The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.
这个协议描述了一种三维(3D)的生成体外肝模型及应用病毒载体系统的研究和开发。该模型是通过与人肝细胞系再植去细胞的大鼠肝脏的细胞外基质获得。该模式允许在血管三维电池系统的研究,随着人们动物替代有潜在危害的实验。另一个优点是模型,这是更接近于人类生理比动物模型的人源化的性质。
在这项研究中,我们证明这种肝模型与腺伴随病毒(AAV载体)衍生的病毒载体的转导。与媒体供应3D模型肝脏灌注电路提供了一种简单的方法来应用向量。该系统允许肝脏的主要代谢参数的监控。对于最后的分析,组织样品可采取确定recellulariza的程度通过组织学技术化。交付的转基因的病毒载体和表达的分布可以通过定量PCR(qPCR的),免疫印迹和免疫组织化学进行分析。在基础研究和在基因治疗应用中的发展的矢量模型的许多应用可以设想,包括新颖的抗病毒治疗学的发展,癌症研究,和病毒载体的研究及其潜在的副作用。
Most current biomedical research relies on one of two approaches, either two-dimensional (2D) cell culture experiments or animal models, which are three-dimensional (3D) by their very nature. However, these approaches have some severe drawbacks. Cells grown in 2D culture have been shown to differ in gene expression patterns and cell physiology from those cultivated under 3D conditions.1 Animal models, in addition to being associated with ethical concerns, often do not model human physiology well. Although the lack of obvious toxic effects of a compound must be confirmed in animal models prior to the first dosing in humans, multiple cases have been documented in which severe, sometimes fatal, adverse effects have occurred in clinical trials.2
To overcome these shortcomings, humanized 3D ex vivo organ models have become important research tools. When cultivated under suitable conditions, cells self-assemble into 3D structures known as spheroids. However, these spheroids lack a vascular system, which limits the distribution of small molecular compounds, large biologics and viral vectors alike. For example, adenoviral vectors only transduced the outer cell layers of spheroids prepared from human glioblastomas.3 A solution to this problem is the use of an organ model containing a vascular system. To this end, the organ of interest can be explanted from an animal, and the animal cells can be replaced by human cells. Various methods for decellularization of animal livers by treatment with detergents or sodium cholate have been described.4-6 The resulting extracellular matrix (ECM) harbors cytokines and growth factors which regulate various cellular processes.7 It can be used as a scaffold for recellularization with human cells to obtain a functional organ model.
In a recent study, we used a humanized 3D liver model to study distribution and transgene expression of an adeno-associated virus (AAV) vector.8 AAV vectors belong to the most promising viral vectors for gene therapeutic applications.9 The first, and to date only, approved gene therapeutic intervention in the Western world uses an AAV vector for the transfer of lipoprotein lipase.10
这里所描述的3D重建肝脏提供了一个模型,研究了人性化的系统病毒载体。与人类肝癌细胞株大鼠肝脏ECM的复育生成血管系统,允许大生物制品的研究。这些结果提供了一个验证的概念,即重构的肝脏模型可以用病毒载体可以有效地转导。
对于此处所示的实验中,肝的每一个叶是由AAV载体转导的。然而,在一些初步实验中,单叶未用细胞重新填充。因此重要的是防止细胞碎片或其它部件从阻塞血管系统。测试所有叶是否可灌注,无毒的染料,如酚红可以通过肝脏模型被刷新。
另一个关键问题是保持肝脏支架无菌。虽然用乙醇或抗生素治疗disadvantageous为血管系统,具有γ射线的细胞外基质的照射保留了容器和灭菌的样品。
此外,外植肝脏的大小有所不同,以便用于recellularization程序和复育时间的细胞的数目可能需要进行调整,以获得可再现的结果。
在本研究中使用的大鼠肝脏是比较大的,并需要大量的细胞和测试试剂( 例如,AAV载体)的。此外,复育过程花了两个多星期。这限制了可使用合理的努力来完成重复的数目。我们目前正在建立小鼠肝脏,这是只有大约一大鼠肝脏体积的第五模式,允许使用较少的细胞和少测试试剂。虽然细胞数的比例的缩小似乎是合理的,确切的数额需要furt待确定她的实验。
该模型的另一个缺点是使用了肝癌HepG2细胞的。实验正在进行开发利用从诱导多能干细胞分化的肝细胞,这将提供一个更生理相关模型。此外,肝由多种细胞类型的除了肝细胞, 如枯否氏细胞和sinosoids。我们假设不同的细胞类型将重新填充它们的天然环境时的ECM与多种细胞类型recellularized。
三维肝模式结合几个优点。常规的体内模型的主要缺点是,动物生理学,从人体生理学很大不同。因此人类患者的治疗的毒副作用可能仍然未被发现。这个缺点可以通过重建与人体细胞的三维肝模型更密切地反映胡生物学被克服男人的患者。
肝脏模型的第二个优点是其对动物福利的贡献。虽然所需的重建实验动物成分,该方法仍然遵循的3R原则(替换,还原,细化)的目的,如剩余的动物可以用来处死,对于其他动物的实验中, 即不需要附加的动物和这种方法完全避免了这是经常与体内实验相关动物的痛苦。球粒的替代工具,研究在3D系统的细胞过程。然而,球状体不血管化,使得大的物质和生物不深入到结构的内部部分。这些问题已经克服了血管的三维肝模型。
在这里描述的实验中,AAV载体进行了研究,因为它们是最有希望的候选基因治疗中应用程序。众多的基因治疗方法的目标是靶向肝脏, 例如,对于具有肝炎病毒或α-1-抗胰蛋白酶缺乏的感染的治疗中,3D肝脏可在这些AAV载体发展的过程中被使用。它是,当然,也适用于其它嗜肝病毒载体, 例如腺病毒载体的研究。此外,它可以被用来研究传染性肝炎病毒如乙型或丙型肝炎病毒。它可以,例如,被用来设计新的抗病毒策略。此外,三维器官模型代表有前途的工具来开发新的细胞生长抑制疗法,治疗癌症,并开展毒理学研究。从长远来看,人工肝可在再生医学作为移植使用。综合来看,3D肝模型提供了一个广泛的感染生物学和应用生物医学研究等领域。
The authors have nothing to disclose.
The authors thank Bernd Krostitz for technical assistance, Radoslaw Kedzierski for initial contributions to the project, Erik Wade for proofreading and giving helpful comments, and Prof. Heike Walles for providing the bioreactor and sharing her valuable experience with organ decellularization. We are also thankful for funding of the project and publication by the Berlin University of Technology.
Incubator | Fraunhofer | / | |
Peristaltic Pump | Fraunhofer | / | |
Flange with groove | Duran | 2439454 | modified by gaffer |
O-Ring Transparent | Duran | 2922551 | |
Quick Release Clamp | Duran | 2907151 | |
Flat Flange Lid | Duran | 2429857 | modified by gaffer |
Screw thread Tube | Duran | 2483802 | modified by gaffer |
Screw thread Tube | Duran | 2483602 | modified by gaffer |
Silicone sealing Ring | Duran | 2862012 | |
Screw Cap | Duran | 2924013 | |
Screw Cap | Duran | 2924008 | |
Screw Cap with aperture | Duran | 2922709 | |
Screw Cap with aperture | Duran | 2922705 | |
Filter | Sarstedt | 831,826,001 | |
Silicone Tubing | VWR | 228-1500 | |
Tube connector | Ismatec | ISM556A | |
Biocompatible Tubing | Ismatec | SC0736 | |
T175 culture flasks | Greiner bio-one | 660 160 | |
RPMI 1640 | BioWest SAS (Th. Geyer) | L0501-500 | |
glutamine | BioWest SAS (Th. Geyer) | X0551-100 | |
Trypsin | BioWest SAS (Th. Geyer) | L0940-100 | |
penicillin/ streptomycin | BioWest SAS (Th. Geyer) | L0022-100 | |
fetal calf serum | cc pro | S-10-M | |
Tissue-Tek O.C.T. | Weckert-Labortechnik | 600001 | |
HepG2 | DSMZ | ACC 180 | |
Cryomold 15x15x5mm | Sakura | 4566 | |
Biopsy punch 4mm | pfm medical | 48401 | |
Nucleospin miRNA | Macherey & Nagel | 740971.10 | |
Nucleospin RNA/DNA Buffer Set | Macherey & Nagel | 740944 |