Hysteresis Effect of Tangential Force Field With Centrifuge on Myoblast: Cultured on Striped Pattern of Micro Ridge for Direction Control

2021 ◽  
Author(s):  
Shigehiro Hashimoto
Keyword(s):  
Force Field ◽  
Tangential Force ◽  
Longitudinal Direction ◽  
Time Lapse ◽  
Stripe Pattern ◽  
Ridge Pattern ◽  
Direction Control ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Abstract Hysteresis effects of the direction of mechanical stimulation on the cell behavior have been examined in vitro. A micro ridge pattern was made on the surface of the scaffold to align the directions of the cells being stimulated. The stripe pattern (0.7 μm heigh, 3 μm wide, and 3 μm interval) was created by the photolithography technique. Three regions, which have the uniform value of the angle between the longitudinal direction of the ridge and the direction of the tangential force, were set: 0, 45, and 90 degrees in each region. Myoblasts (C2C12: mouse myoblast cell line) were used in the experiment. The scaffold plate with cells was set in the tube of a conventional centrifuge placed in an incubator to apply the tangential force field to each cell. After the cell culture for 5 hours with centrifugation, the behavior of each cell was analyzed on time-lapse microscopic images for 10 hours. Experimental results show that cell activities (migration and deformation) are enhanced after stimulation of tangential forces perpendicular to the long axis of myoblasts.

scholarly journals Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Christo J. Botha ◽  
Sarah J. Clift ◽  
Gezina C.H. Ferreira ◽  
Mxolisi G. Masango
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Ethical Issues ◽  
Sesquiterpene Lactones ◽  
Annexin V ◽  
Metabolic Activation ◽  
Suitable Model ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern Africa. The toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. Because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. The objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (C2C12) using methyl-thiazol-tetrazolium (MTT) and lactate dehydrogenase (LDH) assays, annexin V and propidium iodide (PI) flow cytometry and transmission electron microscopy (TEM). Mouse myoblasts were exposed to 2.0 mM, 2.5 mM and 5.0 mM geigerin for 24, 48 and 72 h. A concentration-dependent cytotoxic response was observed. Apoptosis was detected by means of annexin V flow cytometry during the first 24 h and apoptotic bodies were also visible on TEM. According to the LDH and PI flow cytometry results, myoblast cell membranes were not injured. We concluded that the murine myoblast cell line (C2C12) is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts.

scholarly journals A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro

Nutrients ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3727
Author(s):  
Petey W. Mumford ◽  
Shelby C. Osburn ◽  
Carlton D. Fox ◽  
Joshua S. Godwin ◽  
Michael D. Roberts
Keyword(s):  
Cell Line ◽  
Mitochondrial Biogenesis ◽  
C2c12 Myoblast ◽  
Mrna Levels ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Protein Levels ◽  
Myoblast Cell Line ◽  
Rodent Cell ◽  
Myoblast Cell

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.

scholarly journals Cytoprotective potential of anti-ischemic drugs against chemotherapy-induced cardiotoxicity in H9c2 myoblast cell line

2013 ◽  
Vol 63 (4) ◽  
pp. 493-503 ◽  
Author(s):  
Tiam Feridooni ◽  
Chris Mac Donald ◽  
Di Shao ◽  
Pollen Yeung ◽  
Remigius U. Agu
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cancer Drug ◽  
H9c2 Cell ◽  
Drug Induced ◽  
Cardiac Model ◽  
Myoblast Cell Line ◽  
Myoblast Cell ◽  
H9c2 Cell Line

Abstract To investigate potential prevention or attenuation of anti- cancer drug induced cardiotoxicity using anti-ischemic drugs, a rat myoblast (H9c2) cell line was used as our in vitro cardiac model. Irinotecan and doxorubicin were found to be cytotoxic for the H9c2 cell line with IC50 of 30.69 ± 6.20 and 20.94 ± 6.05 mmol L-1, respectively. 5-Flurouracil and cladribine were not cytotoxic and thus IC50 could not be calculated. When 100 mmol L-1 doxorubicin was incubated for 72 hours with 50 mmol L-1 diltiazem, 100 mmol L-1 dexrazoxane and 100 mmol L-1 losartan, respectively, there was a 58.7 ± 10.2, 52.2 ± 11.7 and 44.7 ± 5.4 % reduction in cell death. When 200 mmol L-1 irinotecan was incubated for 72 hours with 100 mmol L-1 dexrazoxane, losartan and diltiazem, respectively, a 27.7 ± 6.9, 25.6 ± 5.1, and 19.1 ± 2.3 % reduction in cell death was observed. Our data suggests that losartan and diltiazem were as effective as dexrazoxane in protecting the cells against irinotecan- and doxorubicin-induced cell toxicity. These findings offer potential uses of anti- -ischemic drugs for ablation of cytotoxicity in response to mitochondrial injury, thereby improving patient outcomes and reducing health-care costs.

scholarly journals Catechol cross-linked antimicrobial peptide hydrogels prevent multidrug-resistant Acinetobacter baumannii infection in burn wounds

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Abidullah Khan ◽  
Miao Xu ◽  
Tengjiao Wang ◽  
Chuangang You ◽  
Xingang Wang ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Acinetobacter Baumannii ◽  
Bacterial Infections ◽  
Skin Surface ◽  
Multidrug Resistant ◽  
Burn Wound ◽  
Burn Wounds ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Abstract Hospital-acquired infections are common in burn patients and are the major contributors of morbidity and mortality. Bacterial infections such as Staphylococcus aureus (S. aureus) and Acinetobacter baumannii (A. baumannii) are difficult to treat due to their biofilm formation and rapidly acquiring resistance to antibiotics. This work presents a newly developed hydrogel that has the potential for treating bacterial wound infections. The hydrogel formulation is based on an antimicrobial peptide (AMP), epsilon-poly-l-lysine (EPL) and catechol, which was cross-linked via mussel-inspired chemistry between the amine and phenol groups. In vitro studies showed that EPL-catechol hydrogels possess impressive antimicrobial and antibiofilm properties toward multidrug-resistant A. baumannii (MRAB). In addition, cytotoxicity study with the clonal mouse myoblast cell line (C2C12) revealed the good biocompatibility of this hydrogel. Furthermore, we created a second-degree burn wound on the mice dorsal skin surface followed by contamination with MRAB. Our results showed that the hydrogel significantly reduced the bacterial burden by more than four orders of magnitude in infected burn wounds. Additionally, there was no significant histological alteration with hydrogel application on mice skin. Based on these results, we concluded that EPL-catechol hydrogel is a promising future biomaterial to fight against multidrug-resistant bacterial infections.

scholarly journals Highly efficient cardiac differentiation and maintenance by thrombin-coagulated fibrin hydrogels enriched with decellularized porcine heart extracellular matrix

2020 ◽  
Author(s):  
Fatemeh Navaee ◽  
Philippe Renaud ◽  
Thomas Braschler
Keyword(s):  
Extracellular Matrix ◽  
Collagen I ◽  
Cardiac Differentiation ◽  
Neonatal Cardiomyocytes ◽  
Decellularized Extracellular Matrix ◽  
Ventricular Tissue ◽  
Key Factor ◽  
Myoblast Cell Line ◽  
Myoblast Cell

AbstractWe provide a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen for the formation of an in-vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin allows gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. We use the system in co-culture with Nor-10 fibroblasts to enhance cardiogenic differentiation of the H9c2 myoblast cell line. The combination of co-culture and appropriate substrate allows to abrogate the use of retinoids, classically considered necessary for cardiogenic H9c2 differentiation. Further enhancement of differentiation efficiency is obtained by 3D embedding. We then proceed with culture of rat neonatal cardiomyocytes in the 3D system. While for H9c2 cells, the collagen content of the dECM was the key factor required for efficient differentiation, the use of dECM-fibrin has specific advantages regarding the culture of neonatal cardiomyocytes. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony and maintenance of spontaneous beating, as compared to various controls including matrigel, pure fibrin and collagen I, but also a fibrin-collagen I blend.

The Effects of a Theacrine-based Supplement on mRNAs Related to Various Metabolic Processes and Sirtuin Activity in vitro

2020 ◽  
Author(s):  
Petey Mumford ◽  
Shelby Osburn ◽  
Michael D. Roberts
Keyword(s):  
Mitochondrial Biogenesis ◽  
Citrate Synthase ◽  
Inflammatory Marker ◽  
C2c12 Myoblast ◽  
Activity Levels ◽  
Mrna Levels ◽  
Expression Of Genes ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Abstract There is evidence in rodents to suggest theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis and the mRNA expression of genes related to various cellular processes in muscle. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~ 2 mM theacrine) for 3 and 24 hours (n=6 treatment wells per time point). Control treatments consisted of cellulose-only treatments at the same time points. Relative to CTL-treated cells, NAD3 treatments increased (p<0.05) Sirt1 mRNA levels at 3 hours, as well as global sirtuin activity at 3 and 24 hours. While NAD3 treatments decreased mRNA levels of Nfe2l2 at 3 hours and increased levels at 24 hours relative to CTL-treated cells (a gene involved in mitochondrial biogenesis, p<0.05), citrate synthase activity levels (a surrogate of mitochondrial density) remained unaltered between treatments. NAD3 treatments for 3 and 24 hours decreased Nlrp3 mRNA levels relative to CTL-treated cells (an inflammatory marker, p<0.05). Additionally, NAD3 treatments decreased Map1lc3b mRNA levels (an autophagy marker) after 24-hour treatments (p<0.05). Although these data are limited to select biomarkers in vitro, these preliminary findings suggest a theacrine-based supplement can modulate various skeletal muscle biomarkers related to sirtuin activity, inflammation, and autophagy. Muscle biopsy studies in humans are needed to confirm these current findings.

Oblique Micro Grooves on Bottom Wall of Flow Channel to Sort Cells

2020 ◽  
Author(s):  
Shigehiro Hashimoto
Keyword(s):  
Flow Direction ◽  
Longitudinal Axis ◽  
Flow Channel ◽  
Bottom Surface ◽  
Rectangular Shape ◽  
Myoblast Cell Line ◽  
Micro Flow ◽  
Myoblast Cell ◽  
Mouse Myoblast

Abstract The movement of a flowing cell near the oblique micro groove on the bottom surface in the micro flow channel has been measured to sort biological cells in vitro. The micro groove of the rectangular shape (4.5 μm depth, and 0.2 mm length) was fabricated on the polydimethylsiloxane (PDMS) disk by the photolithography technique. The angle between the flow direction and the longitudinal axis of the groove is 45 degree. Variation has been made on the width (0.03 mm &lt; w &lt; 0.05 mm) of the groove. A rectangular flow channel (0.05 mm height × 1 mm width × 25 mm length) has been constructed between two transparent PDMS disks. C2C12 (mouse myoblast cell line) was used in the test. A flow velocity (0.1 mm/s &lt; vx &lt; 2.4 mm/s) of the suspension of cells was controlled by the pressure difference between the inlet and the outlet. The shifted distance of each cell along the oblique groove depends on the diameter of the cell. The malnourished cell with the different density can be distinguished by the shifted distance according to the velocity of the cell.

Behavior of Myoblast Under Shear Stress in Couette Type of Flow

2020 ◽  
Author(s):  
Shigehiro Hashimoto
Keyword(s):  
Shear Stress ◽  
Shear Flow ◽  
Flow Direction ◽  
Experimental System ◽  
Longitudinal Axis ◽  
Time Lapse ◽  
Rotating Speed ◽  
Culture Plate ◽  
Myoblast Cell

Abstract Behavior of myoblast has been investigated under the uniform shear flow in vitro. The culture medium was sandwiched with the constant gap between the lower stationary culture plate and the upper rotating parallel plate to make a Couette type of the shear flow. By the rotating speed of the upper disk, the wall shear stress (τ) on the lower culture plate was controlled. C2C12 (mouse myoblast cell) was used in the test. After cultivation without flow for 24 hours for adhesion of cells on the lower plate, τ &lt; 2 Pa was continuously applied on cells for 7 days in the incubator. Behavior of each cell was traced at the time lapse images observed by an inverted phase contrast microscope placed in an incubator. Experimental results show that cells differentiate to myotubes under τ &lt; 2 Pa. Both the cell cycle and the cell length tend to scatter in the wider range, and the longitudinal axis of each cell tends to align to the flow direction by the shear stress of 1 Pa. The experimental system is useful to study quantitative relationships between the shear stress and the cell behavior: deformation, orientation, and differentiation.

Modulation of a potassium conductance in developing skeletal muscle

1995 ◽  
Vol 268 (2) ◽  
pp. C490-C495 ◽  
Author(s):  
S. J. Wieland ◽  
Q. H. Gong
Keyword(s):  
Skeletal Muscle ◽  
Potassium Conductance ◽  
Resting Potential ◽  
Intracellular Signals ◽  
Cell Recording ◽  
Dependent Inhibition ◽  
Myoblast Cell Line ◽  
Myoblast Cell ◽  
Inwardly Rectifying

K+ conductances dominate and potentially modulate the resting potential of skeletal muscle cells. The expression and modulation of a major K+ conductance were examined during in vitro differentiation of the mouse myoblast cell line C2C12. The inwardly rectifying K+ conductance (IKi) increased from unmeasurable levels in undifferentiated myoblasts to approximately 1.56 +/- 0.51 nA (n = 17) in myoballs derived from myotubes at 5 days after induction of differentiation. The inward rectifier was subject to modulation by intracellular signals. Exposure of cytoplasm to guanosine 5'-O-(3-thiotriphosphate) during whole cell recording produced a concentration (5-100 microM)- and time (1-20 min)-dependent inhibition of the mean conductance. Elevation of intracellular free Ca2+ (> 200 nM) also inhibited IKi. These findings demonstrate a potential mechanism for modulation of the resting potential of muscle fibers via the control of skeletal muscle IKi.

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