scholarly journals Improved Tissue Culture Conditions for Engineered Skeletal Muscle Sheets

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Sara Hinds ◽  
Natalia Tyhovych ◽  
Clint Sistrunk ◽  
Louis Terracio
Keyword(s):  
Skeletal Muscle ◽  
Fold Increase ◽  
Culture Conditions ◽  
Neonatal Rat ◽  
Skeletal Myoblasts ◽  
Potential Clinical Utility ◽  
Cell Subpopulations ◽  
The Impact ◽  
Muscle Precursor Cells

The potential clinical utility of engineered muscle is currently restricted by limitedin vitrocapacity of expanded muscle precursor cells to fuse and form mature myofibers. The purpose of this study was to use isotropic skeletal muscle sheets to explore the impact of (1) fibroblast coculture and (2) fibroblast-conditioned media (fCM) onin vitromyogenesis. Muscle sheets were prepared by seeding varying ratios of skeletal myoblasts and fibroblasts on a biomimetic substrate and culturing the resulting tissue in either control media or fCM. Muscle sheets were prepared from two cell subpopulations, (1) C2C12 and NOR-10 and (2) primary neonatal rat skeletal muscle cells (nSKM). In C2C12/Nor-10 muscle sheets fCM conferred a myogenic advantage early in culture; at D1 a statistically significant 3.12 ± 0.8-fold increase in myofiber density was observed with fCM. A high purity satellite cell population was collected from an initially mixed population of nSKMs via cell sorting for positiveα7-integrin expression. On D6, tissue sheets with low fibroblast concentrations (0 & 10%) cultured in fCM had increased average myofiber density (4.8 ± 0.2 myofibers/field) compared to tissue sheets with high fibroblast concentrations (50%) cultured in control media (1.0 ± 0.1 myofibers/field). Additionally, fCM promoted longer, thicker myofibers with a mature phenotype.

scholarly journals Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

2015 ◽  
Vol 60 (3) ◽  
pp. 1226-1233 ◽  
Author(s):  
Petros Ioannou ◽  
Aggeliki Andrianaki ◽  
Tonia Akoumianaki ◽  
Irene Kyrmizi ◽  
Nathaniel Albert ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Cell Culture ◽  
Antifungal Agents ◽  
Fold Increase ◽  
Culture Conditions ◽  
The Other ◽  
Related Factors ◽  
Content Type

The modestin vitroactivity of echinocandins againstAspergillusimplies that host-related factors augment the action of these antifungal agentsin vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against variousAspergillusspecies under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P= 0. 0005). Importantly, the enhanced activity of caspofungin againstAspergillusspp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinatingAspergillushyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin withAspergillushyphae (P< 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery toAspergillushyphae.

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype

1990 ◽  
Vol 259 (6) ◽  
pp. L415-L425 ◽  
Author(s):  
P. E. Roberts ◽  
D. M. Phillips ◽  
J. P. Mather
Keyword(s):  
Epithelial Cell ◽  
Molecular Mechanisms ◽  
Basal Medium ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Number ◽  
Neonatal Rat ◽  
Rat Lung ◽  
Cell Type

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

Abstract 126: Effects of Rosuvastatin on the Protein Composition of Lipoprotein Subfractions

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Scott M Gordon ◽  
Georgina Kemeh ◽  
Michael B Fessler ◽  
Alan T Remaley
Keyword(s):  
Lipoprotein Metabolism ◽  
Protein Composition ◽  
Protein Detection ◽  
Fold Increase ◽  
Cholesterol Content ◽  
Vascular Lesions ◽  
Lipid Lowering ◽  
Lipoprotein Subfractions ◽  
The Impact

Introduction: Statins, by inhibiting HMG-CoA reductase and up regulating hepatic LDL receptors, effectively lower plasma LDL-C by as much as 50%, thus reducing future CVD events. However, the physiological effects of statins are diverse and not all are related to lowering of LDL-C. Goal: The goal of this study was to test our hypothesis that some of these pleiotropic alternative effects from statins may be driven by compositional changes to lipoproteins distinct from their cholesterol content. We, therefore, performed a small clinical pilot study to assess the impact of statins on lipoprotein associated proteins in healthy individuals. Methods: Ten subjects with normal LDL-C (<130 mg/dL) were given rosuvastatin (20 mg/day) for 28 days. Plasma samples collected at baseline and after treatment were used for lipid measurement, nuclear magnetic resonance (NMR) lipoprotein profiling and lipoprotein proteomics. Results: The effects of rosuvastatin treatment on clinical lipid measures and NMR profile were consistent with established findings. Proteomic analysis of FPLC fractions representing LDL, HDL-1 (large) and HDL-2 (small) identified a total of 124 different proteins. Spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool: LDL = 9, HDL-1 = 9 and HDL-2 = 4. These changes included both increases and decreases in proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the treatment was a profound increase in alpha-1-antirypsin (A1AT) spectral counts association with HDL-1 particles. Quantitative measurement by ELISA revealed an average 5.7 fold increase in HDL-1 associated A1AT. Preliminary in vitro studies indicate a potential functional role for A1AT enriched HDL in the formation of neutrophil extracellular traps (NETs), a pro-inflammatory component of vascular lesions. Summary: Based on these results, statins can significantly change the protein composition of both LDL and HDL. Some of these changes, such as the up regulation of A1AT on HDL, may convey anti-inflammatory functionality on lipoproteins and might contribute to some of the non-lipid lowering effects of statins.

scholarly journals Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion

2019 ◽  
Vol 20 (16) ◽  
pp. 3932 ◽  
Author(s):  
Barbara Świerczek-Lasek ◽  
Jacek Neska ◽  
Agata Kominek ◽  
Łukasz Tolak ◽  
Tomasz Czajkowski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Muscle Development ◽  
Embryonic Stem ◽  
Interleukin 4 ◽  
Myogenic Cells ◽  
The Impact

Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging—i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting—and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1, Pax3, and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.

scholarly journals Commercial Biocontrol Agents Reveal Contrasting Comportments Against Two Mycotoxigenic Fungi in Cereals: Fusarium Graminearum and Fusarium Verticillioides

Toxins ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 152 ◽  
Author(s):  
Lucile Pellan ◽  
Noël Durand ◽  
Véronique Martinez ◽  
Angélique Fontana ◽  
Sabine Schorr-Galindo ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Culture Media ◽  
Fusarium Verticillioides ◽  
Inhibition Zone ◽  
Culture Conditions ◽  
Dual Culture ◽  
Growth Inhibition Zone ◽  
Mycotoxigenic Fungi ◽  
The Impact

The aim of this study was to investigate the impact of commercialized biological control agents (BCAs) against two major mycotoxigenic fungi in cereals, Fusarium graminearum and Fusarium verticillioides, which are trichothecene and fumonisin producers, respectively. With these objectives in mind, three commercial BCAs were selected with contrasting uses and microorganism types (T. asperellum, S. griseoviridis, P. oligandrum) and a culture medium was identified to develop an optimized dual culture bioassay method. Their comportment was examined in dual culture bioassay in vitro with both fusaria to determine growth and mycotoxin production kinetics. Antagonist activity and variable levels or patterns of mycotoxinogenesis inhibition were observed depending on the microorganism type of BCA or on the culture conditions (e.g., different nutritional sources), suggesting that contrasting biocontrol mechanisms are involved. S. griseoviridis leads to a growth inhibition zone where the pathogen mycelium structure is altered, suggesting the diffusion of antimicrobial compounds. In contrast, T. asperellum and P. oligandrum are able to grow faster than the pathogen. T. asperellum showed the capacity to degrade pathogenic mycelia, involving chitinolytic activities. In dual culture bioassay with F. graminearum, this BCA reduced the growth and mycotoxin concentration by 48% and 72%, respectively, and by 78% and 72% in dual culture bioassay against F. verticillioides. P. oligandrum progressed over the pathogen colony, suggesting a close type of interaction such as mycoparasitism, as confirmed by microscopic observation. In dual culture bioassay with F. graminearum, P. oligandrum reduced the growth and mycotoxin concentration by 79% and 93%, respectively. In the dual culture bioassay with F. verticillioides, P. oligandrum reduced the growth and mycotoxin concentration by 49% and 56%, respectively. In vitro dual culture bioassay with different culture media as well as the nutritional phenotyping of different microorganisms made it possible to explore the path of nutritional competition in order to explain part of the observed inhibition by BCAs.

scholarly journals Quantitative Analysis Reveals Expansion of Human Hematopoietic Repopulating Cells After Short-term Ex Vivo Culture

1997 ◽  
Vol 186 (4) ◽  
pp. 619-624 ◽  
Author(s):  
Mickie Bhatia ◽  
Dominique Bonnet ◽  
Ursula Kapp ◽  
Jean C.Y. Wang ◽  
Barbara Murdoch ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Fold Increase ◽  
Culture Conditions ◽  
Nonobese Diabetic ◽  
Crucial Component ◽  
Cord Blood Cells ◽  
Ex Vivo Culture

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38− cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38− cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.

Comparison between Progenitor Cells from Mobilized Peripheral Blood (PB) in Healthy Donors and Non-Hodgkin’s Lymphoma (NHL) Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5002-5002
Author(s):  
Eva M. Villaron ◽  
Julia Almeida ◽  
Natalia Lopez-Holgado ◽  
Fermin M. Sanchez-Guijo ◽  
Mercedes Alberca ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Functional Assays ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Cell Subpopulations ◽  
The Impact ◽  
Pbsc Mobilization

Abstract INTRODUCTION: Peripheral blood stem cell (PBSC) mobilization is impaired in patients receiving chemotherapy but, as far as we know there is no data about the impact of chemotherapy on different PB progenitor cell subpopulations. AIM: to ascertain whether or not immature or committed progenitor cell are affected by chemotherapy prior PBSC mobilization in NHL patients. MATERIAL AND METHODS: a total of 27 PB samples from NHL patients and 36 PB samples from healthy donors were studied. Immunophenotypic analysis of CD34+ cell subpopulations was performed using the following four colour combinations of monoclonal antibodies (FITC/PE/PC5/APC): CD90/CD133/CD38/CD34 and CD71/CD13/CD45/CD34. In order to study committed progenitor cells “in vitro”, standard colony-forming assays were used and, in order to investigate the behaviour of the uncommitted progenitors Delta Assays of plastic adherent progenitor cells (PΔ) were performed. RESULTS: The comparison between NHL patients and healthy donors is shown in Table 1. The relationship between data obtained by flow cytometry and cultures was statistically significant (p<0.05, r>0.568) for all the progenitors analysed. Table 1: Results of Immunophenotypic and Functional Assays LNH patients Healthy donors p Data expressed as median (range). 1. Percentage among CD34+ cells. 2. Number of CFU/10 5 planted cells. 3. Number of CFU/10 6 planted cells % CD34 0.16(0.04–3.65) 0.57(0.11–1.81) 0.013 Immunophenotypic Data Erithroid 1 0.05(0.01–0.60) 0.14(0.02–0.42) 0.098 Myelo–monocytic 1 0.11(0.02–2.41) 0.37(0.07–1.18) 0.014 Immature 1 0.01(0.00–0.63) 0.05(0.01–0.19) 0.014 CFU-GM 2 70(4–440) 90(0–904) 0.327 Clonogenic and Delta Assays data BFU-E 2 62(6–172) 85(0–240) 0.046 CFU-Mix 2 18(0–124) 42(0–140) 0.018 CFU Δ3 356(0–3509) 953(90–8320) 0.033 CONCLUSIONS: We can conclude that in NHL, mobilized committed and above all immature progenitors are impaired when compared with healthy subjects, both analysed by immunological and functional assays. Only granulomonocytic progenitors analysed by a functional approach seemed to be preserved.

Arterial Delivery of Genetically Labelled Skeletal Myoblasts to the Murine Heart: Long-Term Survival and Phenotypic Modification of Implanted Myoblasts

1996 ◽  
Vol 5 (1) ◽  
pp. 77-91 ◽  
Author(s):  
Shawn W. Robinson ◽  
Peter W. Cho ◽  
Hyam I. Levitsky ◽  
Jean L. Olson ◽  
Ralph H. Hruban ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myocardial Dysfunction ◽  
C2c12 Cells ◽  
Immunofluorescent Staining ◽  
Major Advance ◽  
Cardiac Muscle Cells ◽  
Term Survival ◽  
Skeletal Myoblasts ◽  
Junction Protein

The ability to replace damaged myocardial tissue with new striated muscle would constitute a major advance in the treatment of diseases that irreversibly injure cardiac muscle cells. The creation of focal grafts of skeletal muscle has been reported following the intramural injection of skeletal myoblasts into both normal and injured myocardium. The goals of this study were to determine whether skeletal myoblast-derived cells can be engrafted into the murine heart following arterial delivery. The murine heart was seeded with genetically labeled C2C12 myoblasts introduced into the arterial circulation of the heart via a transventricular injection. A transventricular injection provided access to the coronary and systemic circulations. Implanted cells were characterized using histochemical staining for β-galactosidase, immunofluorescent staining for muscle-specific antigens, and electron microscopy. Initially the injected cells were observed entrapped in myocardial capillaries. One week after injection myoblasts were present in the myocardial interstitium and were largely absent from the myocardial capillary bed. Implanted cells underwent myogenic development, characterized by the expression of a fast-twitch skeletal muscle sarco-endoplasmic reticulum calcium ATPase (SERCA1) and formation of myofilaments. Four months following injection myoblast-derived cells began to express a slow-twitch/cardiac protein, phospholamban, that is normally not expressed by C2C12 cells in vitro. Most surprisingly, regions of close apposition between LacZ labeled cells and native cardiomyocytes contained structures that resembled desmosomes, fascia adherens junctions, and gap junctions. The cardiac gap junction protein, connexin43, was localized to some of the interfaces between implanted cells and cardiomyocytes. Collectively, these findings suggest that arterially delivered myoblasts can be engrafted into the heart, and that prolonged residence in the myocardium may alter the phenotype of these skeletal muscle-derived cells. Further studies are necessary to determine whether arterial delivery of skeletal myoblasts can be developed as treatment for myocardial dysfunction.

Metabolism of n-hexane by rat liver and extrahepatic tissues and the effect of cytochrome P-450 inducers

1997 ◽  
Vol 16 (3) ◽  
pp. 131-137 ◽  
Author(s):  
Sarah J Crosbie ◽  
PG Blain ◽  
Faith M Williams
Keyword(s):  
Skeletal Muscle ◽  
Rat Liver ◽  
Fold Increase ◽  
Rat Tissues ◽  
Human Cytochrome ◽  
Extrahepatic Tissues ◽  
Mass Spectometry ◽  
Fast Twitch ◽  
Cytochrome P 450

1 The in vitro metabolism ofn-hexane was studied in rat liver, lung, brain and skeletal muscle microsomes and in microsomes prepared from cell lines expressing human cytochrome P-450 2E1 or 2B6. The hydro xylated metabolites ofn-hexane were quantified by gas chromatography-mass spectometry. 2 Rat liver and extensor digitorum longus (EDL, fast- twitch skeletal muscle) microsomes and the CYP 2B6 microsomes produced the pre-neurotoxic metabolite of n-hexane, 2-hexanol as a major metabolite in contrast to the other rat tissues examined. 3 Inhibition of 2- and 3-hexanol production from n- hexane by rat lung microsomes using metyrapone, an inhibitor of cytochrome P-450 2B1 activity, resulted in almost complete inhibition of lung microsomal activ ity. 4 Production of all three hexanols was significantly increased with phenobarbital-induced rat liver micro somes, with a 10-fold increase in 2- and 3-hexanol production. A slight increase in 2-hexanol production with phenobarbital-induced rat EDL and brain micro somes was observed. No increase in n-hexane meta bolism was noted following induction with β- naphthoflavone or with ethanol.

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