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 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 α2, 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 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 α2 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 α2 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 α2, 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 α2 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 α2 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.
All the methodologies described were approved by the Animal Welfare Committee (ORBEA) of the Faculty of Sciences, University of Lisbon, and Direção Geral de Veterinária (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 3 months unless stated otherwise.
2. Sample collection
NOTE: Wild-type C57/BL6 mice were utilized in the study. All techniques were conducted in a laminar flow hood under sterile conditions.
3. Fetal skeletal muscle decellularization
NOTE: All techniques were conducted in a laminar flow hood under sterile conditions. For a detailed schematic representation, see Figure 1A. All steps were performed with agitation in an orbital shaker with a diameter of 120 mm (165 rpm) at 25 °C unless stated otherwise. Add 1% pen/strep to solutions before use. When removing the solutions, aspirate carefully to avoid sample entrapment in the pipette.
4. Decellularization quality assessment
NOTE: DNA quantification, DAPI/methyl green staining, and phalloidin staining were performed to evaluate the presence of residual cell contents after decellularization. Immunohistochemistry and western blot analyses were conducted to assess the retention of key ECM proteins after decellularization.
Table 1: Antibodies and dyes used in immunohistochemistry and western blot analysis and the respective dilutions. Please click here to download this Table.10,22
5. Cell culture in decellularized matrices
NOTE: All the techniques were performed under sterile conditions in a laminar flow hood. All incubations were performed at 37 °C and with 5% CO2.
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 α2 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 α2 subunit reveals the presence of two bands in the native tissue (Figure 2A'), while three bands, with a smaller molecular weight than expected, can be detected in the dECM (Figure 2B'). 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',D').
Figure 1: Fetal skeletal muscle decellularization procedure and evaluation of tissue morphology and DNA content. (A) Schematic representation of the decellularization protocol. (B) Tissue morphology throughout the protocol, from freshly collected to decellularized tissue. Scale bar = 1 mm. (C) Quantification of DNA present in the dECMs compared to the native tissue. Data expressed as mean ± SEM. Student's t-test, two-tailed **p < 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. Please click here to view a larger version of this figure.
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',F'), indicating that this protein is not particularly affected by the decellularization process. However, in the case of collagen I (Figure 2G',H') 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'). While the same three bands are observed in the dECMs (Figure 2J'), the molecular weight of these is lower than expected. Similarly to laminin α2, 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 α2 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.
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) 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 µm. In the native tissue, immunostaining for laminins and collagen IV is present surrounding the myofibers (A,C,I; yellow arrows) 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) 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'-J') Western blot analysis for five ECM proteins in native tissue (A',C',E',G',I') and in dECM samples (B',D',F',H',J'). Both approaches show protein preservation after decellularization. Antibodies used and respective dilutions are listed in Table 1. Abbreviations: LNα2 = laminin α2 chain; LNp = pan-muscle laminins; FN = fibronectin; Col I = collagen I; Col IV = collagen IV; MHC = myosin heavy chain. Please click here to view a larger version of this figure.
Figure 3: dECM recellularization with a myoblast cell line (C2C12 myoblasts). (A) Maximum intensity projection of a confocal image of a 100 µm 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 µm. (B) Maximum intensity projection of a confocal image of a 100 µm stack showing a multinucleated (blue arrows indicate the nuclei) myosin heavy chain-positive myotube (dashed line) formed during culture. Scale bar = 35 µm. (C,D) Immunohistochemistry confocal image showing intra- or peri-cellular staining for laminin α2 chain (yellow arrows), total laminins (magenta arrows in C), and fibronectin (magenta arrows in D) in myoblasts, suggesting de novo protein synthesis. Scale bar = 10 µm. Antibodies used and respective dilutions are listed in Table 1. Abbreviations: pH3 = phospho-histone 3; LNα2 = laminin α2 chain; LNp = pan-muscle laminins; FN = fibronectin; MHC = myosin heavy chain. Please click here to view a larger version of this figure.
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 α2 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 α2 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 α2 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' 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 authors have nothing to disclose.
This work was funded by the Association Française contre les Myopathies (AFM-Téléthon; 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 Luís 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.
12 Well Cell Culture Plate, Flat, TC, Sterile | Abdos Labware | P21021 | |
4′,6-Diamidino-2-phenylindole dihydrochloride | Merck | D8417 | |
4–20% Mini-PROTEAN TGX Precast Gel | Bio-Rad | 4561093 | |
48 Well Cell Culture Plate, Flat, TC, Sterile | Abdos Labware | P21023 | |
96 Well Cell Culture Plate, Flat, TC, Sterile | Abdos Labware | P21024 | |
Bovine Serum Albumin, Fraction V | NZYtech | MB04601 | |
BX60 fluorescence microscope | Olympus | ||
Cryostat CM1860 UV | Leica | ||
Dithiothreitol | ThermoFisher | R0862 | |
DMEM high glucose w/ stable glutamine w/ sodium pyruvate | Biowest | L0103-500 | |
DNase I | PanReac AppliChem | A3778 | |
DNeasy Blood & Tissue Kit | Qiagen | 69506 | |
Ethylenediaminetetraacetic acid (EDTA) | Merck | 108418 | |
Fetal bovine serum | Biowest | S1560-500 | |
Fine tip transfer pipette | ThermoFisher | 15387823 | |
Goat serum | Biowest | S2000-100 | |
Hera Guard Flow Cabinet | Heraeus | ||
Heracell 150 CO2 Incubator | Thermo Scientific | ||
HiMark Pre-stained Protein Standard | Invitrogen | ||
Horse Serum, New Zealand origin | Gibco | 16050122 | |
HRP-α- Rabbit IgG | abcam | ab205718 | |
HRP-α- Rat IgG | abcam | ab205720 | |
HRP-α-Mouse IgG | abcam | ab205719 | |
ImageJ v. 1.53t | |||
Methyl Green | Sigma-Aldrich | 67060 | |
MM400 Tissue Lyser | Retsch | ||
NanoDrop ND-1000 Spectrophotometer | ThermoFisher | ||
Paraformaldehyde, 16% w/v aq. soln., methanol free | Alfa Aesar | 043368-9M | |
Penicillin-Streptomycin (100x) | GRiSP | GTC05.0100 | |
Phalloidin Alexa 488 | Thermo Fisher Sci. | A12379 | |
Polystyrene Petri dish 60x15mm with vents (sterile) | Greiner Bio-One | 628161 | |
Qubit dsDNA HS kit | Thermo Scientific | Q32851 | |
Qubit™ 3 Fluorometer | Invitrogen | 15387293 | |
S6E Zoom Stereo microscope | Leica | ||
Sodium Dodecyl Sulfate | Merck | 11667289001 | |
SuperFrost® Plus adhesion slides | Thermo Scientific | 631-9483 | |
SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Scientific | 15626144 | |
TCS SPE confocal microscope | Leica | ||
Tris-(hidroximetil) aminometano (Tris base) ≥99% | VWR Chemicals | 28811.295 | |
Triton X-100 | Sigma-Aldrich | X100-100ML | |
Trypan Blue Solution, 0.4% | Gibco | 15250061 | |
Trypsin-EDTA (0.05%) in DPBS (1X) | GRiSP | GTC02.0100 | |
TWEEN 20 (50% Solution) | ThermoFisher | 3005 | |
WesternBright PVDF-CL membrane roll (0.22µm) | Advansta | L-08024-001 | |
α-Collagen I | abcam | ab21286 | |
α-Collagen IV | Millipore | AB756P | |
α-Collagen IV | Santa Cruz Biotechnology | sc-398655 | |
α-Fibronectin | Sigma | F-3648 | |
α-Laminin α2 | Sigma | L-0663 | |
α-MHC | D.S.H.B. | MF20 | |
α-Mouse Alexa 488 | Molecular Probes | A11017 | |
α-Mouse Alexa 568 | Molecular Probes | A11019 | |
α-pan-Laminin | Sigma | L- 9393 | |
α-phospho-histone 3 | Merk Millipore | 06-570 | |
α-Rabbit Alexa 568 | Molecular Probes | A21069 | |
α-Rabbit Alexa 488 | Molecular Probes | A11070 | |
α-Rat Alexa 488 | Molecular Probes | A11006 |