Isolation, growth and differentiation of adult rabbit skeletal myoblasts in vitro

1996 ◽  
Vol 18 (4) ◽  
pp. 299-307 ◽  
Author(s):  
Carol E. Torgan ◽  
Mary C. Reedy ◽  
William E. Kraus
Keyword(s):  
Adult Rabbit ◽  
Skeletal Myoblasts

scholarly journals Survival and Proliferation under Severely Hypoxic Microenvironments Using Cell-Laden Oxygenating Hydrogels

2021 ◽  
Vol 12 (2) ◽  
pp. 30
Author(s):  
Shabir Hassan ◽  
Berivan Cecen ◽  
Ramon Peña-Garcia ◽  
Fernanda Roberta Marciano ◽  
Amir K. Miri ◽  
...  
Keyword(s):  
Prolonged Release ◽  
Therapeutic Approaches ◽  
Encapsulated Cells ◽  
Hypoxic Conditions ◽  
Skeletal Myoblasts ◽  
Artificial Tissue ◽  
Delivery Platform ◽  
Living Tissues

Different strategies have been employed to provide adequate nutrients for engineered living tissues. These have mainly revolved around providing oxygen to alleviate the effects of chronic hypoxia or anoxia that result in necrosis or weak neovascularization, leading to failure of artificial tissue implants and hence poor clinical outcome. While different biomaterials have been used as oxygen generators for in vitro as well as in vivo applications, certain problems have hampered their wide application. Among these are the generation and the rate at which oxygen is produced together with the production of the reaction intermediates in the form of reactive oxygen species (ROS). Both these factors can be detrimental for cell survival and can severely affect the outcome of such studies. Here we present calcium peroxide (CPO) encapsulated in polycaprolactone as oxygen releasing microparticles (OMPs). While CPO releases oxygen upon hydrolysis, PCL encapsulation ensures that hydrolysis takes place slowly, thereby sustaining prolonged release of oxygen without the stress the bulk release can endow on the encapsulated cells. We used gelatin methacryloyl (GelMA) hydrogels containing these OMPs to stimulate survival and proliferation of encapsulated skeletal myoblasts and optimized the OMP concentration for sustained oxygen delivery over more than a week. The oxygen releasing and delivery platform described in this study opens up opportunities for cell-based therapeutic approaches to treat diseases resulting from ischemic conditions and enhance survival of implants under severe hypoxic conditions for successful clinical translation.

Isolation and Maintenance of Continuous Cultures of Epithelial Cells from Chemically-injured Adult Rabbit Lung

1990 ◽  
Vol 17 (4) ◽  
pp. 301-315 ◽  
Author(s):  
Sandra Houser ◽  
Lolita Borges ◽  
Dorothy E. Guzowski ◽  
Frank A. Barile
Keyword(s):  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Acute Injury ◽  
Population Doubling ◽  
Adult Rabbit ◽  
Chemical Stimulus ◽  
Rabbit Lung ◽  
Homogeneous Population ◽  
Metabolic Role

Acute alveolar injury commonly occurs as a result of exposure to a variety of toxic stimuli and is characterised by widespread necrosis of the epithelium, followed by regeneration and hyperplasia of type II alveolar epithelial cells. We studied epithelial proliferation in rabbit lung resulting from a chemical stimulus by isolating the mitotically active cells and establishing them in continuous culture. Acute alveolar injury was induced in rabbits with two daily injections of N-nitroso- N-methylurethane (NNNMU). Four to six days after treatment, the lungs were enzymatically digested and a homogeneous monolayer of epithelial cells was established in culture. The cells displayed a typical cobblestone-like arrangement and cuboidal shape, and represented 95% of the cells isolated. The cultures exhibited contact inhibition and achieved a population doubling level of 11 ± 2. Their epithelial origin was confirmed by positive reactions for immunofluorescent cytokeratin, alkaline phosphatase, and the Papanicolaou stain. Ultrastructurally, the cells showed abundant rough endoplasmic reticulum with dilated cisternae, desmosomes, cytokeratin filaments, and lamellar-like structures early in culture. In addition, the cells accumulated paraquat in a manner similar to that shown by established epithelial cell lines. The results demonstrate that regenerating epithelial cells can be isolated and propagated in culture as a relatively homogeneous population. This system, which is designed to further understanding of the metabolic role of epithelial cells following acute injury, may serve as an in vitro model for pulmonary toxicity studies using a minimal number of animals.

Heat Shock Treatment Enhances Graft Cell Survival in Skeletal Myoblast Transplantation to the Heart

Circulation ◽  
2000 ◽  
Vol 102 (suppl_3) ◽  
Author(s):  
Ken Suzuki ◽  
Ryszard T. Smolenski ◽  
Jay Jayakumar ◽  
Bari Murtuza ◽  
Nigel J. Brand ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cell Survival ◽  
Cell Transplantation ◽  
Shock Treatment ◽  
Heat Shock Treatment ◽  
Skeletal Myoblast ◽  
Skeletal Myoblasts ◽  
Myoblast Transplantation ◽  
Hypoxia Reoxygenation

Background —Graft survival after skeletal myoblast transplantation is affected by various pathological processes caused by environmental stress. Heat shock is known to afford protection of several aspects of cell metabolism and function. We hypothesized that prior heat shock treatment of graft cells would improve their survival after cell transplantation. Methods and Results —L6 rat skeletal myoblasts expressing β-galactosidase (β-gal) were subjected to heat shock (42°C, 1 hour). Increased expression of heat shock protein 72 was detected 24 hours later in the heat-shocked cells. After hypoxia-reoxygenation in vitro, lactate dehydrogenase leakage was significantly attenuated in the heat-shocked cells; in addition, the percentage of early apoptosis was lower in this group measured by flow cytometry with annexin V staining. For the in vivo study, 1×10 6 heat-shocked (hsCTx) or normal-cultured (CTx) myoblasts were infused into the explanted rat hearts through the coronary artery followed by heterotopic heart transplantation. β-gal activity was significantly higher in the hsCTx group after cell transplantation, with an estimated 8×10 6 surviving cells per heart in the hsCTx group and 5×10 6 cells in the CTx group on day 28. Discrete loci of grafted cells were globally observed in the myocardium of the hsCTx and CTx groups, with a higher frequency in the hsCTx group. Surviving myoblasts occasionally differentiated into myotubes and had integrated with the native cardiomyocytes. Conclusions —Heat-shocked skeletal myoblasts demonstrated improved tolerance to hypoxia-reoxygenation insult in vitro and enhanced survival when grafted into the heart. Heat shock treatment could be useful in improving graft cell survival in cell transplantation.

scholarly journals miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro

2019 ◽  
Author(s):  
Roza K. Przanowska ◽  
Ewelina Sobierajska ◽  
Zhangli Su ◽  
Kate Jensen ◽  
Piotr Przanowski ◽  
...  
Keyword(s):  
Muscle Function ◽  
Skeletal Muscle Regeneration ◽  
C2c12 Myoblasts ◽  
Skeletal Myoblasts ◽  
Microrna Targets ◽  
Microrna Family ◽  
Differentiation Pathways

AbstractmiR-206, miR-1a-1 and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this microRNA family is hypothesized to play an essential role in differentiation, a triple knockout of the three genes has not been done to test this hypothesis. We report that triple KO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected de-repression of the microRNA targets. Surprisingly, their mitochondrial function is diminished. Triple KO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two triple KO mice survive and grow normally to adulthood with smaller myofiber diameter and diminished physical performance. Thus, unlike other microRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

scholarly journals Hemidesmosome formation in vitro.

1983 ◽  
Vol 97 (3) ◽  
pp. 849-857 ◽  
Author(s):  
I K Gipson ◽  
S M Grill ◽  
S J Spurr ◽  
S J Brennan
Keyword(s):  
Basal Lamina ◽  
Mammalian Species ◽  
Corneal Stroma ◽  
Adult Rabbit ◽  
Lamina Densa ◽  
Nucleation Sites ◽  
Epithelial Sheets ◽  
Free Media ◽  
Molecular Components

Intact, viable sheets of adult rabbit corneal epithelium, 9 mm in diameter, were prepared by the Dispase II method (Gipson, I. K., and S. M. Grill, 1982, Invest. Ophthalmol. Vis. Sci. 23:269-273). The sheets, freed of the basal lamina, retained their desmosomes and stratified epithelial characteristics, but lacked hemidesmosomes (HD). Epithelial sheets were placed on fresh segments of corneal stroma with denuded basal laminae and incubated in serum-free media for 1, 3, 6, 18, or 24 h. Tissue was processed for electron microscopy, and the number of HD/micron membrane, the number of HDs with anchoring fibrils directly across the lamina densa from them, and the number of anchoring fibrils not associated with HDs were counted. After 6 h in culture, the number of newly formed HD was 82% of controls (normal rabbit corneas), and by 24 h the number had reached 95% of controls. At all time periods studied, 80-86% of HDs had anchoring fibrils directly across the lamina densa from them. Anchoring fibrils not associated with HDs decreased with culture time. These data indicate that the sites where anchoring fibrils insert into the lamina densa may be nucleation sites for new HD formation. Corneal epithelial sheets placed on two other ocular basal laminae, Descemet's membrane and lens capsule, had not formed HDs after 24 h in culture. These two laminae do not have anchoring fibrils associated with them. Rabbit epithelial sheets placed on the denuded epithelial basal lamina of rat and human corneas formed new HDs. Thus, at least in these mammalian species, HD formation may involve some of the same molecular components.

Characterization of a 5.4 kb cDNA fragment from the Z-line region of rabbit cardiac titin reveals phosphorylation sites for proline-directed kinases

1995 ◽  
Vol 108 (9) ◽  
pp. 3029-3037 ◽  
Author(s):  
M.G. Sebestyen ◽  
J.A. Wolff ◽  
M.L. Greaser
Keyword(s):  
Skeletal Muscle ◽  
Striated Muscle ◽  
Sequence Data ◽  
Amino Acid Level ◽  
Adult Rabbit ◽  
Cdna Clones ◽  
Protein Fragment ◽  
Cardiac Titin ◽  
Line Region

Titin is an approximately 3 MDa protein that spans from the M- to the Z-line in the sarcomeres of vertebrate striated muscle. The protein is presumably encoded by unusually large mRNAs of 70–80 kb. Although titin has been studied by several laboratories, barely more than half of the cDNA sequence (approximately 45 kb) has been published, most of it obtained from the A-band and M-line region (corresponding to the C-terminal half of the molecule). A special cDNA library was constructed using size selected total RNA from adult rabbit cardiac muscle in order to obtain sequence data from titin's unknown N-terminal region. A monoclonal antibody (T12), which binds to an epitope close to the Z-line, was used to identify initial cDNA clones. Additional overlapping clones were isolated and sequenced yielding a 5.4 kb contig. The encoded polypeptide contains 16 Type-II domains and four unique intervening segments. Polyclonal sera, raised against an expressed protein fragment encoded by the 5′ end of the contig, strongly stained the Z-line of myofibrils of different species. However, the sequence of this fragment is 83% identical at the amino acid level with the previously reported C-terminal (i.e. M-line) end of chicken embryonic skeletal muscle titin. The expressed protein fragment could be phosphorylated in vitro by embryonic skeletal muscle extract and by the purified proline-directed kinase ERK1, presumably at the xSPxR recognition sites located in the first interdomain segment.

Developmental changes in rabbit juxtamedullary proximal convoluted tubule water permeability

1996 ◽  
Vol 271 (4) ◽  
pp. F871-F876 ◽  
Author(s):  
R. Quigley ◽  
M. Baum
Keyword(s):  
Proximal Tubule ◽  
Water Transport ◽  
Water Permeability ◽  
Water Movement ◽  
Adult Rabbit ◽  
Osmotic Water ◽  
Glomerular Filtrate ◽  
Convoluted Tubules ◽  
Insight Into

The mammalian proximal tubule reabsorbs the bulk of the glomerular filtrate in a nearly isosmotic fashion due to the high osmotic water permeability (Pf) of this segment. Although the characteristics of proximal tubule water transport have been studied in the adult proximal tubule, little is known about the neonatal segment. The present study directly measured the Pf and diffusional water permeability (PDW) of neonatal (10 +/- 2 day old) and adult rabbit juxtamedullary proximal convoluted tubules (PCT) using in vitro microperfusion. The Pf of neonatal juxtamedullary PCT was greater than the Pf of adult juxtamedullary PCT. In contrast, the PDW was not different between the two groups. The Pf and PDW values of both neonatal and adult tubules were inhibited to the same degree by p-chloromercuribenzene sulfonate and had identical activation energies. The transepithelial reflection coefficients of NaCl and NaHCO3 were also found to be similar in both the neonatal and adult proximal tubules. Thus neonatal and adult juxtamedullary PCT have many characteristics of water transport that are identical; however, neonatal Pf is three to five times that of the adult value. This difference in Pf with identical PDW values may give an insight into the transepithelial pathway for water movement in the neonatal tubule.

Contribution of LFA-1 and Mac-1 to CD18-dependent neutrophil emigration in a neonatal rabbit model

1996 ◽  
Vol 80 (6) ◽  
pp. 1984-1992 ◽  
Author(s):  
J. M. Graf ◽  
C. W. Smith ◽  
M. M. Mariscalco
Keyword(s):  
Monoclonal Antibody ◽  
Polymorphonuclear Leukocytes ◽  
Rabbit Model ◽  
Transendothelial Migration ◽  
Peritoneal Exudate ◽  
Adult Rabbit ◽  
Primary Mechanism ◽  
Phosphate Buffered Saline

Human neonatal polymorphonuclear leukocytes (PMNs) exhibit decreased mobility, adherence, and transendothelial migration in vitro compared with adult PMNs. These deficits, in part, are due to functional and quantitative defects in neonatal Mac-1 (CD11b/CD18), whereas LFA-1 (CD11a/CD18) function is similar to that found in adults (D.C.Anderson, O.Abbassi, T.K.Kishimoto, J.M.Koenig, L.V.McIntire, and C.W.Smith, J.Immunol. 146: 3372-3379, 1991; C. W. Smith, T. K. Kishimoto, O. Abbassi, B.J.Hughes, R.Rothlein, L.V.McIntire, E.Butcher, and D.C. Anderson, J. Clin. Invest. 87: 609-618, 1991). We tested the hypothesis that the primary mechanism for the neonatal PMN CD18-dependent emigration in vivo is due to LFA-1. Neutrophils from 1-day-old rabbit pups had 32 and 60% of adult rabbit levels of CD11a and CD11b, respectively. Rabbit pups or adult rabbits received the monoclonal antibody (MAb) R7.1 (anti-CD11a) or R15.7 (anti-CD18) or the vehicle phosphate-buffered saline (PBS) before the instillation of intraperitoneal thioglycollate. Six hours later peritoneal exudate was collected. The administration of MAbs R7.1 and R15.7 in adult animals resulted in 60 and 83% inhibition of leukocyte emigration, respectively, compared with PBS-treated animals (P < 0.01). In neonatal animals, R7.1 and R15.7 inhibited leukocyte peritoneal accumulation to the same extent (50 and 60%, respectively) compared with PBS-treated animals (P < 0.01). Adult animals were also treated with the anti-CD11b MAb 198. MAb 198 decreased emigration by 25%, although this was not significant compared with PBS-treated animals. We conclude that although neonatal animals have significantly less neutrophil CD11a, the diminished levels did not contribute to a reduced ability to emigrate to the peritoneum and, like adult animals, neonatal animals primarily utilize LFA-1 for accumulation in this model. The contribution of Mac-1 to neonatal leukocyte emigration remains uncertain.

scholarly journals New 2-aminopyrimidine derivatives and their antitrypanosomal and antiplasmodial activities

2020 ◽  
Vol 151 (9) ◽  
pp. 1375-1385
Author(s):  
Michael Hoffelner ◽  
Usama Hassan ◽  
Werner Seebacher ◽  
Johanna Dolensky ◽  
Patrick Hochegger ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Trypanosoma Brucei ◽  
Phenyl Ring ◽  
Ring Closure ◽  
Causative Organism ◽  
Amino Group ◽  
Trypanosoma Brucei Rhodesiense ◽  
Structural Modifications ◽  
Skeletal Myoblasts

Abstract Novel 2-aminopyrimidine derivatives were prepared from acyclic starting materials, benzylidene acetones and ammonium thiocyanates, via 5 steps, including ring closure, aromatization, S-methylation, oxidation to methylsulfonyl compounds, and formation of guanidines with suitable amines. The prepared compounds differ from each other by the substitutions of their amino group and of their phenyl ring. The 2-aminopyrimidines were tested by use of microplate assays for their in vitro activities against a causative organism of sleeping sickness, Trypanosoma brucei rhodesiense, as well as against a causative organism of malaria, Plasmodium falciparum NF54. Their cytotoxic properties were determined with L-6 cells (rat skeletal myoblasts). Some of the compounds exhibited quite good antitrypanosomal activity, and others showed excellent antiplasmodial activity. The influence of the structural modifications on these activities is discussed. Graphic abstract

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