This work was funded by the Association Fran&#231;aise contre les Myopathies (AFM-T&#233;l&#233;thon; contract no. 23049), the MATRIHEALTH project, and cE3c unit funding UIDB/00329/2020. We would like to thank our donor Henrique Meirelles who chose to support the MATRIHEALTH Project. This work benefitted from the infrastructures of the Faculty of Sciences Microscopy Facility, a node of the Portuguese Platform of BioImaging (reference PPBI-POCI-01-0145-FEDER-022122), and we thank Lu&#237;s Marques for his assistance with image acquisition and processing. Finally, we thank Marta Palma for technical support and our research team for their generous contributions.

<ol>
	<li>Mecham, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+extracellular+matrix.">Overview of extracellular matrix.</a> Current Protocols in Cell Biology. 10, 1-16 (2012).</li><li>Frantz, C., Stewart, K. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> Journal of Cell Science. 123 (24), 4195-4200 (2010).</li><li>Lamand&#233;, S. R., Bateman, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disorders+of+the+extracellular+matrix.">Genetic disorders of the extracellular matrix.</a> Anatomical Record. 303 (6), 1527-1542 (2020).</li><li>Ahmad, K., Shaikh, S., Ahmad, S. S., Lee, E. J., Choi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-talk+between+extracellular+matrix+and+skeletal+muscle:+implications+for+myopathies.">Cross-talk between extracellular matrix and skeletal muscle: implications for myopathies.</a> Frontiers in Pharmacology. 11, 142 (2020).</li><li>Tome, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+muscular+dystrophy+with+merosin+deficiency.">Congenital muscular dystrophy with merosin deficiency.</a> Comptes Rendus de l'Academie des Sciences. Serie III, Sciences de la Vie. 317 (4), 351-357 (1994).</li><li>Gawlik, K. I., Durbeej, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+laminin+and+MDC1A:+pathogenesis+and+treatment+strategies.">Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies.</a> Skeletal Muscle. 1 (1), 9 (2011).</li><li>van Ry, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM-related+myopathies+and+muscular+dystrophies:+pros+and+cons+of+protein+therapies.">ECM-related myopathies and muscular dystrophies: pros and cons of protein therapies.</a> Comprehensive Physiology. 7 (4), 1519-1536 (2017).</li><li>Gawlik, K. I., Durbeej, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+laminin+&#945;2+chain-deficient+mouse+mutants:+advancing+the+research+on+LAMA2-CMD.">A family of laminin &#945;2 chain-deficient mouse mutants: advancing the research on LAMA2-CMD.</a> Frontiers in Molecular Neuroscience. 13, 59 (2020).</li><li>Kuang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merosin-deficient+congenital+muscular+dystrophy.+Partial+genetic+correction+in+two+mouse+models.">Merosin-deficient congenital muscular dystrophy. Partial genetic correction in two mouse models.</a> Journal of Clinical Investigation. 102 (4), 844-852 (1998).</li><li>Nunes, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+fetal+muscle+development+and+JAK-STAT+activation+mark+disease+onset+and+progression+in+a+mouse+model+for+merosin-deficient+congenital+muscular+dystrophy.">Impaired fetal muscle development and JAK-STAT activation mark disease onset and progression in a mouse model for merosin-deficient congenital muscular dystrophy.</a> Human Molecular Genetics. 26 (11), 2018-2033 (2017).</li><li>McCrary, M. W., Bousalis, D., Mobini, S., Song, Y. H., Schmidt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tissues+as+platforms+for+in+vitro+modeling+of+healthy+and+diseased+tissues.">Decellularized tissues as platforms for in vitro modeling of healthy and diseased tissues.</a> Acta Biomaterialia. 111, 1-19 (2020).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Philips, C., Terrie, L., Thorrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+skeletal+muscle:+A+versatile+biomaterial+in+tissue+engineering+and+regenerative+medicine.">Decellularized skeletal muscle: A versatile biomaterial in tissue engineering and regenerative medicine.</a> Biomaterials. 283, 121436 (2022).</li><li>Silva, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparable+decellularization+of+fetal+and+adult+cardiac+tissue+explants+as+3D-like+platforms+for+in+vitro+studies.">Comparable decellularization of fetal and adult cardiac tissue explants as 3D-like platforms for in vitro studies.</a> Journal of Visualized Experiments. (145), e56924 (2019).</li><li>Silva, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+scaffolds+of+fetal+decellularized+hearts+exhibit+enhanced+potential+to+support+cardiac+cells+in+comparison+to+the+adult.">Three-dimensional scaffolds of fetal decellularized hearts exhibit enhanced potential to support cardiac cells in comparison to the adult.</a> Biomaterials. 104, 52-64 (2016).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270 (5639), 725-727 (1977).</li><li>Burattini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C2C12+murine+myoblasts+as+a+model+of+skeletal+muscle+development:+morpho-functional+characterization.">C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization.</a> European Journal of Histochemistry. 48 (3), 223-233 (2004).</li><li>Murphy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caesarean+section+and+fostering.">Caesarean section and fostering.</a> Methods in Molecular Biology. 18, 177-178 (1993).</li><li>Prieto, D., Aparicio, G., Machado, M., Zolessi, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+DNA-specific+stain+methyl+green+in+the+fluorescent+labeling+of+embryos.">Application of the DNA-specific stain methyl green in the fluorescent labeling of embryos.</a> Journal of Visualized Experiments. (99), e52769 (2015).</li><li>Lichtman, J. W., Conchello, J. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nature Methods. 2 (12), 910-919 (2005).</li><li>Jonkman, J., Brown, C. M., Wright, G. D., Anderson, K. I., North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial:+guidance+for+quantitative+confocal+microscopy.">Tutorial: guidance for quantitative confocal microscopy.</a> Nature Protocols. 15 (5), 1585-1611 (2020).</li><li>Paulsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.T.S.+Biosynthesis,+tissue+distribution+and+isolation+of+laminins.">A.T.S. Biosynthesis, tissue distribution and isolation of laminins.</a> The Laminins. , 1-26 (1994).</li><li>Fedecostante, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recellularized+native+kidney+scaffolds+as+a+novel+tool+in+nephrotoxicity+screening.">Recellularized native kidney scaffolds as a novel tool in nephrotoxicity screening.</a> Drug Metabolism and Disposition: the Biological Fate of Chemicals. 46 (9), 1338-1350 (2018).</li><li>Wagner, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+decellularization+and+recellularization+of+normal+versus+emphysematous+human+lungs.">Comparative decellularization and recellularization of normal versus emphysematous human lungs.</a> Biomaterials. 35 (10), 3281-3297 (2014).</li><li>Brouki Milan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+and+preservation+of+human+skin:+A+platform+for+tissue+engineering+and+reconstructive+surgery.">Decellularization and preservation of human skin: A platform for tissue engineering and reconstructive surgery.</a> Methods. 171, 62-67 (2020).</li><li>Lamand&#233;, S. R., Bateman, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+VI+disorders:+Insights+on+form+and+function+in+the+extracellular+matrix+and+beyond.">Collagen VI disorders: Insights on form and function in the extracellular matrix and beyond.</a> Matrix Biology. 71, 348-367 (2018).</li><li>Baptista, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+whole+organ+decellularization+for+the+generation+of+a+vascularized+liver+organoid.">The use of whole organ decellularization for the generation of a vascularized liver organoid.</a> Hepatology. 53 (2), 604-617 (2011).</li><li>White, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+detergents+on+the+tissue+decellularization+process:+a+ToF-SIMS+study.">The impact of detergents on the tissue decellularization process: a ToF-SIMS study.</a> Acta Biomaterialia. 50, 207-219 (2017).</li><li>Nakayama, K. H., Batchelder, C. A., Lee, C. I., Tarantal, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+tissue+engineering+with+decellularized+rhesus+monkey+kidneys:+age-related+differences.">Renal tissue engineering with decellularized rhesus monkey kidneys: age-related differences.</a> Tissue Engineering. Part A. 17 (23-24), 2891-2901 (2011).</li><li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+in+vivo+validation+of+tissue-engineered,+small-diameter+vascular+grafts+from+decellularized+aortae+of+fetal+pigs+and+canine+vascular+endothelial+cells.">Development and in vivo validation of tissue-engineered, small-diameter vascular grafts from decellularized aortae of fetal pigs and canine vascular endothelial cells.</a> Journal of Cardiothoracic Surgery. 12 (1), 101 (2017).</li><li>Wu Young, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+fetal+matrix+suppresses+fibrotic+gene+expression+and+promotes+myogenesis+in+a+rat+model+of+volumetric+muscle+loss.">Decellularized fetal matrix suppresses fibrotic gene expression and promotes myogenesis in a rat model of volumetric muscle loss.</a> Plastic and Reconstructive Surgery. 146 (3), 552-562 (2020).</li></ol>

The authors have no conflicts of interest to disclose.

The ECM is a complex network of macromolecules that is present in all tissues and plays a crucial role in regulating cell behavior and function2. The ECM acts as a physical scaffold for cells to attach to and provides cues that actively modulate cellular processes such as proliferation, motility, differentiation, and apoptosis. Thus, proper formation and maintenance of the ECM is essential for both development and homeostasis1.
While 2D cell culture models have been widely used, they are increasingly being replaced by more advanced 3D platforms. This is because 2D cultures lack the chemical and physical cues that affect cell behavior, while 3D cultures are considered a more realistic alternative for studying molecular and cellular dynamics in native tissues11. Decellularization of tissues results in the production of scaffolds that more closely mimic the microenvironments of biological tissues, as demonstrated in a number of studies across various tissue or organ systems14,15,23,24,25. The ability of dECMs to replicate native tissue microenvironments holds great potential for research on normal development, various disease states, and the effect of drugs or toxins on tissues11.
The protocol used in this study builds on the protocol developed by Silva et al. for decellularizing fetal heart tissue14 as a starting point. It involves a combination of a hypotonic buffer, treatment with an anionic detergent (SDS), and a DNase treatment. One of the main challenges in decellularization protocols is finding a balance between removing cells and preserving the ECM protein composition. Given our focus on using this 3D cell culture system to study the early stages of LAMA2-CMD, special attention was paid to preserving laminin 211 during decellularization. The protocol of Silva et al.14 led to the loss of laminin &#945;2 chain immunoreactivity in the decellularized fetal muscles; this could be due to the concentration of SDS used. Therefore, alternatives to the 0.2% SDS detergent solution step were tested, such as lower concentrations of SDS (0.1%, 0.05%, and 0.02%) and the substitution of SDS with different concentrations of Triton X-100 (0.5% and 0.2%). The best results were achieved by using 0.05% SDS detergent treatment for 24 h. This concentration effectively removed cell contents while preserving laminin &#945;2 chain immunoreactivity after decellularization. This protocol reproducibly produces acellular dECMs that are free of cellular residues, including DNA.
The protocol used in this study preserves both the interstitial matrix proteins (fibronectin and collagen I) and basement membrane proteins (laminins and collagen IV). Future studies should assess whether collagen VI is also preserved, as it is also a player in muscular dystrophies26. It is known that SDS can disrupt protein ultrastructure and damage collagens12; for fetal skeletal muscle, it was important to use a low concentration of SDS (0.05%) to maintain laminin &#945;2 immunoreactivity. However, the western blot results show that the decellularized samples display more bands after immunodetection of laminins and collagens compared to the native tissue, indicating that some protein degradation occurred as a result of the decellularization process27.
Importantly, the recellularization experiments demonstrate that these matrices are reliable scaffolds that consistently support cell adhesion, proliferation, and differentiation. SDS has been reported to be cytotoxic28, and therefore the washing steps included in the protocol are crucial if the matrices are to be used for recellularization. These scaffolds were effectively colonized by C2C12 cells, indicating their suitability as a model system for 3D culture of cells. The observation of intracellular and pericellular staining for ECM proteins surrounding the C2C12 cells further suggests that the cells are actively contributing to their microenvironment within the dECMs. Additionally, when placed in differentiation medium, the C2C12 cells differentiated, fused, and formed myotubes within the dECMs.
A significant challenge in this procedure is the manipulation of the samples throughout the protocol. The samples are very small and soft, requiring care and skill in handling to prevent entrapment in the fine-tip pipette and loss of the samples. The best results are obtained when starting the protocol with fresh tissue. Using frozen, stored samples can impede cellular content removal and lead to increased protein degradation, hindering protein detection and reducing decellularization efficiency.
Only a few studies have reported the decellularization of fetal tissues15,29,30,31. Specifically, regarding the decellularization of fetal skeletal muscle, only one previous study has reported on the decellularization of composite samples of dermis, subcutaneous tissue, and panniculus carnosus31. To the best of the authors&#39; knowledge, this is the first time that a decellularization protocol for isolated fetal mouse skeletal muscle has been established. This protocol can serve as a foundation for the creation of similar protocols for fetal muscle tissues of other species, such as pigs and humans13.
The present protocol for decellularizing E18.5 mouse skeletal muscle is very similar to the procedure of Silva et al.14, who applied it to an E18 mouse heart. The only difference is the concentration of SDS used, which is considerably lower than the one used for the fetal heart (0.05% vs. 0.2%), possibly due to different physical properties of these two fetal tissues.
The development of this in vitro model not only allows for study of the processes involved in normal fetal muscle development, but also enables the parallel investigation of early onset muscular dystrophies and myopathies, such as LAMA2-CMD, which manifests as a myogenesis defect at E18.5 in the dyW mouse model10. However, it should be noted that this system is limited in that it only includes the ECM and muscle cells and does not contain other cell types such as neurons, endothelial cells, and fibroblasts. The relevance of these additional cell types may vary depending on the disease, and modifications to the culture system may be needed to include them. Overall, the use of dECMs as described in this study can be applied in the study of various early onset muscular dystrophies and myopathies.

The extracellular matrix (ECM) is a major constituent of tissues, representing their non-cellular component. This three-dimensional (3D) structure not only provides physical support for cells, but also plays a crucial role in the biochemical processes involved in the development of organisms1. The formation of a tissue-specific ECM occurs during development, as a result of the complex interactions between cells and their niches, influenced by various intra- and extracellular stimuli. The ECM is a highly dynamic structure that undergoes chemical and mechanical rearrangements in a temporal-spatial manner and directly impacts cell fate2. One of the most notable characteristics of the ECM is its functional diversity, as each tissue ECM displays a unique combination of molecules that provide different topologies and properties that are tailored to the cells it contains1.
ECM signaling and support are crucial for development and homeostasis, and when disrupted can lead to multiple pathological conditions3,4. One example is LAMA2-deficient congenital dystrophy (LAMA2-CMD), which is the most common form of congenital muscular dystrophy. The LAMA2 gene encodes for the laminin &#945;2 chain, which is present in laminin 211 and laminin 221, and when mutated can lead to LAMA2-CMD5. Laminin 211 is the main isoform found in the basement membrane surrounding skeletal muscle fibers. When laminin 211 is abnormal or absent, the link between the basement membrane and muscle cells is disrupted, leading to the onset of the disease6. Patients with LAMA2-CMD show a mild to severe phenotype depending on the type of mutation in the LAMA2 gene.
When the function of the laminin &#945;2 protein is affected, patients can experience severe muscle hypotonia at birth and develop chronic inflammation, fibrosis, and muscle atrophy, leading to a reduced life expectancy. To date, no targeted treatments have been developed and therapeutic approaches are limited to alleviating the symptoms of the disease7. Therefore, understanding the underlying molecular mechanisms involved in the onset of this disease is crucial for developing appropriate therapeutic strategies6,8. Previous work using the dyW mouse9, a model for LAMA2-CMD, suggests that the onset of the disease starts in utero, specifically during fetal myogenesis10. A better understanding of how the fetal myogenesis defect emerges would be a game changer in generating novel therapeutic approaches for LAMA2-CMD.
In vitro systems provide a controlled environment for studying cell-cell and cell-ECM interactions, but 2D culture models lack the complexity of native tissues. Decellularization of tissues produces tissue- and developmental stage-specific acellular ECM scaffolds that more accurately mimic the natural cell microenvironment compared to 2D models and engineered/synthetic scaffolds. Decellularized matrices (dECMs) have the potential to preserve the molecular and mechanical cues of the host tissue, making them better alternative models for understanding in vivo processes11.
There are various techniques, reagents, and conditions that can be used for decellularization12,13. In this study, a decellularization protocol for the fetal mouse heart, described by Silva et al.14,15, is adapted to fetal mouse skeletal muscle and found to retain all tested ECM components (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV). The protocol includes three steps: cell lysis by osmotic shock (hypotonic buffer), plasma membrane dissolution and protein dissociation (0.05% sodium dodecyl sulfate [SDS]), and enzymatic destruction of DNA (DNase treatment). To our knowledge, this is the first established protocol for decellularizing mouse fetal skeletal muscle.
To use this 3D in vitro system for studying LAMA2-CMD, it is crucial to maintain the laminin &#945;2 chain after decellularization. Therefore, an optimization protocol was implemented where different detergents (SDS and Triton X-100) and concentrations (0.02%, 0.05%, 0.1%, 0.2%, and 0.5%) were tested (data not shown). The optimal choice for cell removal and preservation of the laminin &#945;2 protein was found to be 0.05% SDS. C2C12 cells, a well-established myoblast cell line16,17, were used to seed the dECMs. These cells invade the dECM, proliferate, and differentiate inside these scaffolds, synthesizing new ECM proteins. The successful production of this 3D in vitro model offers a new approach to understanding the molecular and cellular processes involved in fetal myogenesis, the onset of LAMA2-CMD, and can be extended to other muscle diseases where the communication between the ECM and skeletal muscle cells is disrupted.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >12 Well Cell Culture Plate, Flat, TC, Sterile</td>
			<td >Abdos Labware</td>
			<td >P21021</td>
			<td ></td>
		</tr>
		<tr >
			<td >4&#8242;,6-Diamidino-2-phenylindole dihydrochloride</td>
			<td >Merck</td>
			<td >D8417</td>
			<td></td>
		</tr>
		<tr >
			<td >4&#8211;20% Mini-PROTEAN TGX Precast Gel</td>
			<td >Bio-Rad</td>
			<td >4561093</td>
			<td></td>
		</tr>
		<tr >
			<td >48 Well Cell Culture Plate, Flat, TC, Sterile</td>
			<td >Abdos Labware</td>
			<td >P21023</td>
			<td></td>
		</tr>
		<tr >
			<td >96 Well Cell Culture Plate, Flat, TC, Sterile</td>
			<td >Abdos Labware</td>
			<td >P21024</td>
			<td></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumin, Fraction V</td>
			<td >NZYtech</td>
			<td >MB04601</td>
			<td></td>
		</tr>
		<tr >
			<td >BX60 fluorescence microscope</td>
			<td >Olympus</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat CM1860 UV</td>
			<td >Leica </td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dithiothreitol</td>
			<td >ThermoFisher</td>
			<td >R0862</td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM high glucose w/ stable glutamine w/ sodium pyruvate</td>
			<td >Biowest</td>
			<td >L0103-500</td>
			<td></td>
		</tr>
		<tr >
			<td >DNase I</td>
			<td >PanReac AppliChem</td>
			<td >A3778</td>
			<td></td>
		</tr>
		<tr >
			<td >DNeasy Blood &#38; Tissue Kit </td>
			<td >Qiagen</td>
			<td >69506</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA)</td>
			<td >Merck</td>
			<td >108418</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal bovine serum</td>
			<td >Biowest</td>
			<td >S1560-500</td>
			<td></td>
		</tr>
		<tr >
			<td >Fine tip transfer pipette</td>
			<td >ThermoFisher</td>
			<td >15387823</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat serum</td>
			<td >Biowest</td>
			<td >S2000-100</td>
			<td></td>
		</tr>
		<tr >
			<td >Hera Guard Flow Cabinet</td>
			<td >Heraeus</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Heracell 150 CO2 Incubator</td>
			<td >Thermo Scientific</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >HiMark Pre-stained Protein Standard</td>
			<td >Invitrogen</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Horse Serum, New Zealand origin</td>
			<td >Gibco</td>
			<td >16050122</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-&#945;- Rabbit IgG</td>
			<td >abcam</td>
			<td >ab205718</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-&#945;- Rat IgG</td>
			<td >abcam</td>
			<td >ab205720</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-&#945;-Mouse IgG</td>
			<td >abcam</td>
			<td >ab205719</td>
			<td></td>
		</tr>
		<tr >
			<td >ImageJ v. 1.53t</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Methyl Green</td>
			<td >Sigma-Aldrich</td>
			<td >67060</td>
			<td></td>
		</tr>
		<tr >
			<td >MM400 Tissue Lyser</td>
			<td >Retsch</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >NanoDrop ND-1000 Spectrophotometer </td>
			<td >ThermoFisher</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde, 16% w/v aq. soln., methanol free</td>
			<td >Alfa Aesar</td>
			<td >043368-9M</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin (100x) </td>
			<td >GRiSP</td>
			<td >GTC05.0100</td>
			<td></td>
		</tr>
		<tr >
			<td >Phalloidin Alexa 488 </td>
			<td >Thermo Fisher Sci.</td>
			<td >A12379</td>
			<td></td>
		</tr>
		<tr >
			<td >Polystyrene Petri dish 60x15mm with vents (sterile)</td>
			<td >Greiner Bio-One</td>
			<td >628161</td>
			<td></td>
		</tr>
		<tr >
			<td >Qubit dsDNA HS kit</td>
			<td >Thermo Scientific</td>
			<td >Q32851</td>
			<td></td>
		</tr>
		<tr >
			<td >Qubit&#8482; 3 Fluorometer</td>
			<td >Invitrogen</td>
			<td >15387293</td>
			<td></td>
		</tr>
		<tr >
			<td >S6E Zoom Stereo microscope</td>
			<td >Leica</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Dodecyl Sulfate</td>
			<td >Merck</td>
			<td >11667289001</td>
			<td></td>
		</tr>
		<tr >
			<td >SuperFrost&#174; Plus adhesion slides</td>
			<td >Thermo Scientific</td>
			<td >631-9483</td>
			<td></td>
		</tr>
		<tr >
			<td >SuperSignal West Pico PLUS Chemiluminescent Substrate</td>
			<td >Thermo Scientific</td>
			<td >15626144</td>
			<td></td>
		</tr>
		<tr >
			<td >TCS SPE confocal microscope</td>
			<td >Leica </td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Tris-(hidroximetil) aminometano (Tris base) &#8805;99%</td>
			<td >VWR Chemicals</td>
			<td >28811.295</td>
			<td></td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td >Sigma-Aldrich</td>
			<td >X100-100ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan Blue Solution, 0.4%</td>
			<td >Gibco</td>
			<td >15250061</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin-EDTA (0.05%) in DPBS (1X)</td>
			<td >GRiSP</td>
			<td >GTC02.0100</td>
			<td></td>
		</tr>
		<tr >
			<td >TWEEN 20 (50% Solution)</td>
			<td >ThermoFisher</td>
			<td >3005</td>
			<td></td>
		</tr>
		<tr >
			<td >WesternBright PVDF-CL membrane roll (0.22&#181;m)</td>
			<td >Advansta</td>
			<td >L-08024-001</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Collagen I</td>
			<td >abcam</td>
			<td >ab21286</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Collagen IV</td>
			<td >Millipore</td>
			<td >AB756P</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Collagen IV</td>
			<td >Santa Cruz Biotechnology</td>
			<td >sc-398655</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Fibronectin</td>
			<td >Sigma</td>
			<td >F-3648</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Laminin &#945;2 </td>
			<td >Sigma</td>
			<td >L-0663</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-MHC</td>
			<td >D.S.H.B.</td>
			<td >MF20</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Mouse Alexa 488</td>
			<td >Molecular Probes</td>
			<td >A11017</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Mouse Alexa 568</td>
			<td >Molecular Probes</td>
			<td >A11019</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-pan-Laminin</td>
			<td >Sigma</td>
			<td >L- 9393</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-phospho-histone 3</td>
			<td >Merk Millipore</td>
			<td >06-570</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Rabbit Alexa 568</td>
			<td >Molecular Probes</td>
			<td >A21069</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Rabbit Alexa 488</td>
			<td >Molecular Probes</td>
			<td >A11070</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;-Rat Alexa 488</td>
			<td >Molecular Probes</td>
			<td >A11006</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of 3d decellularized matrices from fetal mouse skeletal muscle for cell culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

The goal of the decellularization protocol is to produce dECMs that closely resemble the composition of native tissue. To determine the effectiveness of the decellularization process, various methods were employed, including examination of tissue morphology, measurement of DNA levels, staining for F-actin, and analysis of key ECM components using immunohistochemistry and western blotting techniques. Specifically, five major ECM components of skeletal muscle tissues were analyzed.
Throughout the protocol, samples change in appearance (Figure 1B1-4). After isolation, the muscle tissue appears reddish due to the presence of myoglobin (Figure 1B1). After incubation in the hypotonic buffer, which lyses cells and removes most of the cytoplasmatic proteins, the samples turn white (Figure 1B2). SDS, an anionic detergent commonly used in tissue decellularization12, effectively removes cytoplasmic and nuclear material, as well as membranes. As a result, the samples become more transparent after SDS treatment (Figure 1B3). However, high concentrations or prolonged exposure to this detergent can cause protein denaturation, loss of glycosaminoglycans, and disruption of collagen fibers12. The optimal balance between cell removal and preservation of the ECM is achieved using 0.05% SDS for 24 h, as shown in Figure 2 and Figure 3. Finally, DNA is removed through DNase treatment, resulting in transparent dECM scaffolds that are slightly smaller than after SDS treatment (Figure 1B4).
The success of the decellularization protocol is indicated by the amount of residual DNA present in the matrices after the process. DNA quantification reveals a nearly 100% decrease in the dECMs compared to the native tissue (Figure 1C). DAPI staining also confirms the absence of DNA in the dECMs (Figure 2B,D,F,H,J).
The presence of five major ECM proteins was evaluated by immunohistochemistry and by western blot after decellularization and compared to the native tissue. Immunostaining for the laminin &#945;2 subunit (Figure 2A,B) and total laminins (Figure 2C,D) shows that the dECMs display a tubular staining for these proteins (arrows in Figure 2B,D), similar to the laminin matrix surrounding myotubes in the native tissue (arrows in Figure 2A,C). Western blot analysis for laminin &#945;2 subunit reveals the presence of two bands in the native tissue (Figure 2A&#39;), while three bands, with a smaller molecular weight than expected, can be detected in the dECM (Figure 2B&#39;). This may be the result of protein degradation or removal during the decellularization process. Western blot analysis for total laminins shows some fragmentation of these proteins in the dECM compared to samples from the native tissue (Figure 2C&#39;,D&#39;).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65069/65069fig01.jpg" /> 
Figure 1: Fetal skeletal muscle decellularization procedure and evaluation of tissue morphology and DNA content.&#160;(A)&#160;Schematic representation of the decellularization protocol. (B)&#160;Tissue morphology throughout the protocol, from freshly collected to decellularized tissue. Scale bar = 1 mm. (C)&#160;Quantification of DNA present in the dECMs compared to the native tissue. Data expressed as mean &#177; SEM. Student&#39;s&#160;t-test, two-tailed **p&#160;&#60; 0.01. The decellularization protocol efficiently removes nuclear content producing acellular dECMs. Abbreviations: dECMs = decellularized matrices; PBS = phosphate-buffered saline; SDS = sodium dodecyl sulfate; NT = native tissue.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65069/65069fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Immunostaining for fibronectin (Figure 2E,F) and collagen I (Figure 2G,H) shows the presence of these proteins in the interstitial space between cells in the native tissue (arrows in Figure 2E,G) and a similar staining in the dECMs (arrows Figure 2F,H). Western blot analysis shows similar bands for fibronectin in both conditions (Figure 2E&#39;,F&#39;), indicating that this protein is not particularly affected by the decellularization process. However, in the case of collagen I (Figure 2G&#39;,H&#39;) there are fewer bands in the dECMs, indicating some degree of degradation. Immunostaining for collagen IV shows a tubular staining pattern in the native tissue (arrow in Figure 2I) and a similar staining in the dECM, although the tubular structures are narrower (arrow in Figure 2J). Western blot analysis for collagen IV reveals the presence of three bands in the native tissue (Figure 2I&#39;). While the same three bands are observed in the dECMs (Figure 2J&#39;), the molecular weight of these is lower than expected. Similarly to laminin &#945;2, this can be the result of protein degradation or removal during the decellularization.
dECMs are then seeded with C2C12 myoblasts and cultured for 8 days in complete culture medium. C2C12 cells colonize the dECMs and proliferate, as shown by phospho-histone 3 nuclear staining (arrows in Figure 3A). After an additional 4 days of culture in differentiation medium, C2C12 cells differentiate and fuse into multinucleated (blue arrows in Figure 3B) myotubes expressing a myosin heavy chain (dashed line in Figure 3B). Interestingly, intracellular and/or pericellular staining for the laminin &#945;2 chain (yellow arrows in Figure 3C,D), total laminins (magenta arrow in Figure 3C), and fibronectin (magenta arrow in Figure 3D) can be detected in C2C12 cells cultured in complete culture medium, suggesting that these cells are able to synthesize these ECM proteins de novo, therefore contributing to the formation of their niche. These results demonstrate that the decellularization protocol generates a dECM microenvironment where C2C12 myoblasts can proliferate, differentiate, and form multinucleated myotubes.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65069/65069fig02.jpg" /> 
Figure 2: Assessment of five ECM proteins in native versus decellularized E18.5 fetal muscle tissue. Immunohistochemistry on sections of native tissue (A,C,E,G,I)&#160;and staining of dECMs (B,D,F,H,J) with DAPI, a DNA marker, phalloidin detecting F-actin, and for ECM proteins. Scale bar = 15 &#181;m. In the native tissue, immunostaining for laminins and collagen IV is present surrounding the myofibers (A,C,I; yellow arrows)&#160;and staining for fibronectin and collagen I is detected in the interstitial space between myofibers (E,G; yellow arrows). In the dECMs, staining for laminins and collagen IV (B,D,J; yellow arrows)&#160;show a tubular pattern, surrounding the spaces left by the myofibers after decellularization. Fibronectin and collagen I immunostaining of dECMs is consistent with their presence in the interstitial space (F,H; yellow arrows). (A&#39;-J&#39;) Western blot analysis for five ECM proteins in native tissue (A&#39;,C&#39;,E&#39;,G&#39;,I&#39;) and in dECM samples (B&#39;,D&#39;,F&#39;,H&#39;,J&#39;).&#160;Both approaches show protein preservation after decellularization. Antibodies used and respective dilutions are listed in Table 1. Abbreviations: LN&#945;2 = laminin &#945;2 chain; LNp = pan-muscle laminins; FN = fibronectin; Col I = collagen I; Col IV = collagen IV; MHC = myosin heavy chain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65069/65069fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65069/65069fig03.jpg" /> 
Figure 3: dECM recellularization with a myoblast cell line (C2C12 myoblasts). (A)&#160;Maximum intensity projection of a confocal image of a 100 &#181;m stack of recellularized matrix, colonized with C2C12 cells labelled with methyl green staining DNA, phalloidin detecting F-actin, and anti-pH3 antibody, a proliferation marker (magenta arrows). Scale bar = 35 &#181;m. (B)&#160;Maximum intensity projection of a confocal image of a 100 &#181;m stack showing a multinucleated (blue arrows indicate the nuclei) myosin heavy chain-positive myotube (dashed line) formed during culture. Scale bar = 35 &#181;m. (C,D)&#160;Immunohistochemistry confocal image showing intra- or peri-cellular staining for laminin &#945;2 chain (yellow arrows), total laminins (magenta arrows in C), and fibronectin (magenta arrows in D) in myoblasts, suggesting de novo protein synthesis.&#160;Scale bar = 10 &#181;m. Antibodies used and respective dilutions are listed in Table 1. Abbreviations: pH3 = phospho-histone 3; LN&#945;2 = laminin &#945;2 chain; LNp = pan-muscle laminins; FN = fibronectin; MHC = myosin heavy chain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65069/65069fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture at JoVE.com

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

preparation-3d-decellularized-matrices-from-fetal-mouse-skeletal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.2K Views.  University of Lisbon. In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

Video: Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...