Sections were in that case washed with 2 M NaCl/50 mM EDTA/1x protease inhibitors/20 mM Tris, pH 7
Sections were in that case washed with 2 M NaCl/50 mM EDTA/1x protease inhibitors/20 mM Tris, pH 7.4 for 10 min at 4C followed by 100 mM NaCl/20 mM Tris for 1 h at 4C. long range scanner. The above images are amplitude images with a scan size of 5 x 5 m2 and scale Doxycycline monohydrate bar2m. Clean coverslips washed with ethanol were used as control glass.(TIFF) pone.0127675.s002.tiff (2.6M) GUID:?FD2D7354-8B0C-4112-8D9B-6BD291F38149 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, Doxycycline monohydrate well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to Doxycycline monohydrate collagen and fibronectin. Doxycycline monohydrate Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and Doxycycline monohydrate organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct Rabbit Polyclonal to ACOT2 muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates. Introduction Skeletal muscle tissue comprises 40C50% of the human body mass and is essential for body movement, metabolism and thermoregulation. Although muscle has excellent regenerative capacity, when an injury results in a significant loss of muscle, natural repair processes are unable to bridge the gap between the remaining segments of the skeletal muscle fibres, leading to loss of muscle tissue and functional deficit. One approach for treating volumetric muscle loss is tissue engineering, where the use of biological scaffolds composed of extracellular matrix (ECM) derived from animal tissue is being explored. Natural ECM, secreted by resident tissue cells, should provide the optimal physical, chemical and biological cues to support regeneration of that tissue. Scaffolds of native ECM have been used to regenerate heart [1] and liver [2] and decellularized porcine small intestinal submucosa (SIS) ECM has been used in human and animal models to reconstruct skin [3, 4], urinary bladder [5], abdominal wall defects [6, 7], rotator cuff tendon [8], and load bearing skeletal muscle [9]. Decellularized skeletal muscle is an ideal source of muscle specific ECM and provides a system to study the role of ECM in muscle regeneration. Skeletal muscle has three layers of ECM: the endomysium surrounds individual fibres and is in intimate contact with myofibres and satellite cells, while the perimysium, and epimysium surround groups of myofibres and the entire muscle, respectively. The ECM provides both structural support and biochemical cues that direct muscle formation. Collagens I and III are the major structural proteins in skeletal muscle ECM, whereas collagen VI is an essential component of the satellite cell niche and contributes to the regulation of satellite cell self-renewal [10]. Laminins containing the 2 2 chain are located in the skeletal muscle basement membrane and are required for myotube formation and the prevention of apoptosis [11, 12]. Fibronectin binds collagen and laminin and contributes to myoblast adhesion, migration and differentiation [13], as well as being.