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

Summary

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.

Abstract

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.

Introduction

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 fate

Protocol

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.

1. Prepare 10x phosphate-buffered saline (10x PBS) by adding sodium chloride (NaCl) at 137 mM, potassium ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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