Name: generation of porcine testicular organoids with testis specific architecture using microwell culture
Uploaded: 2019-10-03T15:00:00
Description: Watch this Scientific Journal Video about Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture at JoVE.com

这项工作得到了NIH/NICHD HD091068-01对伊娜·多布林斯基博士的支持。

注：从1周大的小猪身上获得睾号，作为商业猪的副产品。卡尔加里大学动物护理委员会批准了睾兽的采购。
1. 组织消化酶溶液的制备
注：需要三种不同的酶溶液，包括两种不同的胶原酶IV溶液（溶液A，B）和脱氧核糖核酸酶I（DNase I）溶液。
<ol><li>要制备溶液A，在25mL的高葡萄糖Dulbeco的改性鹰中分部（DMEM）中溶解20毫克胶原酶IV S（材料表）...

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我们已经建立了一个简单的方法，允许一致，可重复地生成大量的睾丸器官与组织结构，类似于睾丸在体内12。该方法是利用猪睾细胞开发的，但更广泛地适用于小鼠、非人类灵长类动物和人类睾人。已报告生产睾丸类有机物8、9、10、11的多种不同方法。Baert等人通...

近年来，人们对三维（3D）有机体的兴趣重新抬头。不同的器官，如肠1，胃2，胰腺3，4，肝脏5，和大脑6已经成功地衍生成3D器官系统。与单层培养系统7相比，这些有机物与体内器官具有建筑和功能上的相似性，在研究组织微环境方面更具生物学相关性。结果，睾丸类物开始引起兴趣，以及8，9，10，11，12。迄今报告的大多数方法都是复杂的，非高通量10，并且需要添加ECM蛋白8，10。这种复杂性还会导致重现性问题。需要一种简单且可重复的方法，允许生成具有细胞关联（类似于体内睾丸）的睾丸器官。
我们最近报告有一个系统，以解决这些问题的要求12。以猪为模型，我们在微井系统中采用了离心强制聚集方法。在微井系统中，每口井都含有大量相同的小微井13。这允许生成大量均匀大小的球体。微井系统能够生成大量具有睾号特异性架构的均匀有机体。该系统简单，不需要添加ECM蛋白。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm ultra low attachment tissue culture dish</td>
			<td>Corning</td>
			<td>#CLS3262</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm tissue culture dish</td>
			<td>Corning</td>
			<td>#353803</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrwell 400</td>
			<td>Stemcell Technologies</td>
			<td>#34411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>#07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type IV from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>#C5138</td>
			<td>referred as Collagenase IV S</td>
		</tr>
		<tr>
			<td>Collagenase type IV Worthington</td>
			<td>Worthington-Biochem</td>
			<td>#LS004189</td>
			<td>referred as Collagenase IV W</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>#DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/F12</td>
			<td>Gibco</td>
			<td>#11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>#D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>#D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>R&#38;D Systems</td>
			<td>#236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainers 70 &#181;m</td>
			<td>FisherScientific</td>
			<td>#352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainers 40 &#181;m</td>
			<td>FisherScientific</td>
			<td>#352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>#12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>Gibco</td>
			<td>#41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>#P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine testicular tissue</td>
			<td>Sunterra Farms Ltd (Alberta, Canada)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>#SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>#T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of porcine testicular organoids with testis specific architecture using microwell culture

有机体是由多种细胞类型组成的三维结构，能够概括组织结构和体内器官的功能。有机物的形成开辟了基础研究和转化研究的不同途径。近年来，睾丸器官在男性生殖生物学领域引起了兴趣。睾丸类有机物允许研究细胞-细胞相互作用、组织发育和生殖细胞利基微环境，并促进高通量药物和毒性筛选。需要一种方法，以可靠和可重复地生成睾丸器官与睾丸特定的组织结构。微井培养系统包含密集的金字塔形微井阵列。从青春期前睾丸中提取的睾丸细胞被离心到这些微孔中，并培养成具有睾丸特异性组织架构和细胞关联的睾丸器官。通过这一过程可以产生数千个均质有机体。这里报道的协议将引起研究男性生殖的研究人员的广泛兴趣。

从微孔中自组织成球体的1周老猪睾的分离细胞（图1A，图2），具有划定和独特的外部（显性上皮）和内部隔间（插曲） （图 1B，图 2）.两个隔间由胶原蛋白 IV+ve基底膜隔开。UCH-L1+ve生殖细胞和GATA4+saSertoli细胞位于地下室膜的外部隔间内（<st...

Watch this Scientific Journal Video about Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture at JoVE.com

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

在这里，我们提出了一个协议，用于使用市售的微孔培养系统，用睾丸特定组织架构的可重复生成猪睾丸器官。

使用微孔培养法生成具有睾丸特定结构的猪睾丸组织

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...

该协议是重要的，因为它允许睾丸有机体，它可以用作一个平台，研究睾丸形态发生和高特罗波药物和毒性筛查。该技术的主要优点是，它具有高度可重复性，需要相对较少的细胞数量，并可用于生成大量具有睾丸特异性结构的睾丸器官。该方法可用于研究其他系统，如多能干细胞或胰岛细胞与必要的优化。展示睾丸组织酶消化程序将是安娜沃伊格特，一个博士生从我的实验室。将睾丸收集到无菌烧杯中，用含有1%青霉素链霉素的PBS清洗。洗涤后将睾丸转移到100毫米组织培养皿中，PBS含有1%青霉素链霉素。使用自动剪剪刀和钳子去除阴道和表皮。将分离的睾丸转移到新的100毫米的盘子中，用含有1%青霉素链霉素的PBS彻底清洗。沿着直轴直接切割睾丸。然后用两个钳子将睾丸从图尼卡中剥离出来，并放入含有1毫升DMEM的新的100毫米盘中，配以1%青霉素链霉素。用无菌剪刀将去皮的睾丸切成一到两毫米的组织碎片。切后使用无菌钳子去除结缔组织的白色片段。将碎块转移到溶液A中，放入50毫升的管子中，用DMEM将其加到50毫升，获得每毫升0.4毫克的浓度，用于可乐酶4S，每毫升浓度为0.8毫克，用于11月4W。将含有组织碎片的管子放入37摄氏度的水浴中30分钟。每五分钟轻轻反转一次管子。30分钟后，在90倍G下离心管，在25摄氏度下休息1.5分钟。丢弃上一杯，使用溶液B的40毫升，用DMEM加50毫升，获得每毫升可乐酶4W的浓度为1.2毫克。将管子放入37摄氏度的水浴中30分钟，每5分钟轻轻反转一次管。在 90 倍 G 下离心管，在 25 摄氏度下中断 1.5 分钟。之后丢弃上经剂，用1%青霉素链霉素用PBS洗一次。小心地从顶部收集管，放入新的 50 毫升管中。用 PBS 向上顶管，高达 50 毫升。将管块以 90 倍 G 离心，在 25 摄氏度下休息 1.5 分钟。丢弃上一液，并添加新鲜的 PBS 洗两次额外的时间。上次 PBS 洗涤后，取出上经液，并在五毫升 PBS 中重新悬浮半阴性管。然后，在管子中加入15毫升0.25%的三辛埃德普辛EDTA。将管子放在37摄氏度的水浴中，每两分钟轻轻倒置一次。五分钟后，用组织培养板上的10微升样品，评估在显微镜下将小管的酶消化到单个细胞。每两分钟将样品放在显微镜下观察。如果大多数单个细胞可以检测到停止反应与5毫升FBS和过滤通过70微米网格，然后通过40微米网格。将单个细胞在500倍G下离心，在25摄氏度下中断5分钟。在50毫升富集介质中重新悬浮，并使用血细胞计计算可行细胞的数量。将2000万这种起始细胞种群转移到两个超低附着100毫米培养皿中，并在37摄氏度、5%的二氧化碳和21%的氧气下孵育，为期两天。每100毫米组织培养培养皿中剩余的2000万至2500万个细胞，总体积为8毫升富集培养培养皿。将盘子放入培养箱中，1.5小时后确保大多数Sertoli细胞附着在盘子上。稍微倾斜两个板，收集并结合超级纳特到一个新的100毫米板，并放回孵化器。一小时后，再次将两个板的超自然剂组合成一个新的100毫米板。将盘子放回孵化器过夜。然后收集两个分数中丰富的生殖细胞，非粘附分数和微粘附分数。收集上一级作为非粘附分数。对于稍微粘附的馏分，用PBS轻轻清洗板，并在室温下用两毫升1至20稀释0.25%Trypsin EDTA进行5分钟的治疗。通过添加两毫升富集介质停止反应，并收集在非粘附分数管中。将起始细胞制备和富集的生殖细胞结合在管中，获得含有25%生殖细胞的工作细胞制剂。在500倍G下离心，在25摄氏度下休息5分钟。丢弃上一级，将细胞重新悬浮在20毫升的有机形成介质中，达到每毫升240万个细胞的浓度。在一个新的管子中加入含有细胞的溶液的一毫升。在微井板的每个井中加入0.5毫升表面活性剂冲洗溶液。确保井中没有气泡。去除气泡，将板在2000倍G下离心，在25摄氏度下破裂两分钟。在低放大率倒置显微镜下观察板，以验证气泡是否已从微孔中去除。用盖子盖住盘子，在室温下孵育30分钟。处理完成后，取出冲洗液，并立即用无菌水或PBS清洗板。在每个井中加入0.5毫升的有机形成介质，并在2000次G下离心，在25摄氏度的温度下休息两分钟，以清除任何捕获的气泡。在低放大率倒置显微镜下观察井，以验证气泡是否已清除。加入 0.5 毫升的工作细胞悬浮液，通过上下移液轻轻混合。在500倍G下离心，在25摄氏度下休息5分钟。使用倒置显微镜验证细胞是否聚集在微孔内。将盘子转移到细胞培养箱和培养机构五天。每隔一天更换一半的介质。为了恢复器官，使用宽嘴移液器轻轻地移液介质向上和向下。这允许器官从微井中出来。收集器官，修复和执行免疫细胞化学的生殖细胞标记，Sertoli细胞标记，围膜阿米德细胞标记和Leydig细胞标记，并在共生显微镜下可视化。在这项研究中，从一周大的猪睾丸中分离出细胞，这些细胞在微井中自行培养成球体，具有划界和独特的外部和内部隔间。两个隔间由胶原蛋白IV正底膜隔开。GATA4阳性的Sertoli细胞和UCHL1阳性生殖细胞位于地下室膜的外部隔间中。aSMA正围管状阿米德细胞沿地下室膜内侧进行局部化，而CYP450阳性的Leydig细胞位于中间体的中心。这种结构类似于在原地条件，其中Leydig细胞，围管状的阿米德细胞，位于内层，在间层隔间和生殖细胞，Sertoli细胞位于半尼弗上皮。重要的是要确保用于生成器官的细胞具有最佳质量。在酶消化的三位一体阶段，应注意。按照这个程序，器官可以长期培养，以研究生殖细胞分化或分析基因表达和分泌的蛋白质或激素。使用睾丸特异性细胞关联创建器官的能力为研究对睾丸发育和生殖细胞功能重要的细胞-细胞相互作用提供了一种新颖的体外系统。

Research

JoVE Journal

Medicine

Teratoma Generation in the Testis Capsule

畸胎瘤代睾丸胶囊

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

科研

医学

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

使用Active波尔图腔静脉分流术猪肝脏采购和原位移植技术

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

器官空调，媒体和应用的产生对乳腺癌转移行为研究器官特异性的影响

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

药品再利用产生假设使用“RE：精药品”系统

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

肌肉失衡: 运动人群功能性偏心肌筋强度的检测与训练

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...