Summary

小鼠胚胎肾的解剖与培养

Published: May 17, 2017
doi:

Summary

该方案描述了一种从小鼠胚胎中分离和培养元肾小球的方法。

Abstract

该方案的目的是描述一种用于解剖,分离和培养小鼠睾丸的初步方法。

在哺乳动物肾脏发育期间,两个祖细胞组织,输尿管芽和间质间质介导并相互诱导细胞机制,最终形成肾脏的收集系统和肾单位。由于哺乳动物胚胎生长在宫内,因此观察者无法接近,已经开发出器官培养。通过这种方法,可以研究肾脏器官发生期间的上皮 – 间质相互作用和细胞行为。此外,可以研究先天性肾脏和泌尿生殖道畸形的起源。经仔细解剖后,将met ric r are are are are onto onto that that on。。。。。。。。。。。。。。。。。。。。。。。但是,必须意识到条件是人造并可能影响组织中的新陈代谢。此外,由于外植体中存在的细胞外基质和基底膜,测试物质的渗透可能受到限制。

器官文化的一个主要优点是实验者可以直接进入器官。该技术便宜,简单,并且可以进行大量的修改,例如添加生物活性物质,遗传变异体的研究以及先进成像技术的应用。

Introduction

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other’s cell morphogenesis.

In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Saxén, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Saxén’s technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.

Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.

In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

Protocol

根据瑞典法规和欧盟立法(2010/63 / EU)维护小鼠。所有程序均按照瑞典伦理委员会的准则进行(许可证C79 / 9,C248 / 11和C135 / 14)。海德堡大学涉及动物科目的程序已获得卡尔斯鲁厄大学和海德堡大学动物福利干事的批准。 1.培养试剂和材料的制备注意:使用层流罩以尽量减少污染。 在解剖当天,通过在无菌磷酸盐缓冲盐水(PBS)中将其与抗人Fc?…

Representative Results

肾脏肾脏成纤维细胞源于怀孕的黑-65近交小鼠E11.5,并进行培养。 3天后,输尿管芽分支达5次,最终导致T型输尿管芽的分枝。拍摄每个外植体,并对片段和端点的数量进行量化,以确定分枝代数并计算每个分枝的端点数( 图1 )。 ImageJ( rd代;只有8%的处理的外植体达到第 4代,而对照外植体的比例为35%)( 图1b )。因此,每个…

Discussion

该手稿描述了一种从小鼠胚胎中分离出发育中的甲基化成核细胞并培养器官基因的方法。这种方法是由Grobstein 8和Saxén9,10开发的一种标准技术,并被许多其他11,12修改和修改。该方法的成功主要取决于解剖的持续时间,随着剥离时间的延长,外植体存活和诱导电位降低。在清洁周围组织的肾脏基础时,还要注意不要损伤间质。间质间质的损伤通常是外植体生长?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

作者感谢Leif Oxburgh和Derek Adams慷慨分享他们的知识,Leif Oxburgh对手稿有帮助的评论,StefanWölfl和UlrikeMüller的技术支持和Saskia Schmitteckert,Julia Gobbert,Sascha Weyer和Viola Mayer在实验室。这项工作得到了“生物学家公司” (CP)发展部门的支持。

Materials

DMEM/F-12 Thermo Fisher Scientific 21331020
Penicillin-Streptomycin (10,000 U/mL) Thermo Fisher Scientific 15140148
GlutaMAX Supplement Thermo Fisher Scientific 35050061
DPBS, calcium, magnesium Thermo Fisher Scientific 14040117 use for dissection
holo-Transferrin human Sigma-Aldrich T0665
Insulin-Transferrin-Selenium (ITS -G) (100X) Thermo Fisher Scientific 41400045
Paraformaldehyde Sigma-Aldrich 158127
Amphotericin B solution Sigma-Aldrich A2942
Triton X-100 Sigma-Aldrich X100
Sodium azide Sigma-Aldrich S8032
Thimerosal Sigma-Aldrich T5125
Propyl gallate Sigma-Aldrich 2370
Mowiol 4-88 Sigma-Aldrich 81381
Glycerol Sigma-Aldrich G5516
Biotinylated Dolichorus Biflorus Agglutinin Vector Laboratories B-1035
Alexa488 conjugated Streptavidin Jackson Immuno Research 016-540-084
Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF R&D Systems 496-EB
Recombinant Human IgG1 Fc, CF R&D Systems 110-HG-100
Goat Anti-Human IgG Fc Antibody R&D Systems G-102-C
Phosphate buffered saline tablets Sigma-Aldrich P4417 use for fixation and immunostaining
Dumont #5, biologie
tips, INOX, 11cm
agnthos.se 0208-5-PS 2 pairs of forceps are needed
Iris scissors, straight, 12cm agnthos.se 03-320-120
Dressing Forceps,
straight, delicate, 13cm
agnthos.se 08-032-130
Petri dishes Nunclo Delta treated Thermo Fisher Scientific 150679
TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 µm, 13 mm, white, plain MerckMillipore TMTP01300
Nunclon Multidishes
4 wells, flat bottom
Sigma-Aldrich D6789-1CS
Microscope cover glass24x50mm thickn. No.1.5H 0.17+/-0.005mm nordicbiolabs 107222
Cover glasses No.1.5, 18x18mm nordicbiolabs 102032
Slides ~76x26x1, 1/2-w. ground plain nordicbiolabs 1030418
VWR Razor Blades VWR 55411-055
50 mL centrifuge tubes Sigma-Aldrich CLS430828
15 mL centrifuge tubes Sigma-Aldrich CLS430055
Whatman prepleated qualitative filter paper, Grade 113V, creped Sigma-Aldrich WHA1213125
Fixed stage research mircoscope Olympus BX61WI
Black 6 inbred mice, male, C57BL/6NTac Taconic B6-M
Black 6 inbred mice,female, C57BL/6NTac Taconic B6-F
Greenough Stereo Microscope Leica Leica S6 E

Referencias

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Aresh, B., Peuckert, C. Dissection and Culture of Mouse Embryonic Kidney. J. Vis. Exp. (123), e55715, doi:10.3791/55715 (2017).

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