Development and charaterisation of 3 dimensional culture models for zebrafish (Danio rerio) skeletal muscle cells

2.50
Hdl Handle:
http://hdl.handle.net/10547/556396
Title:
Development and charaterisation of 3 dimensional culture models for zebrafish (Danio rerio) skeletal muscle cells
Authors:
Vishnolia, Krishan Kumar
Abstract:
Zebrafish (Danio rerio) have been extensively used over the past two decades to study muscle development, human myopathies and dystrophies, due to its higher degree of homology with human disease causing genes and genome. Despite its unique qualities, zebrafish have only been used as an in-vivo model for muscle development research, due to the limitations surrounding lack of a consistent isolation and culture protocol for zebrafish muscle progenitor cells in-vitro. Using different mammalian myoblast isolation protocols, a novel and robust protocol has been developed to successfully isolate and culture zebrafish skeletal muscle cells repeatedly and obtain differentiated long multi nucleated zebrafish myotubes. Commitment to myogenic lineage was confirmed by immuno-staining against muscle specific protein desmin, and expression pattern of different genetic markers regulating myogenesis. In order to recapitulate the in-vivo bio-physiological environment for zebrafish skeletal muscle cells in-vitro, these cells were successfully cultured in tissue engineered three dimensional (3D) constructs based on fibrin and collagen models. Maturation of tissue engineered collagen and fibrin based constructs was confirmed using the basic parameters described in the literature i.e. collagen three times greater contraction from the original width (Mudera, Smith et al. 2010) and fibrin constructs tightly coiled up to 4mm of diameter (Khodabukus, Paxton et al. 2007). In-vitro characterisation of zebrafish skeletal muscle cells showed hypertrophic growth of muscle mass compared to hyperplasic growth in-vivo as suggested for fish species in literature (Johnston 2006), which is different from human and other mammals. Comparative analysis of zebrafish muscle cells cultured in monolayer against cultured in 3D tissue engineered constructs showed significant increase in fusion index, nuclei per myotube (two-fold) and myotubes per microscopic frame (two-fold). Cells cultured in tissue engineered construct closely resembled in-vivo muscle in terms of their unidirectional orientation of myotubes. These tissue engineered 3D zebrafish skeletal muscle models could be used for various purposes such as drug screening, effect of different temperature extremes, studying underlined pathways involved in human diseases; and with further refinements it would potentially replace the need for studies on live fish in these areas.
Citation:
Vishnolia, K.K. (2014) 'Development and charaterisation of 3 dimensional culture models for zebrafish (Danio rerio) skeletal muscle cells'. PhD thesis. University of Bedfordshire.
Publisher:
University of Bedfordshire
Issue Date:
Sep-2013
URI:
http://hdl.handle.net/10547/556396
Type:
Thesis or dissertation
Language:
en
Description:
A thesis submitted to the University of Bedfordshire, in partial fulfilment of the requirements for the degree of Doctor of Philosophy
Appears in Collections:
PhD e-theses

Full metadata record

DC FieldValue Language
dc.contributor.authorVishnolia, Krishan Kumaren
dc.date.accessioned2015-06-04T12:09:39Zen
dc.date.available2015-06-04T12:09:39Zen
dc.date.issued2013-09en
dc.identifier.citationVishnolia, K.K. (2014) 'Development and charaterisation of 3 dimensional culture models for zebrafish (Danio rerio) skeletal muscle cells'. PhD thesis. University of Bedfordshire.en
dc.identifier.urihttp://hdl.handle.net/10547/556396en
dc.descriptionA thesis submitted to the University of Bedfordshire, in partial fulfilment of the requirements for the degree of Doctor of Philosophyen
dc.description.abstractZebrafish (Danio rerio) have been extensively used over the past two decades to study muscle development, human myopathies and dystrophies, due to its higher degree of homology with human disease causing genes and genome. Despite its unique qualities, zebrafish have only been used as an in-vivo model for muscle development research, due to the limitations surrounding lack of a consistent isolation and culture protocol for zebrafish muscle progenitor cells in-vitro. Using different mammalian myoblast isolation protocols, a novel and robust protocol has been developed to successfully isolate and culture zebrafish skeletal muscle cells repeatedly and obtain differentiated long multi nucleated zebrafish myotubes. Commitment to myogenic lineage was confirmed by immuno-staining against muscle specific protein desmin, and expression pattern of different genetic markers regulating myogenesis. In order to recapitulate the in-vivo bio-physiological environment for zebrafish skeletal muscle cells in-vitro, these cells were successfully cultured in tissue engineered three dimensional (3D) constructs based on fibrin and collagen models. Maturation of tissue engineered collagen and fibrin based constructs was confirmed using the basic parameters described in the literature i.e. collagen three times greater contraction from the original width (Mudera, Smith et al. 2010) and fibrin constructs tightly coiled up to 4mm of diameter (Khodabukus, Paxton et al. 2007). In-vitro characterisation of zebrafish skeletal muscle cells showed hypertrophic growth of muscle mass compared to hyperplasic growth in-vivo as suggested for fish species in literature (Johnston 2006), which is different from human and other mammals. Comparative analysis of zebrafish muscle cells cultured in monolayer against cultured in 3D tissue engineered constructs showed significant increase in fusion index, nuclei per myotube (two-fold) and myotubes per microscopic frame (two-fold). Cells cultured in tissue engineered construct closely resembled in-vivo muscle in terms of their unidirectional orientation of myotubes. These tissue engineered 3D zebrafish skeletal muscle models could be used for various purposes such as drug screening, effect of different temperature extremes, studying underlined pathways involved in human diseases; and with further refinements it would potentially replace the need for studies on live fish in these areas.en
dc.language.isoenen
dc.publisherUniversity of Bedfordshireen
dc.subjectC131 Applied Cell Biologyen
dc.subjectzebrafishen
dc.subjectDanio rerioen
dc.subjectskeletal muscle cellsen
dc.subjectcell cultureen
dc.titleDevelopment and charaterisation of 3 dimensional culture models for zebrafish (Danio rerio) skeletal muscle cellsen
dc.typeThesis or dissertationen
dc.type.qualificationnamePhDen_GB
dc.type.qualificationlevelPhDen
dc.publisher.institutionUniversity of Bedfordshireen
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