Summary

胎儿小鼠骨骼肌3D脱细胞基质的制备用于细胞培养

Published: March 03, 2023
doi:

Summary

在这项工作中,优化了脱细胞方案以获得胎鼠骨骼肌的脱细胞基质。C2C12成肌细胞可以在这些基质中定植,增殖和分化。这种 体外 模型可用于研究骨骼肌疾病(如肌肉萎缩症)背景下的细胞行为。

Abstract

细胞外基质(ECM)在为细胞提供结构支持和传递对各种细胞过程很重要的信号方面起着至关重要的作用。二维(2D)细胞培养模型过度简化了细胞与ECM之间的复杂相互作用,因为缺乏完整的三维(3D)支持会改变细胞行为,使其不足以理解 体内 过程。ECM组成和细胞 – ECM相互作用的缺陷是导致各种不同疾病的重要因素。

一个例子是LAMA2先天性肌营养不良症(LAMA2-CMD),其中功能性层粘连蛋白211和221的缺失或减少可导致严重的肌张力低下,可在出生时或出生后不久检测到。先前使用该疾病的小鼠模型的研究表明,其发病发生在胎儿肌生成期间。本研究旨在开发一种3D 体外 模型,允许研究肌肉细胞和胎儿肌肉ECM之间的相互作用,模拟天然微环境。该协议使用从E18.5小鼠胎儿解剖的深背部肌肉,用低渗缓冲液,阴离子洗涤剂和DNase处理。与天然组织相比,所得的脱细胞化基质(dECM)保留了测试的所有ECM蛋白(层粘连蛋白α2,总层粘连蛋白,纤连蛋白,胶原蛋白I和胶原蛋白IV)。

当C2C12成肌细胞接种在这些dECM上时,它们穿透并定植了dECM,从而支持了它们的增殖和分化。此外,C2C12细胞产生ECM蛋白,有助于重塑其在dECM中的生态位。这种 体外 平台的建立为揭示LAMA2-CMD发病过程中涉及的过程提供了一种新的有前途的方法,并有可能适应其他骨骼肌疾病,其中ECM和骨骼肌细胞之间的沟通缺陷有助于疾病进展。

Introduction

细胞外基质(ECM)是组织的主要成分,代表其非细胞成分。这种三维(3D)结构不仅为细胞提供物理支持,而且在生物体发育所涉及的生化过程中起着至关重要的作用1。组织特异性ECM的形成发生在发育过程中,这是细胞与其壁龛之间复杂相互作用的结果,受到各种细胞内和细胞外刺激的影响。ECM是一种高度动态的结构,以时空方式经历化学和机械重排,直接影响细胞命运2。ECM最显着的特征之一是其功能多样性,因为每个组织ECM都显示出独特的分子组合,这些分子提供了针对其所含细胞量身定制的不同拓扑结构和特性1。

ECM信号传导和支持对于发育和体内平衡至关重要,当中断时可导致多种病理状况34。一个例子是LAMA2缺陷性先天性营养不良症(LAMA2-CMD),这是先天性肌营养不良症最常见的形式。LAMA2基因编码层粘连蛋白α2链,该链存在于层粘连蛋白211和层粘连蛋白221中,当突变时可导致 LAMA2-CMD 5。层粘连蛋白211是在骨骼肌纤维周围的基底膜中发现的主要亚型。当层粘连蛋白211异常或不存在时,基底膜和肌肉细胞之间的联系被破坏,导致疾病的发作6。LAMA2-CMD患者表现出轻度至重度表型,具体取决于 LAMA2 基因突变的类型。

当层粘连蛋白α2蛋白的功能受到影响时,患者在出生时会出现严重的肌肉张力减退,并发生慢性炎症、纤维化和肌肉萎缩,导致预期寿命缩短。迄今为止,尚未开发出有针对性的治疗方法,治疗方法仅限于缓解疾病的症状7。因此,了解参与这种疾病发作的潜在分子机制对于制定适当的治疗策略至关重要68。先前使用LAMA2-CMD模型dy W小鼠9的工作表明,该疾病的发作始于子宫内,特别是在胎儿肌发生期间10。更好地了解胎儿肌生成缺陷是如何出现的,将是为LAMA2-CMD产生新治疗方法的游戏规则改变者。

体外 系统为研究细胞-细胞和细胞-ECM相互作用提供了受控环境,但2D培养模型缺乏天然组织的复杂性。组织的去细胞化产生组织和发育阶段特异性无细胞ECM支架,与2D模型和工程/合成支架相比,它更准确地模拟天然细胞微环境。脱细胞基质(dECM)有可能保留宿主组织的分子和机械线索,使其成为理解 体内 过程的更好替代模型11

有多种技术、试剂和条件可用于脱细胞1213。在这项研究中,由Silva等人描述的胎鼠心脏脱细胞方案1415适用于胎鼠骨骼肌,并发现保留所有测试的ECM成分(层粘连蛋白α2,总层粘连蛋白,纤连蛋白胶原蛋白I和胶原蛋白IV)。该方案包括三个步骤:渗透休克细胞裂解(低渗缓冲液),质膜溶解和蛋白质解离(0.05%十二烷基硫酸钠[SDS])和DNA的酶促破坏(DNase处理)。据我们所知,这是第一个建立的小鼠胎儿骨骼肌去细胞化的方案。

为了使用该3D体外系统研究LAMA2-CMD,在脱细胞后维持层粘连蛋白α2链至关重要。因此,实施了优化方案,其中测试了不同的洗涤剂(SDS和Triton X-100)和浓度(0.02%,0.05%,0.1%,0.2%和0.5%)(数据未显示)。细胞去除和保存层粘连蛋白α2蛋白的最佳选择是0.05%SDS。C2C12细胞是一种成熟的肌母细胞系1617用于接种dECM。这些细胞侵入dECM,在这些支架内增殖和分化,合成新的ECM蛋白。这种3D体外模型的成功生产提供了一种新方法来理解胎儿肌生成,LAMA2-CMD的发作所涉及的分子和细胞过程,并且可以扩展到ECM和骨骼肌细胞之间的通信被破坏的其他肌肉疾病。

Protocol

所描述的所有方法均已获得里斯本大学理学院动物福利委员会(ORBEA)和兽医协会(DGAV;ref. 0421/000/000/2022)的批准,并符合欧洲指令2010/63 / EU。 1. 脱细胞缓冲液和试剂的制备 注意:除非另有说明,否则在脱细胞方案期间使用的所有溶液都应通过高压灭菌灭菌并储存长达3个月。 通过添加 10 mM 的氯化钠 (NaCl)、2.68 mM 的氯化钾 (KCl)?…

Representative Results

去细胞化方案的目标是产生与天然组织组成非常相似的dECM。为了确定去细胞化过程的有效性,采用了各种方法,包括检查组织形态,测量DNA水平,染色F-肌动蛋白以及使用免疫组织化学和蛋白质印迹技术分析关键ECM组分。具体而言,分析了骨骼肌组织的五种主要ECM成分。 在整个协议中,样品的外观会发生变化(图1B 1-4</str…

Discussion

ECM是一个复杂的大分子网络,存在于所有组织中,在调节细胞行为和功能方面起着至关重要的作用2。ECM充当细胞附着的物理支架,并提供主动调节细胞过程(如增殖,运动,分化和凋亡)的线索。因此,ECM的正确形成和维持对于发育和体内平衡都至关重要1。

虽然2D细胞培养模型已被广泛使用,但它们正越来越多地被更先进的3D平台所取代?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作由法国反肌病协会(AFM-Téléthon;合同号23049),MATRIHEALTH项目和cE3c单位资助UIDB / 00329 / 2020资助。我们要感谢我们的捐助者Henrique Meirelles选择支持MATRIHEALTH项目。这项工作得益于科学学院显微镜设施的基础设施,该设施是葡萄牙生物成像平台的一个节点(参考PPBI-POCI-01-0145-FEDER-022122),我们感谢路易斯·马克斯在图像采集和处理方面的帮助。最后,我们感谢玛尔塔·帕尔马的技术支持和我们的研究团队的慷慨贡献。

Materials

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

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Gameiro dos Santos, P., Soares, A. R., Thorsteinsdóttir, S., Rodrigues, G. Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture. J. Vis. Exp. (193), e65069, doi:10.3791/65069 (2023).

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