to 88 years, at time of orthopedic surgery. Sex of donors did not influence muscle culture parameters. In contrast, aging tended to affect muscle cell yield (age group 50-59 years vs 70-79 years, P < 0.08), but not myogenic cell abilities to proliferate and to fuse into myotubes. The anatomic origin of muscle samples used for culture appeared to influence culture parameters. In contrast with other tested muscles, the tensor fasciae muscle gave both a good cell yield (174 +/- 25 10(3) cells per gram) and homogeneous proliferation and differentiation rates. Storage of the muscle sample at 4 degrees C in transport medium was associated with a very high cell yield when processing was done in early hours after biopsy (277 +/- 50 10(3) cells/g), a high and stable cell yield when processing was done from day 1 to day 3 after biopsy (185 +/- 15 10(3) cells/g), and a poor cell yield when processing was done after day 4 (111 +/- 13 10(3) cells/g). Storage of muscle biopsy samples at 4 degrees C for 1 to 4 days was associated with good proliferation and fusion rates. In conclusion, these data validate a convenient procedure of primary human muscle cell culture, using tensor fasciae muscle biopsy, which is easily done at time of orthopedic surgery, obtained from men and women of all ages (if possible less than 70 years to obtain good cell yield), and allowing of 1-3 days of storage before processing that may compensate uncertainty of the exact time of availability of muscle samples for the scientist.
Patterns of repair of dystrophic mouse muscle: Studies on isolated fibers
Developmental Dynamics, 1999
central to the regeneration that occurs after injury or disease of muscle and is vital to the success of myoblast transplantation to treat inherited myopathies. However, we lack a detailed knowledge of the mechanisms of this muscle repair. Here, we have used a novel combination of techniques to study this process, marking MPC with nuclear-localizing LacZ and tracing their contribution to regeneration of muscle fibers after grafting into preirradiated muscle of the mdx nu/nu mouse. In this model system, there is muscle degeneration, but little or no regeneration from endogenous MPC. Incorporation of donor MPC into injected muscles was analyzed by preparing single viable muscle fibers at various times after cell implantation. Fibers were either stained immediately for -gal, or cultured to allow their associated satellite cells to migrate from the fiber and then stained for -gal. Marked myonuclei were located in discrete segments of host muscle fibers and were not incorporated preferentially at the ends of the fibers. All branches on host fibers were also found to be composed of myonuclei carrying the -gal marker. There was no significant movement of donor myonuclei within myofibers for up to 7 weeks after MPC implantation. Although donor-derived dystrophin was usually located coincidentally with donor myonuclei, in some fibers, the dystrophin protein had spread further along the mosaic myofibers than had the myonuclei of donor origin. In addition to repairing segments of the host fiber, the implanted MPC also gave rise to satellite cells, which may contribute to future muscle repair. Dev Dyn 1999;216:244-256.
Cell differentiation, 1980
Satellite cells were isolated from skeletal muscles of adult normal and dystrophic mice (C57/6J/dy strain) by sequential digestion of tissue fragments with collagenase, hyaluronidase and trypsin. These cells exhibit in culture similar behaviour to that of embryonic myoblasts, undergoing an initial duplicative period lasting about 2--3 days, followed by a shorter phase (1--2 days) of rapid cell fusion. During the duplicative phase most of the satellite cells appear round-shaped, whereas embryonic myoblasts appear typically spindle-shaped: both cell types actively incorporate [3H]thymidine. During the subsequent days of culture an increasing number of satellite cells becomes spindle-shaped; afterwards the cells contact each other and fuse into multinucleated myotubes. The majority of spindle-shaped satellite cells is unable to incorporate [3H]thymidine, thus behaving as post-mitotic cells. Concomitantly with satellite cell fusion, an increase of about 80-fold of creatine phosphokinase...
The Journal of cell biology, 1990
Dystrophin deficiency in skeletal muscle of the x-linked dystrophic (mdx) mouse can be partially remedied by implantation of normal muscle precursor cells (mpc) (Partridge, T. A., J. E. Morgan, G. R. Coulton, E. P. Hoffman, and L. M. Kunkel. 1989. Nature (Lond.). 337:176-179). However, it is difficult to determine whether this biochemical "rescue" results in any improvement in the structure or function of the treated muscle, because the vigorous regeneration of mdx muscle more than compensates for the degeneration (Coulton, G. R., N. A. Curtin, J. E. Morgan, and T. A. Partridge. 1988. Neuropathol. Appl. Neurobiol. 14:299-314). By using x-ray irradiation to prevent mpc proliferation, it is possible to study loss of mdx muscle fibers without the complicating effect of simultaneous fiber regeneration. Thus, improvements in fiber survival resulting from any potential therapy can be detected easily (Wakeford, S., D. J. Watt, and T. A. Patridge. 1990. Muscle & Nerve.) Here, we h...