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Bioengineering

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Published: July 25, 2016
doi:
10.3791/54148
, ,
1Center for Vaccine Development, Department of Pediatrics,University of Maryland School of Medicine, 2Mucosal Immunology and Biology Research Center,Massachusetts General Hospital for Children

Summary

细胞在三维（3-D）的环境中生长代表的2-D的环境中（ 例如 ，瓶或菜）在细胞培养的显着改进。这里，我们描述根据通过旋转壁上容器（RWV）生物反应器提供微重力培养人肠粘膜的多细胞三维器官模型的发展。

Abstract

因为在一个三维（3-D）的环境中生长的细胞具有弥合的2-D的环境中的细胞培养的许多间隙（ 例如 ，瓶或碗碟）的潜力。事实上，人们普遍认识到，在烧瓶或菜肴生长细胞倾向于去分化并失去其最初来源的组织的专门特征。目前，主要有两种类型的3-D培养系统的模拟天然细胞外基质（ECM），其中将细胞接种到支架：（一）静态模型和使用生物反应器（B）中的模型。第一个突破是静态的三维模型。使用生物反应器，如旋转壁上容器3-D模型（RWV）生物反应器是一个较新的发展。在RWV生物反应器的原始概念是美国宇航局约翰逊航天中心在90年代初开发的，被认为是克服静态模型的局限性，如缺氧，坏死核心的发展。该RWV生物反应器可以规避日是通过提供流体动力学，允许营养物和氧的高效的扩散问题。这些生物反应器组成，其用于支持和旋转培养容器的两个不同的格式，可以通过通气源类型而不同的转子碱：（1）缓慢车削外侧容器（STLVs）与在中心同轴氧合器，或（2通过一台，硅橡胶气体转移膜）高宽比容器（HARVs）与充氧。这些血管允许有效的气体传递，同时避免气泡形成和随之而来的湍流。这些条件导致在层流和最小的剪切力模型降低了培养容器内的重力（失重）。在这里，我们描述由RWV生物反应器提供了一种肠上皮细胞系和原代人淋巴细胞，微重​​力下培养的内皮细胞和成纤维细胞构成的人体肠道黏膜的多细胞三维器官模型的发展。</ P>

Introduction

在构建三维模型首次突破据报道，在80年代初，当科学家们开始研究不同类型的支架（ 例如 ，层粘连蛋白，胶原蛋白I型，Ⅳ型胶原和纤维连接蛋白）和生长因子鸡尾酒改善细胞-细胞和的“静态”3-D模型1-7的ECM相互作用。此后，随着这些模型的主要问题一直是在营养物质的传递和氧的介质和组织构建8内的限制。在对比中，其接收从周围的血管网络的营养素的稳定流量和氧的体内环境的细胞，这些模型的静态性质阻碍他们的向细胞的有效分配。例如，在体外静态模型生成大小超过几毫米细胞聚集体将总是开发缺氧，坏死芯件9。该RWV生物反应器可以解决这个问题通过提供流体动力学，允许营养物和氧10-12的有效扩散。然而，到目前为止，使用RWV生物反应器的工作已被限制为包含一种或两种的细胞类型13-17。此外，代替类似于天然组织的空间取向，这些细胞形成细胞聚集体。这些限制的主要原因是缺乏能够把电池于一体的综合时尚支架。在RWV生物反应器迄今所使用的支架组成，除了少数例外16-18，主要用于合成微珠，管状圆筒或小片13-15,19-23的。这些是刚性的材料，其组成和灵活性不能被操纵，并且其中细胞附着到其表面上。因此，这是不可能的，这些模型将提供在其中评估系统，以综合的方式，（ 例如 ，成纤维细胞，免疫细胞和内皮细胞）的各种细胞成分如基质细胞那个SHOULD被支架内分散地模拟人体组织。

在这里，我们描述了一种肠上皮细胞系和原代人淋巴细胞，内皮细胞构成的人体肠道黏膜的多细胞三维器官模型的开发，和成纤维细胞24。这些细胞培养微重力下的生物反应器RWV提供13,25-30。在我们的3-D模型，则ECM具有许多不同的特性，例如类似培养基的重量摩尔渗透压浓度（ 例如 ，在培养过程中可以忽略的扩散限制），并掺入细胞和其它相关的细胞外基质蛋白的能力，以及在在生物反应器24中使用适当的刚度。生物系统是非常复杂的，而且在过去的几年里，一直在研究黏膜对与周围环境细胞的相互作用的考试的重点的转变，而不是隔离和研究它们ñ。特别是，细胞-细胞相互作用的影响肠细胞的存活和分化的重要性是有据可查的31-34。具体来说，上皮细胞和自己的优势之间的交流对上皮细胞扩增和分化35深远的影响。事实上，它已被广泛接受，不仅细胞 – 细胞，而且细胞 – 细胞外基质相互作用是上皮细胞中的3-D培养模型的维持和分化至关重要。以前的研究已经表明，肠ECM蛋白如胶原蛋白I 24,36,37，层粘连蛋白38和纤连蛋白39在影响肠上皮细胞获得类似于天然粘膜空间定向工具。因此，新技术的发展，象我们的3-D模型24，可以模仿所需肠道的表型多样性如果研究人员打算重新创建复杂的细胞和结构建筑和肠道微环境的功能。这些模型表示在新的口服药物和疫苗候选的开发和评估的一个重要工具。

Protocol

伦理声明：从参与协议号HP-00040025-1志愿者收集的所有血液样本。马里兰机构审查委员会的批准大学的这个协议和授权的血液样本的收集从包括在这个手稿研究健康志愿者。这项研究的目的是解释志愿者，以及所有的志愿者给予知情，抽血前签署同意书。 注：请参阅表1为中补充的准备。 见表2为三维培养基的制备。 1.培养容器的制备旋50毫?…

Representative Results

先前我们已经设计了人肠黏膜包括肠上皮细胞系和原代人淋巴细胞，微重力条件下24（ 图1）下培养的内皮细胞和成纤维细胞的多细胞三维器官模型。成纤维细胞和内皮细胞包埋在我矩阵附加肠基底膜蛋白45（ 即 ，层粘连蛋白，胶原蛋白IV，纤连蛋白和硫酸肝素蛋白聚糖）富含胶原，并加入到RWV生物反应器。经过10 – 15天，组织染色…

Discussion

在这个手稿，我们描述了人肠黏膜由倍数细胞类型，包括原代人淋巴细胞，成纤维细胞和内皮细胞，以及肠上皮细胞系24的生物工程化的模型的开发。在这个3-D模型，细胞在富含胶原蛋白的细胞外基质中的微重力条件下24培养。

如先前所描述，该模型的主要特征是：（i）以模仿上皮组织单层组织的能力，（ⅱ）上皮细胞，紧密连接，?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported, in part, by NIAID, NIH, DHHS federal research grants R01 AI036525 and U19 AI082655 (CCHI) to MBS and by NIH grant DK048373 to AF. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy And Infectious Diseases or the National Institutes of Health.

Materials

Quad Rotator/Independent Rotating Wall Vessel (RWV) bioreactor  Synthecon RCCs-4DQ For up to 4 vessels. Models with more or less vessels are also available.
Disposable 50 ml-vessel Synthecon D-405 Box with 4 vessels
HCT-8 epithelial cells  ATCC CCL-244
CCD-18Co Fibroblasts  ATCC CRL-1459
Human Umbilical Vein Endothelial Cells ATCC CRL-1730 HUVEC
Fibroblast Growth Factor-Basic  Sigma F0291 bFGF
Stem Cell Factor  Sigma S7901 SCF
Hepatocyte Growth Factor  Sigma H1404 HGF
Endothelin 3 Sigma E9137
Laminin Sigma L2020 Isolated from mouse Engelbreth-Holm-Swarm tumor
Vascular Endothelial Growth Factor  Sigma V7259 VEGF
Leukemia Inhibitory Factor  Santa Cruz sc-4377 (LIF
Adenine Sigma A2786
Insulin Sigma I-6634
3,3',5-triiodo-L-thyronine  Sigma T-6397 T3
Cholera Toxin Sigma C-8052
Fibronectin BD 354008 Isolated from human plasma
apo-Transferrin Sigma T-1147
Heparin Sigma H3149
Heparan sulfate  proteoglycan Sigma H4777 Isolated from basement membrane of mouse  Engelbreth-Holm-Swarm tumor
Collagen IV Sigma C5533 Isolated from human placenta
Heat-inactivated fetal bovine serum  Invitrogen 10437-028
D-MEM, powder Invitrogen 12800-017
10% formalin–PBS  Fisher Scientific SF100-4
Bovine type I collagen  Invitrogen A1064401
Trypsin-EDTA  Fisher Scientific MT25-052-CI
Sodium pyruvate Invitrogen 11360-070
Gentamicin  Invitrogen 15750-060
Penicillin/streptomincin  Invitrogen 15140-122
L-Glutamine Invitrogen 25030-081
Hepes Invitrogen 15630-080
Ham's F-12 Invitrogen 11765-054
Basal Medium Eagle Invitrogen 21010-046 BME
RPMI-1640 Invitrogen 11875-093
Endothelial Basal Medium Lonza CC-3156 EBM-2
Endothelial cell growth supplement Millipore 02-102 ECGS
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Tags

3D Organotypic ModelHuman Intestinal MucosaMicrogravityRotating Wall Vessel BioreactorsEndothelial CellsFibroblastsCollagenLamininFibronectinHeparin Sulfate ProteoglycanCell Culture

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Salerno-Goncalves, R., Fasano, A., Sztein, M. B. Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity. J. Vis. Exp. (113), e54148, doi:10.3791/54148 (2016).
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