Name: preparation of 3d decellularized matrices from fetal mouse skeletal muscle for cell culture
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Description: Watch this Scientific Journal Video about Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture at JoVE.com

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.

All the methodologies described were approved by the Animal Welfare Committee (ORBEA) of the Faculty of Sciences, University of Lisbon, and Dire&#231;&#227;o Geral de Veterin&#225;ria (DGAV; ref. 0421/000/000/2022), and are in accordance with the European Directive 2010/63/EU.
1. Preparation of decellularization buffers and reagents
NOTE: All solutions used during the decellularization protocol should be sterilized by autoclaving and stored for up to ...

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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 cult...

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 th...

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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 ...

To our knowledge, this protocol is the first to produce decellularized matrices from fetal skeletal muscle, providing a novel approach to study myopathies that start developing in utero. This protocol generates a developmental and tissue-specific metrics that can be used to study muscle cell behavior and interactions with extracellular matrix in a controlled environment. LAMA2-CMD is a congenital muscle dystrophy that starts manifesting at birth.This model can help unravel the pattern mechanisms involved in its onset and lead to new target therapies. This protocol can be adapted to different tissues and disease models. Different complexity levels can be achieved depending on matrix and cell types, demonstrating the versatility of the system.This is a very straightforward protocol. The most challenging part is the collection and the handling of the samples. But, with practice, technical skills can easily be improved.Begin by transferring one euthanized fetus at a time to a Petri dish containing ice-cold PBS. After excising the skin and limbs, cut through the ventral side of the ribcage. Remove the sternum and underlying organs.Position the fetuses dorsal side up and remove the cervical portion of the vertebral column. Next, excise the dorsal fat deposits and the deep back muscle connective tissue. Now, anchor the ribcage using surgical forceps.With a microscalpel, carefully scrape to detach the deep back muscles from the surrounding tissue. Store the tissues in PBS in a 12-well cell culture plate at four degrees Celsius for future use. For a longer period, store the tissue in an empty microcentrifuge tube at minus 80 degrees Celsius.On the first day, use whole epaxial muscle masses. Add three milliliters of hypotonic buffer containing 1%penicillin and streptomycin to each well of a 12-well plate. Add a muscle tissue fragment to each well and incubate overnight for 18 hours with agitation.On the second day, remove the buffer with a fine-tip pipette. Now wash the samples three times with three milliliters of PBS with a one-hour agitation each time. After discarding the PBS, incubate the samples in three milliliters of 0.05%SDS detergent solution for 24 hours with agitation.On the third day, use a fine tip-pipette to remove the SDS detergent and wash the fragments thrice with three milliliters of hypotonic wash buffer with a 20-minute agitation each time. Replace the solution of the wells with two milliliters of DNase solution and incubate the tissue fragments at 37 degrees Celsius for three hours with agitation. After removing the DNase solution, wash the fragments thrice with three milliliters of PBS with a 20 minute agitation each time, leaving the last wash overnight.To a T-25 flask seeded with subconfluent C2C12 cells, add 500 microliters of trypsin and resuspend in one milliliter of complete medium. Mix 10 microliters of the suspension with 10 microliters of trypan blue dye and load into a hemocytometer to count cell number and estimate viability. In a laminar flow hood, place the decellularized matrices, or dECMs, in a Petri dish containing PBS with 1%penicillin and streptomycin.Using a microscalpel and tweezers, separate the matrices into small pieces. Transfer the fragments into the wells of a 96-well plate, placing three to four pieces per well. Add 200 microliters of prewarmed complete culture medium into each well and incubate the plate for two hours at 37 degrees Celsius with 5%carbon dioxide.Discard the medium in the well plate and add 200 microliters of complete culture medium containing 50, 000 viable C2C12 cells to each well. Incubate the well plate for two days. Transfer the dECMs with the cells to a 48 well-plate containing 400 microliters of complete culture medium.Aspirate the medium carefully every two days to prevent matrix detachment and replenish with the fresh medium until the eighth day. For differentiation, replace the complete culture medium with a differentiation medium and incubate for four days until the 12th day. The muscle tissue was reddish immediately post-isolation and turned white due to cell lysis after incubation in hypotonic buffer.SDS causes the muscles to turn transparent. After DNase treatment, a smaller, transparent dECM was obtained. DNA quantification reveals a nearly 100%decrease in the dECMs compared to the native tissue.The dECMs and native tissue displayed similar tubular staining for laminin alpha two and laminin protein. Western blot analysis of the native tissue displayed two bands for the laminin alpha two subunit, while three smaller bands were detected in the dECM. For total laminins, dECM samples showed protein fragmentation when compared to the native tissue.Fibronectin and collagen I were present in the interstitial space between cells of the native tissue and the dECMs. Similar fibronectin bands were present in both samples, indicating that it is not affected by decellularization. Fewer collagen I bands were observed in the dECMs.For collagen IV, both samples showed similar tubular staining. However, the molecular weight of the dECM bands was lower. C2C12 cells colonized and proliferated in the dECMs and fused into multi-nucleated myotubes.These expressed myosin-heavy chain protein after four days of incubation in differentiation medium. Intracellular and pericellular staining was observed for laminin alpha two chain, total laminins, and fibronectin. Decellularized fetal mouse skeletal scaffolds can be used to grow cells in co-culture systems, bringing it closer to the in vivo situation, and therefore contributes to understanding muscle disease.

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