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.

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</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 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><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&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride</td><td>Merck</td><td>D8417</td><td></td></tr><tr><td>4&amp;ndash;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 &amp;amp; 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-&amp;alpha;- Rabbit IgG</td><td>abcam</td><td>ab205718</td><td></td></tr><tr><td>HRP-&amp;alpha;- Rat IgG</td><td>abcam</td><td>ab205720</td><td></td></tr><tr><td>HRP-&amp;alpha;-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&amp;trade; 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&amp;reg; 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) &amp;ge;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&amp;micro;m)</td><td>Advansta</td><td>L-08024-001</td><td></td></tr><tr><td>&amp;alpha;-Collagen I</td><td>abcam</td><td>ab21286</td><td></td></tr><tr><td>&amp;alpha;-Collagen IV</td><td>Millipore</td><td>AB756P</td><td></td></tr><tr><td>&amp;alpha;-Collagen IV</td><td>Santa Cruz Biotechnology</td><td>sc-398655</td><td></td></tr><tr><td>&amp;alpha;-Fibronectin</td><td>Sigma</td><td>F-3648</td><td></td></tr><tr><td>&amp;alpha;-Laminin &amp;alpha;2 </td><td>Sigma</td><td>L-0663</td><td></td></tr><tr><td>&amp;alpha;-MHC</td><td>D.S.H.B.</td><td>MF20</td><td></td></tr><tr><td>&amp;alpha;-Mouse Alexa 488</td><td>Molecular Probes</td><td>A11017</td><td></td></tr><tr><td>&amp;alpha;-Mouse Alexa 568</td><td>Molecular Probes</td><td>A11019</td><td></td></tr><tr><td>&amp;alpha;-pan-Laminin</td><td>Sigma</td><td>L- 9393</td><td></td></tr><tr><td>&amp;alpha;-phospho-histone 3</td><td>Merk Millipore</td><td>06-570</td><td></td></tr><tr><td>&amp;alpha;-Rabbit Alexa 568</td><td>Molecular Probes</td><td>A21069</td><td></td></tr><tr><td>&amp;alpha;-Rabbit Alexa 488</td><td>Molecular Probes</td><td>A11070</td><td></td></tr><tr><td>&amp;alpha;-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 ...

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Research

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Developmental Biology

1.3K Views.  University of Lisbon. Voor zover wij weten, is dit protocol het eerste dat gedecellulariseerde matrices uit foetale skeletspieren produceert, wat een nieuwe benadering biedt om myopathieën te bestuderen die zich in de baarmoeder beginnen te ontwikkelen. Dit protocol genereert een ontwikkelings- en weefselspecifieke metriek die kan worden gebruikt om spiercelgedrag en interacties met extracellulaire matrix in een gecontroleerde omgeving te bestuderen. LAMA2-CMD is een aangeboren spierdystrofie die zich bij de gebo...

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

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Bioengineering

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

This protocol provides methods for generating 3D engineered cardiac and skeletal muscle tissues and describes their use in preclinical drug screening modalities. The described methods utilize a magnetic sensing system to facilitate the simultaneous assessment of 24 tissues in parallel.

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and ...

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

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