Myoblasts, culture

In the study of the effect of beryllium on fibroblast and myoblast cultures, anomalous mitoses were observed and the cell division was stopped in the metaphase, which was greatly extended. Practically no telophase and anaphase were observed. Beryllium was demonstrated histochemically in the liver and particularly in chromosomes of dividing cells. In the case of fibroblasts, there was a remarkable inhibition of the synthesis of nude-oproteins. Damage to the mechanism of the RNA synthesis is assumed to occur by action of beryllium. Beryllium is bound to DNA in the same way as magnesium, however, its affinity is many times higher. The complex of magnesium with DNA can be easily hydrolysed, whereas the complex with beryllium is stable. It was thus demonstrated that beryllium modifies physico-chemical characteristics of DNA. 

We conclude from these observations that there is an obligatory requirement for ADPRT activity in the terminal differentiation of primary chick myoblast cultures [15,16]. 

Stem-Straeter, J., Bach, A.D., Stangenherg, L., Eoerster, V.T., Horch, R.E., Stark, G.B., Beier, J.P., 2005. Impact of electrical stimulation on three-dimensional myoblast cultures -a real-time RT-PCR study. J. Cell Mol. Med. 9, 883—892. 

Contrasting with rodents, BAT is found in small amounts in adult humans. It has been proposed that skeletal muscle rather than BAT may play a pivotal role in energy homeostasis in adult humans. The authors also demonstrated that cultured human skeletal muscle myoblasts express D2 and high levels of TGR5, and a number of common bile acids (cholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid) were able to increase cAMP levels concomitant with increased D2 activity (Figure 7.4). Taurocholic acid was also able to... 

Skeletal myoblasts are viewed as an attractive alternative by some [76]. The first therapeutic trials used skeletal myoblasts obtained under sterile conditions and local anesthesia from 0.5- to 5.0-g muscle biopsy specimens. Individual cells were isolated by digestion with trypsin and collagenase, washed to remove red blood cells and debris, plated, and cultured to obtain the numbers necessary for therapeutic use. 

Breese, T.W. and Admassu, W. (1999) Feasibility of culturing c2cl2 mouse myoblasts on glass microcarriers in a continuous stirred tank bioreactor. Bioprocess Eng. 20, 463-468... 

There are several limitations in using primary cell lines from patients with mitochondrial disease (e.g., fibroblasts and myoblasts) (Jl). They are not immortal, and usually grow very slowly in regular culture media. Some of the cell lines do not retain a fully physiological, differentiated phenotype in culture. In addition, they tend to lose mutant mtDNA as a result of mitotic segregation (Jl). Most importantly, one cannot rule out the effect of the nuclear background on the phenotypic expression of mitochondrial dysfunction under examination. 

The first adenoviral (Ad)-mediated gene delivery studies for GSDII were performed in GSDII patient fibroblasts, myoblasts, and myotubes (Nicolino et al., 1998 Pauly et al., 1998, 2001). Several important findings came from these early studies including the ability to achieve overexpression of GAA from Ad vectors of up to 19-20-fold over untreated normal cells (Nicolino et al., 1998 Pauly et al., 1998), localization of the Ad-delivered GAA protein to the lysosomes (Pauly et al., 1998, 2001), clearance of accumulated glycogen in treated cells (Nicolino et al., 1998 Pauly et al., 1998), secretion of the 110 kDA precursor form into the culture media (Nicolino et al., 1998 Pauly et al., 1998), and M6P receptor-mediated uptake of GAA secreted from transduced cells by GAA-deficient cells (Nicolino et al., 1998 Pauly et al., 1998). 

Skeletal muscle cells are capable of regeneration and repair more readily than cardiac myocytes owing to the so-called satellite cells that can be readily mobilized at the time of injury (16). They are capable of easy expansion in culture can be harvested easily from the autologous host and are fairly resistant to hypoxia (17). The major drawback is that as the skeletal myocytes mature, they no longer form gap junctions and become electrically isolated, predisposing to re-entry arrhythmias (18). In addition, despite some encouraging data (19), the viability of dissociated myoblasts in suspension injected into myocardial scar deprived of appropriate trophic environment has been called into question (20), In our hands, skeletal myoblasts injected into canine myocardium could not be identified four weeks after implantation, and the needle tract caused fibrosis and scarring (Fig. 2). 

Another small pilot of 12 patients undergoing CABG with EFs between 25% and 45% and nonviable scar showed no need for AICD implantation and improvement in EF from mean of 35-53% at three months (P = 0.002) (52). It is unclear what contributed to this lower incidence of inducible VT) although the use of autologous patient plasma for muscle culture may have decreased the degree of inflammation around skeletal myoblasts, potentially caused by culture in fetal bovine serum. The suggestion that the risk of VT can be reduced by the use of autologous plasma is also shown in... 

Cultured cells are described as being fibroblast-like (spindle shaped) or epithelial-like (polygonal). These names are not very useful as the shape of cells varies depending on the medium and cell density. Furthermore, as cells from an increasingly varied tissue source are being cultured, cells are best described with regard to their origin, e.g. neuronal cells, myoblasts, lymphocytes etc. Many tissues contain two or more types of cell and this can lead to confusion. 

Thus McCoy s medium 5A (McCoy et al., 1959) has been used as a standard medium for cloning cells. It is based on BME amino acids and the vitamins from medium 199 (Appendix 1 Table 18). This was modified further to form RPMI (Rosswell Park Memorial Institute)-1629 (Appendix 1 Table 17) for long-term culture of leukaemic myoblasts (Armstrong, 1966). 

The added attraction of the transition from myoblast to myotube is the synchrony with which differentiation occurs in vitro. Myogen-esis will occur in primary cultures of skeletal muscle (e.g. 6.12) but can also be induced in diploid myoblast lines (Richter and Yaffe, 1970) which has allowed the selection of mutants (Chapter 13) that exhibit drug resistance or temperature-sensitive differentiation (Loomis et al., 1973 Somers et al., 1975). Holtzer et al. (1975) and Fiszman and Fuchs (1975) have developed a myoblast line transformed with a temperature-sensitive virus. At the permissive temper-... 

In our previous study we found that cultured human myoblasts, fusing myoblasts and myotubes constitutively secrete interleukin-6 (IL-6) (Prelovsek et al., 2006). This cytokine is known as the major eytokine released from the skeletal musele under various eonditions (Febbraio and Pedersen, 2005). We also found that IL-6 secretion is stimulated by the major proinflammatory agents like tumor necrosis factor (TNF)-a and endotoxin lipopolysaccharide (EPS). Sinee IL-6 is a potent stimulator of myoblast... 

FIGURE 45.2. IL-6 secretion from myoblasts, fusing myoblasts, and myotubes. Cultured human myoblasts, fusing myoblasts, and myotubes were added DFP at the 10 M concentration. The secreted IL-6, calculated per 100,000 nuclei, was determined 24 h later with ELISA kit (Endogen, Rockford, IL, USA). Significant difference (Student I-test, p < 0.05 n — 3) between control and treated cultures was observed at all stages studied but was most prominent in the myoblasts. 

FIGURE 45.3. The effects of DFP on the HSP 27 and HSP 70 levels in myoblast, myoblasts in fusion, and myotube cultures. Levels of HSP 27 and 70 were determined 24 h after addition of DFP at the 10 M concentration. They were quantitated by Western blot with Chemi Genius Biolmaging System (Syngen, Cambridge, UK). Significant difference (Student t-test, p < 0.05 n — 4) between control and DFP-treated cultures was observed in all determinations except for HSP 70 in myoblasts. 

Austin, L., Burgess, A.W. (1991). Stimulation of myoblast proliferation in culture by leukaemia inhibitory factor and other cytokines. J. Neurol Sci. 101 193-7. 

Gmhic, Z., Komel, R., Walker, W.F., Miranda, A.F. (1995). Myoblast fusion and innervation with rat motor nerve alter distribution of acetylcholinesterase and its mRNA in cultures of human muscle. Neuron 14 317-27. 

Primary cultures of hepatocyte and tubule cells are prepared by collagenase perfusion and dissociation of the liver or kidney into cell suspensions. Primary rat myocardial cells can be similarly prepared by using trypsin to dissociate the cells. Rhythmic contractions of myocardial cells can be observed in such cultures. Changes in these contractions serve as another end point for the measurement of toxicity.The L6 rat skeletal muscle myoblast cell line has been used to screen compounds for potential muscle irritating and damaging activity. ... 

Shahar A, Mizrahi A, Reuveny S, Zinman T Shainberg A (1985) Differentiation of myoblasts with nerve cells on microcarriers in culture. Developments in Biological Standardization 60 263-268. 

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