Name: efficient vascularization of kidney organoids through intracelomic transplantation in chicken embryos
Uploaded: 2023-02-17T13:10:00
Description: Watch this Scientific Journal Video about Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos at JoVE.com

我们感谢George Galaris（LUMC，莱顿，荷兰）在鸡胚注射方面的帮助。我们感谢Saskia van der Wal-Maas（荷兰莱顿LUMC解剖与胚胎学系），Conny van Munsteren（荷兰莱顿LUMC解剖与胚胎学系），Manon Zuurmond（LUMC，莱顿，荷兰）和Annemarie de Graaf（LUMC，莱顿，荷兰）的支持。M. Koning由'Nephrosearch Stichting tot steun van het wetenschappelijk onderzoek van de afdeling Nierziekten van het LUMC'提供支持。这项工作部分得到了莱顿大学基金“Jaap de Graeff-Lingling Wiyadhanrma Fund”GWF2019-02 的支持。这项工作得到了再生医学跨境（RegMedXB）和Health Holland，Top Sector Life Sciences & Health的合作伙伴的支持。C.W. van den Berg和T.J. Rabelink由诺和诺德基金会干细胞医学中心（reNEW）支持，诺和诺德基金会干细胞医学中心由诺和诺德基金会资助（NNF21CC0073729）支持。

根据荷兰法律，这项研究不需要动物福利委员会的批准。
1. 制备用于移植的hiPSC来源的肾类器官
<ol><li>使用高里等人开发的协议将hiPSCs与肾脏类器官分化4，18，31。按照该协议在具有0.4μm孔的聚酯细胞培养插入物（细胞培养插入物）上培养类器官，直到分化的第7 + 12天。每个细胞培养插入片?...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-edited+human+kidney+organoids+reveal+mechanisms+of+disease+in+podocyte+development.">Gene-edited human kidney organoids reveal mechanisms of disease in podocyte development.</a> Stem Cells. 35 (12), 2366-2378 (2017).</li><li>Takasato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organoids+from+human+iPS+cells+contain+multiple+lineages+and+model+human+nephrogenesis.">Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.</a> Nature. 526 (7574), 564-568 (2015).</li><li>Hale, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+organoid-derived+human+glomeruli+for+personalised+podocyte+disease+modelling+and+drug+screening.">3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening.</a> Nature Communications. 9 (1), 5167 (2018).</li><li>Vanslambrouck, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+metanephric+specification+to+functional+proximal+tubule+enables+toxicity+screening+and+infectious+disease+modelling+in+kidney+organoids.">Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.</a> Nature Communications. 13 (1), 5943 (2022).</li><li>Tanigawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+from+nephrotic+disease-derived+iPSCs+identify+impaired+NEPHRIN+localization+and+slit+diaphragm+formation+in+kidney+podocytes.">Organoids from nephrotic disease-derived iPSCs identify impaired NEPHRIN localization and slit diaphragm formation in kidney podocytes.</a> Stem Cell Reports. 11 (3), 727-740 (2018).</li><li>Forbes, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-iPSC-derived+kidney+organoids+show+functional+validation+of+a+ciliopathic+renal+phenotype+and+reveal+underlying+pathogenetic+mechanisms.">Patient-iPSC-derived kidney organoids show functional validation of a ciliopathic renal phenotype and reveal underlying pathogenetic mechanisms.</a> American Journal of Human Genetics. 102 (5), 816-831 (2018).</li><li>Freedman, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+kidney+disease+with+CRISPR-mutant+kidney+organoids+derived+from+human+pluripotent+epiblast+spheroids.">Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.</a> Nature Communications. 6, 8715 (2015).</li><li>Cruz, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+cystogenesis+reveals+a+critical+role+of+microenvironment+in+human+polycystic+kidney+disease.">Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease.</a> Nature Materials. 16 (11), 1112-1119 (2017).</li><li>Gupta, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+injury+and+repair+in+kidney+organoids+reveals+that+homologous+recombination+governs+tubular+intrinsic+repair.">Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair.</a> Science Translational Medicine. 14 (634), eabj4772 (2022).</li><li>Hiratsuka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid-on-a-chip+model+of+human+ARPKD+reveals+mechanosensing+pathomechanisms+for+drug+discovery.">Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery.</a> Scencei Advances. 8 (38), eabq0866 (2022).</li><li>Dorison, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organoids+generated+using+an+allelic+series+of+NPHS2+point+variants+reveal+distinct+intracellular+podocin+mistrafficking.">Kidney organoids generated using an allelic series of NPHS2 point variants reveal distinct intracellular podocin mistrafficking.</a> Journal of the American Society of Nephrology. , (2022).</li><li>Eremina, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular-specific+alterations+of+VEGF-A+expression+lead+to+distinct+congenital+and+acquired+renal+diseases.">Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases.</a> The Journal of Clinical Investigation. 111 (5), 707-716 (2003).</li><li>Kitamoto, Y., Tokunaga, H., Tomita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+is+an+essential+molecule+for+mouse+kidney+development:+glomerulogenesis+and+nephrogenesis.">Vascular endothelial growth factor is an essential molecule for mouse kidney development: glomerulogenesis and nephrogenesis.</a> The Journal of Clinical Investigation. 99 (10), 2351-2357 (1997).</li><li>Sison, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+structure+and+function+require+paracrine,+not+autocrine,+VEGF-VEGFR-2+signaling.">Glomerular structure and function require paracrine, not autocrine, VEGF-VEGFR-2 signaling.</a> Journal of the American Society of Nephrology. 21 (10), 1691-1701 (2010).</li><li>Ryan, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+deficiencies+in+renal+organoids+and+ex+vivo+kidney+organogenesis.">Vascular deficiencies in renal organoids and ex vivo kidney organogenesis.</a> Developmental Biology. 477, 98-116 (2021).</li><li>vanden Berg, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+subcapsular+transplantation+of+PSC-derived+kidney+organoids+induces+neo-vasculogenesis+and+significant+glomerular+and+tubular+maturation+in+vivo.">Renal subcapsular transplantation of PSC-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo.</a> Stem Cell Reports. 10 (3), 751-765 (2018).</li><li>Sharmin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cell-derived+podocytes+mature+into+vascularized+glomeruli+upon+experimental+transplantation.">Human induced pluripotent stem cell-derived podocytes mature into vascularized glomeruli upon experimental transplantation.</a> Journal of the American Society of Nephrology. 27 (6), 1778-1791 (2016).</li><li>Bantounas, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functioning+nephrons+by+implanting+human+pluripotent+stem+cell-derived+kidney+progenitors.">Generation of functioning nephrons by implanting human pluripotent stem cell-derived kidney progenitors.</a> Stem Cell Reports. 10 (3), 766-779 (2018).</li><li>vanden Berg, C. W., Koudijs, A., Ritsma, L., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+size-selective+glomerular+sieving+in+transplanted+human+induced+pluripotent+stem+cell-derived+kidney+organoids.">In vivo assessment of size-selective glomerular sieving in transplanted human induced pluripotent stem cell-derived kidney organoids.</a> Journal of the American Society of Nephrology. 31 (5), 921-929 (2020).</li><li>Asai, R., Bressan, M., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avians+as+a+model+system+of+vascular+development.">Avians as a model system of vascular development.</a> Methods in Molecular Biology. 2206, 103-127 (2021).</li><li>Jankovic, B. 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在这份手稿中，展示了鸡胚胎中hiPSC衍生的肾类器官的细胞内移植方案。移植后，类器官通过灌注的血管进行血管化，这些血管由人类器官来源和鸡来源的EC的组合组成。它们遍布整个类器官并侵入肾小球结构，使EC和足细胞之间的相互作用成为可能。先前表明，这导致类器官肾小球和管状结构的成熟增强28。移植非常有效，每个胚胎需要~5分钟，胚胎唯一需要的维护是定期补充培...

人类诱导的多能干细胞（hiPSC）来源的肾脏类器官已被证明具有发育研究的潜力1，2，3，4，毒性筛查5，6和疾病建模5，7，8，9，10，11，12，13.然而，由于缺乏血管网络，它们对这些和最终临床移植目的的适用性受到限制。在胚胎肾脏发育过程中，足细胞、系膜细胞和血管内皮细胞 （EC） 相互作用形成肾小球的复杂结构。没有这种相互作用，由足细胞、肾小球基底膜（GBM）和EC组成的肾小球滤过屏障就无法正常发育14，15，16。尽管体外的肾脏类器官确实含有一些EC，但这些EC无法形成适当的血管网络并随着时间的推移而减少17。因此，类器官仍然不成熟也就不足为奇了。小鼠移植诱导肾脏类器官18，19，20，21的血管化和成熟。不幸的是，这是一个劳动密集型过程，不适合分析大量类器官。
一个多世纪以来，鸡胚一直被用来研究血管形成和发育22。它们易于接近，需要低维护，缺乏功能齐全的免疫系统，并且在打开蛋壳23，24，25，26后可以正常发育。类器官在其脉络膜尿囊膜（CAM）上的移植已被证明可导致血管形成27。然而，CAM上的移植持续时间以及移植物的成熟水平受到CAM形成的限制，CAM形成需要直到胚胎第7天才能完成。因此，最近开发了一种通过鸡胚胎中鞘内移植来有效血管化和成熟肾脏类器官的方法28。自 1930 年代以来，众所周知，鸡胚胎的天体腔是胚胎组织分化的有利环境29，30。它可以在胚胎发育的早期进入，并允许移植物在各个方向上相对无限地扩张。
本文概述了在第 4 天鸡胚胎的天体腔中移植 hiPSC 衍生的肾类器官的方案。该方法在8天内诱导类器官的血管形成和增强成熟。在牺牲胚胎之前注射荧光标记的晶状体凝集素（LCA），可以通过共聚焦显微镜可视化类器官内的灌注血管。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;m filter: Whatman Puradisc 30 syringe filter 0.2 &#181;m</td>
			<td>Whatman</td>
			<td>10462200</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm glass bottom dishes&#160;</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>A5177-5EA</td>
			<td>Contains silicone tubes, mouth piece and connector</td>
		</tr>
		<tr>
			<td>Confocal microscope: Leica White Light Laser Confocal Microscope&#160;</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps, simple type. Titanium, curved, with fine sharp tips</td>
			<td>Hammacher Karl</td>
			<td>HAMMHTC091-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps, simple type. Titanium, straight, with fine sharp tips</td>
			<td>Hammacher Karl</td>
			<td>HAMMHTC090-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Wild Heerbrugg</td>
			<td>355110</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors, curved, OP-special, extra sharp/sharp</td>
			<td>Hammacher Karl</td>
			<td>HAMMHSB391-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey-&#945;-mouse Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-212-02</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Donkey-&#945;-sheep Alexa Fluor 647</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21448</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Double edged stainless steel razor blades</td>
			<td>Electron Microsopy Sciences</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium (DPBS-/-)</td>
			<td>ThermoFisher Scientific</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium (DPBS+/+)</td>
			<td>ThermoFisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg cartons or custom made egg holders&#160;</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertilized white leghorn eggs (Gallus Gallus Domesticus)&#160;</td>
			<td>Drost Loosdrecht B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Elbanton BV</td>
			<td>ET-3 combi</td>
			<td></td>
		</tr>
		<tr>
			<td>Lotus Tetragonolobus lectin (LTL) Biotinylated</td>
			<td>Vector Laboratories</td>
			<td>B-1325</td>
			<td>dilution 1:300</td>
		</tr>
		<tr>
			<td>Micro scissors, straight, sharp/sharp, cutting length 10 mm</td>
			<td>Hammacher Karl</td>
			<td>HAMMHSB500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcapillaries: Thin wall glass capillaries 1.5 mm, filament</td>
			<td>World Precision Instruments</td>
			<td>TW150F-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument Company</td>
			<td>Model P-97</td>
			<td>We use the following settings: Heat 533, Pull 60, Velocity 150, Time 200</td>
		</tr>
		<tr>
			<td>Microscalpel holder: Castroviejo blade and pins holder, 12 cm, round handle, conical 10 mm jaws.</td>
			<td>Euronexia</td>
			<td>L-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium: Prolong Gold Antifade Mountant&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Olivecrona dura dissector 18 cm&#160;</td>
			<td>Reda</td>
			<td>41146-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm&#160;</td>
			<td>Heathrow Scientific</td>
			<td>HS234526B</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin 5,000 U/mL</td>
			<td>ThermoFisher Scientific</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Perforated spoon&#160;</td>
			<td>Euronexia</td>
			<td>S-20-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60 x 15 mm&#160;</td>
			<td>CELLSTAR</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipettes&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>PP89SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified mouse anti-human CD31 antibody</td>
			<td>BD Biosciences</td>
			<td>555444</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>Rhodamine labeled Lens Culinaris Agglutinin (LCA)</td>
			<td>Vector Laboratories</td>
			<td>RL-1042</td>
			<td>This product has recently been discontinued. Vectorlabs does still produce Dylight 649 labeled LCA (DL-1048-1) and fluorescein labeled LCA (FL-1041-5)</td>
		</tr>
		<tr>
			<td>Sheep anti-human NPHS1 antibody</td>
			<td>R&#38;D systems</td>
			<td>AF4269</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>Sterile hypodermic needles, 19 G</td>
			<td>BD microlance</td>
			<td>301500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Alexa Fluor 405</td>
			<td>ThermoFisher Scientific</td>
			<td>S32351</td>
			<td>dilution 1:200</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD Emerald</td>
			<td>307731</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent tape&#160;</td>
			<td>Tesa</td>
			<td>4124</td>
			<td>Available at most hardware stores</td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire, 0.25 mm dia&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>010404.H2</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient vascularization of kidney organoids through intracelomic transplantation in chicken embryos

源自人类诱导的多能干细胞的肾脏类器官含有肾单位样结构，在一定程度上类似于成人肾脏中的结构。不幸的是，由于缺乏功能性脉管系统，因此体 外成熟有限，它们的临床适用性受到阻碍。在鸡胚胎的天体腔中移植肾脏类器官通过灌注血管诱导血管形成，包括肾小球毛细血管的形成，并增强其成熟。这种技术非常有效，可以移植和分析大量的类器官。本文描述了鸡胚胎中肾类器官的细胞内移植的详细方案，然后注射荧光标记的凝集素以染色灌注的脉管系统，并收集移植的类器官进行成像分析。该方法可用于诱导和研究类器官血管化和成熟，以寻找在 体外 增强这些过程和改善疾病建模的线索。

图1A总结了hiPSCs分化为肾类器官、受精鸡蛋孵化、肾类器官移植、LCA注射和类器官收集的方法和时间表。协调类器官分化和鸡蛋孵化的时间很重要，在孵化前 15 天开始分化。孵化第 0、3、4 和 12 天的操作由时间线下方的照片说明。类器官在分化的第7 + 12天移植到第4天（HH 23-24）鸡胚中。移植后8天将LCA注射到胚胎的静脉系统中，以染色灌注的脉管系统，然后牺牲胚胎并取回类...

Watch this Scientific Journal Video about Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos at JoVE.com

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

在这里，我们提出了在鸡胚胎的天体腔中移植肾脏类器官的详细方案。该方法在8天内诱导类器官的血管形成和增强成熟，可用于以有效的方式研究这些过程。

通过鸡胚胎的腹内移植对肾脏类器官进行有效的血管化

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain ...

体外培养的肾脏类器官仍然不成熟，限制了它们的适用性。并且这种腔内移植方法诱导血管形成并增强类器官的成熟。该程序非常有效，允许在一次实验中移植和分析大量类器官。在制备HIPSC衍生的肾脏类器官并孵化受精腿角卵后，在孵化的第三天在蛋壳中创建一个窗口。将一小块透明胶带放在鸡蛋的尖头上。用一把解剖剪刀的锋利末端敲击透明胶带中间的蛋壳，在蛋壳上打一个小孔。接下来，将 19 毫升注射器上的 45 号针头以 45 度角插入孔中。从卵子中吸出两到三毫升白蛋白，以降低卵子内的胚胎。用第二块透明胶带隐藏孔，然后将一大块透明胶带放在铅笔上，标记为鸡蛋朝上的一侧。用一把解剖剪刀的锋利末端敲击透明胶带中间的蛋壳，在蛋壳上打一个洞。用弯曲的解剖剪刀从这个洞的蛋壳上剪一个小圆形窗口。通过此窗口查看以找到胚胎。然后，扩大窗口以优化对胚胎的访问。用镊子去除任何可能落在胚胎顶部的大块蛋壳。用塑料移液管将几滴含有钙和镁的DPBS滴在胚胎上，然后用蛋壳将DPBS吸入移液管中，从而取出较小的碎片。使用塑料移液管，向鸡蛋中加入三滴DPBS，并补充0.5%青霉素链霉素。用一大块透明胶带小心地密封窗口，然后将鸡蛋放回孵化器中，直到孵化的第 4 天。用弯曲的解剖剪刀从窗户上剪下胶带。将鸡蛋放在橡胶架或鸡蛋盒上的解剖显微镜下。鸡胚现在处于汉堡-汉密尔顿阶段 23 至 24，左侧躺着，右侧面向观众。找到胚胎的右翼和腿芽，因为将在这两个肢芽之间评估腔肠。在右翼和腿芽之间的区域，用两对解剖镊子握住它们并轻轻地将它们拉向相反的方向，在卵黄膜、绒毛膜和羊膜上连续开一个开口。检查腔腔是否畅通无阻。用一对解剖镊子轻轻抓住翅膀和肢芽之间的体壁边缘，并将其稍微拉向观察者。腔腔必须清晰可见。小心地将钝而纤细的器械插入腔腔。使用硬脑膜解剖器将半个类器官放在蛋内尿囊顶部。使用解剖镊子，小心地抓住体壁的边缘并将其稍微拉向观察者，以使腔肠的开口可见。用钝钨丝和微型手术刀支架轻轻地将类器官移向并通过体壁上的开口进入腔。稍微颅骨推动类器官，使其卡在腔内。现在在翼芽后面可以看到它。使用塑料移液管将三滴DPBS添加到鸡蛋中。用一大块透明胶带小心地密封窗口，然后将鸡蛋放回孵化器中，直到孵化的第 12 天。用弯曲的解剖剪刀从窗户上剪下胶带。将鸡蛋放在橡胶架中的解剖显微镜下。评估脉管系统并定位静脉，静脉可通过略亮的红色与动脉区分开来。为了改善对脉管系统的访问，请用弯曲的解剖剪刀剪开窗户，小心地扩大窗户。选择静脉后，将微毛细管针尖以 0 到 20 度角插入静脉。通过轻轻地将尖端从一侧移动到另一侧来确保针头在静脉中。轻轻稳定地吹入注射系统以注射晶状体凝集素。然后将鸡蛋放回孵化器中 10 分钟，让它循环。将鸡蛋放入长凳上的橡胶架中。然后，用弯曲的解剖剪刀切开胚胎周围的膜。用穿孔的勺子从鸡蛋中舀出胚胎，并立即用剪刀将胚胎斩首。在解剖显微镜下，将胚胎的身体放入培养皿中。将胚胎仰卧在培养皿中并展开四肢。使用镊子沿纵轴小心地打开胚胎的腹壁。在胚胎内找到类器官。类器官最常附着在右肝叶上，无论是在肋骨尖端还是颅骨。因此，建议从查看这些位置开始。找到类器官后，用微型剪刀将其从胚胎中取出。将类器官放入鸡组织中，鸡组织不可避免地附着在解剖显微镜下的培养皿中。用双刃不锈钢剃须刀片尽可能多地去除鸡肉组织。非移植肾类器官和移植肾类器官的免疫荧光图像均包含肾小球和肾小管结构。在未移植的类器官中，存在一些人类内皮细胞。在移植的类器官中，整个类器官中可见灌注的血管网络。盒装区域的放大图像显示了在移植类器官中可以区分的三种类型的内皮细胞：灌注的人内皮细胞、未灌注的人内皮细胞和灌注的鸡源性内皮细胞。在未移植的类器官中，内皮细胞围绕肾小球结构，但不会侵入它们。然而，在移植的类器官中，肾小球结构通过灌注的毛细血管形成血管化。移植的类器官可以根据实验目的使用不同的技术进行分析。与未移植对照的直接比较可以深入了解血管形成和成熟过程。

Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

科研

发育生物学

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

鸡胚全视网膜外植体电穿孔和化学试剂处理

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

In Ovo Electroporation in the Chicken Auditory Brainstem

In Ovo Electroporation in the Chicken Auditory Brainstem

在奥运电击鸡听觉脑干中

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

Tracing Gene Expression Through Detection of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-galactosidase Activity in Whole Mouse Embryos

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

一种从人类多能干细胞中生成肾类器官的简化方法

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...