We gratefully acknowledge Dr. Hans Clevers (Hubrecht Institute for Developmental Biology and Stem Cell Research, Netherlands) for the kind gift of TOPflash cells.
This project was supported in part by NIH-R01DK083325 (MAH); NIH P30 DK078392 (Digestive Health Center); NIH UL1RR026314 (CTSA).

注：伦理学声明：在使用CCHMC本文所述被批准的IRB人体组织的所有试验（IRB＃2012年至2858年;＃2014-0427）。从供体处CCHMC获得用于组织收集，储存和使用的样本的知情同意书。 1.准备文化注：所有试剂都列在表1中 。 <ol><li>准备EDTA原液如下：准备0.5M的乙二胺四乙酸，pH值为8（EDTA）在超纯H 2 O，过滤消毒用0.22微米的过滤器。可选，存储EDTA原液在室温下去。 </li><li>制备螯合缓冲如下：混合2％山梨糖醇，1％蔗糖，1％牛血清白蛋白组分V（BSA）和1×庆大霉素/在Dulbecco氏磷酸盐两性霉素溶液缓冲盐水不含Ca 2+和Mg 2+（DPBS），过滤灭菌与0.22微米的过滤器。准备螯合BUFFER新鲜。 </li><li>制备的Wnt-3A条件培养基如下：的Wnt-3A条件培养基是使用细胞线L的Wnt-3A根据制造商的说明（ATCC，CRL-2647），制成在内部。补充了2mM谷氨酰胺，10毫米的HEPES，100U / ml的青霉素，100μg/ ml链霉素，1 N2添加剂，1 B27添加剂，1％BSA和过滤灭菌用0.22μm的过滤器中的介质。 注：测试每一批的使用TOPFLASH试验的Wnt活性。根据制造商的说明使用稳定的HEK293 TOPFLASH细胞系（汉斯Clevers实验室）与Renilla荧光素酶测定试剂盒。正常化用100ng / ml人重组Wnt-3A的TOPFLASH测定。确认至少有10倍的变化与对照组相比的条件培养基的活性。分新鲜的Wnt-3A条件培养基到10ml等份在15ml锥形管中，并冷冻在-20℃下进行长达6个月。商店解冻等分长达5天4°C时不丧失活性。 </li><li>公关epare人类minigut介质如下：补编高级DMEM / F12与2mM谷氨酰胺，10mM的HEPES，100U / mL的青霉素，100μg/ mL链霉素，1 N2添加剂，1 B27添加剂，1％BSA和过滤介质灭菌0.22微米的过滤器。 注：在-20℃下进行长达3个月除以新鲜人minigut介质到10ml等分试样在15ml锥形管中并冷冻。商店解冻等分长达5天4°C时不丧失活性。 </li><li>制备完整的人minigut介质如下：准备隐窝培养物或培养基改变人类minigut介质（见1.4），补充了50％的Wnt-3A条件培养基（见1.3），1微克/毫升的R-脊椎蛋白1（1前新鲜：1000稀释1毫克/毫升的股票），100毫微克/毫升的头蛋白（1：1000稀释100克/毫升的股票），50 ng / mL的EGF（1：10,000稀释500微克/毫升的股票），500 Nm的-83 ，10μMSB202190：（稀释500兆库存1000 1）（1：3000稀释的30mM库存）-01，10nM的〔亮氨酸〕15胃泌素1（1：10,000稀释的100μM的股票），10毫烟酰胺（1：稀释100 1M的股票）和1mM的N-乙酰半胱氨酸（1：1000稀释的1M股票）。 注：存储完整的人类minigut媒体2天，4℃而不丧失活性。 </li></ol>从整个组织2地穴隔离注意：从组织采集，它是必不可少的，以保持在盐水中的样品。建议保持组织上的冰块在运输过程中。应尽快进行样品地穴隔离的准备。 注：所有试剂都列在表1中 ，工具，设备和消耗品列于表2。 <ol><li>在开始实验前准备好所有的试剂。解冻在冰上和基底膜基质预先孵育，在37℃在CO 2培养箱24孔板中。 注：另外，用15微升/孔的24的中心作出的薄层基底膜基质的-嗯板，并将其放置在CO 2培养箱中在37℃。此兼性步骤保持基底膜基质作为聚合过程中下降。 </li><li>与洗组织冰冷Dulbecco氏磷酸盐缓冲盐水不含Ca 2+和Mg 2+（DPBS）。继续进行直到DPBS的内容是清楚的。 </li><li>用0.2毫米直径minutien销，固定在组织上的硅氧烷涂层玻璃培养皿填充用冰冷的DPBS。伸展和针组织扁平的粘膜面朝上。 </li><li>在解剖显微镜下，从粘膜下层和结缔组织使用微解剖剪和细点弯钳除去覆粘膜（ 图2A，B）。 </li><li>伸展和针的解剖粘膜平放在硅氧烷涂覆玻璃培养皿与粘膜面朝上。剩余的粘膜下层和结缔组织可以被丢弃或者用于进一步的实验（ 图2C）。 </li><li>平缓刮掉粘膜与弯钳的表面上。这一步是必要的，以改善该制剂的质量。 <ol><li>对于小肠，轻轻刮去粘膜去除绒毛。 </li><li>对于结肠，轻轻刮去粘膜去除粘液和碎屑。 </li></ol></li><li>用冰冷的缓冲螯合洗粘膜3-4次，以除去绒毛和碎片。 </li><li>盖上新鲜烹制2mM的EDTA螯合缓冲（200微升0.5M的EDTA在49.6毫升螯合缓冲）的粘膜。 </li><li>广场上冰的培养皿中轻轻摇晃在水平轨道摇床30分钟。 </li><li>洗组织3-4次，用冰冷的缓冲螯合无EDTA中。在洗涤后，离开粘膜冰冷螯合缓冲器。 </li><li>处理使用弯曲和细镊子解剖显微镜下的粘膜。轻轻刮去黏膜释放使用弯钳肠腺。 </li><li>轻轻用圆周率从培养皿去除隐窝悬浮佩特并转移到一个50ml锥形管中。 注：检查组织，以确保几乎所有的隐窝已从粘膜去除。 </li><li>通过150微米过滤隐窝暂停网2倍。 注意：检查显微镜（ 图2D）下流通的地穴富集。 </li><li>离心地穴暂停5分钟50 XG，4℃。弃去上清液。 </li><li>重悬在5ml冰冷缓冲螯合沉淀。 </li><li>从步骤2.15计数每从悬浮液10微升滴隐窝数。转移所需要的电镀到5毫升的圆底管隐窝数。使用24孔盘200〜500隐窝每口井建立enteroids或colonoids。 </li><li>离心机地下室部分为10分钟，在150克，4℃。去除上清。 </li><li>使用隐窝后续文化。 </li></ol>从活检3.地穴隔离<ol><li>准备前的所有试剂开始实验。解冻在冰上和基底膜基质预先孵育，在37℃在CO 2培养箱24孔板中。 </li><li>与洗活检冰冷Dulbecco氏磷酸盐缓冲盐水不含Ca 2+和Mg 2+（DPBS）。 </li><li>使用直径0.1mm minutien销，固定在活检上硅氧烷涂层玻璃培养皿填充用冰冷的DPBS。伸展和针粘膜平与粘膜一侧朝上（ 图2E）。 </li><li>轻轻刮去黏膜弯钳的表面，以去除绒毛和碎片。这一步是必要的，以改善该制剂的质量。 </li><li>用冰冷的缓冲螯合洗活检3-4次，以除去绒毛和碎片。 </li><li>盖上新鲜烹制2mM的EDTA螯合缓冲（200微升0.5M的EDTA在49.8毫升螯合缓冲）活检。 </li><li>广场上冰的培养皿中轻轻摇晃在水平轨道摇床30分钟。 </li> &lt;LI&gt;洗净切片3-4次用冰冷的螯合缓冲无EDTA。洗涤后，将留在冰冷的缓冲螯合活检。 </li><li>处理使用弯曲和细镊子解剖显微镜下的活检。轻轻刮去粘膜用弯钳松开肠腺。 </li><li>轻轻用移液管从培养皿中去除隐窝悬浮并转移到50ml锥形管中。 注：检查组织，以确保几乎所有的隐窝已从粘膜去除。 </li><li>过滤器通过一个150微米的尼龙地穴悬吊网2倍。 注意：检查在显微镜下的流过的地穴富集。 </li><li>离心地穴暂停5分钟50 XG，4℃。弃去上清液。 </li><li>重悬在1ml冰冷缓冲螯合沉淀。转让墓穴悬挂到1.5 ml离心管。 </li><li>离心机地穴分数在150 XG，4℃，10分钟。去除上清。&lt;/ li&gt; <li>使用隐窝后续文化。 </li></ol> 4.地穴文化基底膜基质<ol><li>采用预冷枪头，悬浮颗粒隐窝（从步骤2.18或3.15）在基底膜基质（200〜500隐窝/ 50微升基底膜基质）。 </li><li>申请50微升隐窝悬浮液以每孔基底膜基质上的预热板。慢慢喷射在井的中心基底膜基质。 </li><li>将24孔板在37℃，5％CO 2的培养箱中30分钟，以使基底膜基质的完全聚合。 </li><li>覆盖基底膜基质用500微升完整的人类minigut介质中添加2.5μMCHIR99021（1：4000稀释10毫米股票）和2.5μMThiazovivin（1：4000稀释10毫米股票）。 </li><li>孵育所述板在37℃，5％CO 2的培养箱中培养。 </li><li>用新鲜完整的呼玛更换介质ñminigut中每2天。 </li></ol> 5.传代培养Enteroids和Colonoids的。 注：通道Enteroids和Colonoids初步电镀后，每7〜10天。通常，分割1以及3〜4个孔。 <ol><li>在实验开始前，准备所有试剂。解冻在冰上和基底膜基质预先孵育，在37℃在CO 2培养箱24孔板中。 </li><li>使用无菌针头和覆盖用1毫升冰冷的DPBS中取出介质。 </li><li>吸管来回1000微升提示。转移在一个新的15毫升锥形管中的溶液中。 </li><li>加入2 ml人minigut培养基补充有每1ml培养基中的5％FBS中。 </li><li>离心5分钟，该溶液在50×g离心，4℃。弃去上清液。 </li><li>重悬沉淀用2ml的细胞解离酶补充有10μM的Y-27632（1：1000稀释的10mM储备）。孵育，持续5分钟在37℃在水 - 浴中。 </li><li>分离使用3毫升鲁尔锁注射器配备了一个18-G填充/钝针的细胞团块。轻轻吸取的溶液来回使用注射器10倍。 </li><li>离心机暂停5分钟，在500 XG，4℃。弃去上清液。 </li><li>用预冷的枪头，重悬在基底膜基质细胞沉淀。 </li><li>申请50微升隐窝悬浮液以每孔基底膜基质上的预热板。慢慢喷射在井的中心基底膜基质。 </li><li>将24孔板在37℃，5％CO 2的培养箱中20分钟，以使基底膜基质的完全聚合。 </li><li>覆盖基底膜基质用500微升完整的人类minigut介质辅以10μMY-27632（1：1000稀释10毫米股票）。 </li><li>孵育所述板在37℃，5％CO 2的培养箱中培养。 </li><li>术后第2天（：1000稀释10毫米的股票1），更换新鲜完整的人类minigut培养基中添加10μMY-27632的培养基中。此后，用新鲜的，完整的人类minigut中更换培养基隔日1次。 </li></ol> 6.冷冻培养Enteroids和Colonoids的注：通常冻结一口井成2-3冷冻管。 <ol><li>重复步骤5.1至5.8。 </li><li>悬浮用冷冻介质中的颗粒。转移1毫升冷冻液到标记离心管中。将离心管中含有500毫升异丙醇的冷冻集装箱。 </li><li>在冷冻集装箱转移到80℃的冷冻24小时，然后转移到离心管液氮储存。 注：存储Enteroids和Colonoids长达1年。 </li></ol>

<ol>
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The authors have nothing to disclose.

这种方法提供了一个完整的系统复制肠上皮细胞系和上皮动态构成来研究肠上皮生物学的有用工具。本文介绍的方法是改编自原鼠研究佐藤和Clevers 22有效地导致人类enteroids和colonoids。在这里，我们手动拾取隐窝通过显微解剖，以避免任何细胞污染物。此方法允许隐窝的直接可视化，并通过“摇动”导致稠度加班相比原来隐窝集合。其他小组开发出了类似的工艺使用略有不同的方法尤其是胶原酶25取代螯合通过EDTA。除了 ​​在隐窝集合的差异，这些工艺使用一个所需生长人类enteroids中培养22限定的介质。为了增加在隐窝播种生长效率，我们增加GSK3抑制剂（CHIR99021）对于前两天12。 组织或活检的处理是很重要的，并隐窝隔​​离应尽快组织到达在实验室进行的。然而，延迟隐窝分离和培养可进行到组织收集后24小时（数据未示出）如前对小鼠组织26进行说明。肠组织应放置在一个锥形管完全填充用DPBS，以避免组织破坏，并应保持在4℃。延迟准备允许组织装运，但在运输过程中应避免温度变化。所需的初始隐窝电镀的总时间是约2小时，用15至30分钟来处理所述组织和1至2小时，以分离和板隐窝。组织的显微切割是一种清洁的隐窝制备的一个关键因素和谓词。然而，地穴释放握手中各种协议描述的可能22,23。 尽管与鼠enteroid（enteroids）系统的相似性，人类enteroids需要特定的分子，以提高和维持其随时间增长。生长因子，EGF，头蛋白，R-脊椎蛋白被类似地使用与鼠上皮组织体。然而，使用的Wnt-3A是至关重要的。我们注意到，形成以及生长效率大于使用的Wnt-3A条件培养基比人类重组蛋白质。与此同时，我们展示了使用的糖原合酶激酶3（CHIR99021）12抑制剂改善培养条件。重组生长因子可通过的Wnt-3A，R脊椎蛋白，头蛋白和调节介质代替。阿的Wnt-3A表达L-细胞系可购（ATCC）。其它基团已经开发的R-脊椎-1- 23,27，Noggin- 19，和的Wnt-3A / R-spondin3 / Noggin- 28表达细胞系。两个小分子抑制剂在培养中使用了我直径和所必需的培养29的维护。 A-83-01是转化生长因子β和激活素/交点受体的选择性抑制剂（激活素样激酶4，5，7）和SB202190是p38促分裂原活化蛋白激酶抑制剂（MAPK）。这两种抑制剂已被分别用来维持人类诱导多能干细胞自我更新和建立人类天真多能干细胞30-32。此外，烟酰胺，烟酰胺腺嘌呤二核苷酸的前体，需要保持enteroids和colonoids膨胀在一个长期的方式22,29。 在EDTA螯合是重要的一步，因为它决定了从地穴编制的产量。我们已经成功了2mM EDTA处理。然而，EDTA浓度可以从2mM的进行修改，以对组织类型为15mM。在这种情况下，温育时间，必须凭经验确定。最初的电镀后，隐窝将全面-Up和最终形成enteroids。然而，enteroids或colonoids往往表现出“干”型形成球体几乎没有分化的细胞。在这种情况下，分化可以通过抽出的Wnt-3A，烟酰胺和p38蛋白抑制剂来启动。使用的Notch抑制剂诸如DAPT或DBZ有助于enteroids 22内增强的分化。 该模型概括肠生理学与来自干细胞区室所产生的含有吸收和分泌分化谱系持续隐窝出芽事件以及绒毛状上皮域。有趣的是，该系统不包含任何间充质细胞，并使用特定的介质的条件，以满足利基信号要求。 像的小鼠模型，人类enteroids可以从分离的肠上皮细胞中产生，以测试这些细胞以用作干细胞的能力。塞弗RAL研究使用的分化标志物（CD44，CD24和CD166）和EPHB2阳性细胞群的细胞来丰富与干性12,23,33。总之，这些研究表明人类enteroids培养物的效用，用于测试的干性。其他研究者使用该模型来调查肠道疾病，如感染性腹泻病，囊性纤维化，或结肠直肠癌22,34-37。这些研究表明，人类enteroids构成具有可能性的可靠的人类疾病模型走向个性化的筛选。人类enteroids可以使用DNA转染或感染病毒颗粒38转基因。这提供了一个有力的工具，以在人上皮组织体或正确的基因突变中研究基因的特定功能。近日，施万克和他的同事证明的可能性，以编辑的基因组与CRISPR / Cas9制度和纠正CFTR基因交流造成的基因突变ystic纤维化24。人类enteroids构成宝贵的系统来研究肠干细胞和上皮粘膜生物学，并作为一个新的实验系统，研究正常和异常消化道生理学。

胃肠道的衬里上皮细胞是在不断更新。这个过程是由肠干细胞（ISCS），它连续地产生子代迅速更换肠上皮，因为它翻转的增殖触发。增殖隔室包括所述ISC的被限制在隐窝的底部。综合监控系统产生的后代最终分化成吸收或分泌谱系。走出地下室，并到绒毛或表面上皮细胞逐步分化，因为他们去角质前向上迁移到管腔1。 ISC的产生所有肠上皮细胞类型，包括肠，微皱细胞，肠内分泌细胞，杯状细胞，绒头细胞和帕内特细胞。结肠的特点是多为结肠和杯状细胞组成的长形墓穴，有散肠内分泌和一簇细胞2。 体外 CULTURE系统构成有前途的工具，学习ISC维护和肠组织的动态平衡。然而，这是难以依靠组织培养技术的生理条件不充分再现和上皮微环境经常改变3,4。在ISC领域的一个重大进步是建立组织培养技术来维护和使用已定义的生长因子来替代正常的肠道利基信号扩大个体鼠ISC的。长期培养条件由Sato等人描述的，在其中单隐窝或从肠道上皮分离的干细胞的生长，以形成3维的上皮结构，包括多个隐窝样结构域5-7。这些立体结构发生裂变的事件不断扩大。有趣的是，所有的具体来源的组织中的肠细胞类型产生和以及被挤压成的内腔8。使用该系统的变型，上皮组织体可以从胃，小肠和结肠中产生。更具体地，从小肠上皮组织体是enteroids 9，以及那些从结肠是colonoids 9,10。这些上皮类器官培养系统已被用于测试分离的单细胞的充当干细胞在体外培养 ，从而检测分离的细胞5,6,10-15的“干细胞”的能力。其他研究者已经使用既enteroids和colonoids研究个体的上皮细胞16-21的功能。因此，enteroid和colonoid培养物可以被用于评估两个干和非干细胞的功能，并提供新的洞察肠子内基本细胞相互作用。 在2011年，佐藤和同事产生来自人小肠和结肠22,23衍生上皮组织体的长期培养。除了在介质组合物中，人上皮enteroids差异和colonoids表现出相同的特点及其小鼠对应。此外，他们可以从患病组织诸如巴雷特食管，腺瘤或腺癌，和囊性纤维化22,24产生。人类enteroids构成宝贵的系统来研究肠干细胞和上皮粘膜生物学，并作为一个新的实验系统，研究正常和异常消化道生理3。 这里，我们描述的方法来建立enteroids和colonoids来自人小肠和结肠隐窝（ 图1）。在这种方法审查，我们强调从整个组织活检地下室集合。我们概括的培养方式，这对于成功的生长和维持人类enteroids和colonoids和由该模型所进行的可能的实验策略是至关重要的。

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:480px;">Name of Reagent</td>
			<td style="width:195px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dulbecco&#8217;s Phosphate buffered saline Ca2+, Mg2+ free (DPBS)</td>
			<td>Life technologies; Gibco</td>
			<td>14190-144</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Fischer Scientific</td>
			<td>BP439-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fischer Scientific</td>
			<td>BP220-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA) Fraction V</td>
			<td>Fischer Scientific</td>
			<td>BP1600-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gzntamycin/Amphotericin B solution</td>
			<td>Life technologies; Gibco</td>
			<td>R-015-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wnt-3A conditionned medium</td>
			<td>in house</td>
			<td>-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Advanced DMEM/F12</td>
			<td>Life technologies; Gibco</td>
			<td>12634-028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES 1M</td>
			<td>Life technologies; Gibco</td>
			<td>15630-080</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX (glutamine)</td>
			<td>Life technologies; Gibco</td>
			<td>35050-061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies; Gibco</td>
			<td>15140-148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Life technologies; Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement</td>
			<td>Life technologies; Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotidamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel, GFR, Phenol free (basement membrane matrix)</td>
			<td>Corning</td>
			<td>356231</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant Noggin</td>
			<td>R&#38;D</td>
			<td>6057-NG/CF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant R-Spondin</td>
			<td>Preprotech</td>
			<td>120-38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant EGF</td>
			<td>Sigma-Aldrich</td>
			<td>E9644-.2MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503-1MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067-5MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human [Leu]15-Gastrin 1</td>
			<td>Sigma-Aldrich</td>
			<td>G9145-.1MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazovivin</td>
			<td>Stemgent</td>
			<td>04-0017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express Enzyme (1X), phenol red (cell dissociation enzyme)</td>
			<td>Life technologies; Gibco</td>
			<td>12605-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Life technologies; Gibco</td>
			<td>10082-147</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CTS Synth-a-Freeze Medium (freezing medium)</td>
			<td>Life technologies; Gibco</td>
			<td>A13713-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L Wnt-3A cell line</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla luciferase assay</td>
			<td>Promega</td>
			<td>E2710</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant Wnt-3A</td>
			<td>R&#38;D</td>
			<td>5036-WN/CF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HEK293 TOPflash cell line</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

establishment of human epithelial enteroids and colonoids from whole tissue and biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

图2D示出的新鲜分离的隐窝从整个组织（ 图2D）的典型例子。隐窝从活检中分离的数目比在整个组织中低。使用标准容量活检钳针，我们通常在单次执行两种活检叮咬。每个活检咬结果在10 mm 2的表面与每个活检平均50至100隐窝（ 图2F）。 文化基底膜基质后，隐窝围捕，形成enterospheres的小肠和colonospheres结肠癌。地穴萌芽通常发生在5〜6天，播种后。然而这是不寻常地看到任enteroids（enteroids）或colonoids（colonoids）形成在基底膜基质球体（ 图3A-C;电影1）。的传代可以在7天之内完成，这取决于enteroids的大小。该enteroids或colonoids establ从活检ished进行文化同发展。然而，由于在播种隐窝密度较低，传代通常在10至12天的文化做了（图4a，b）所示。 Enteroids和colonoids培养扩大可重复的方式。 既enteroids和colonoids呈现腔侧，并衬有上皮（ 图5A，B）。增生细胞可在enteroids内被观察和位于芽尖端（ 图5C，D）的范围内。沾上E-cadherin的（ECAD）enteroids的共焦成像显示上皮细胞（ 图5E）。 既enteroids和colonoids可以从患者的遗传/先天性疾病获得的组织来确定。 图6示出了由于在上皮细胞广告先天性突变代表enteroids从囊性纤维化（ 图6A）和一个簇绒肠病的患者不断增加HESION分子基因（EpCAM的）（ 图6B）。旁边的遗传缺陷，该enteroids没有表现出在基础条件的差异。 <img alt="图1" src="/files/ftp_upload/52483/52483fig1highres.jpg" /> 图1.工作流的隐窝解离和代人enteroids和文化colonoids的。地穴（从人体小肠或结肠）由EDTA螯合隔离。培养隐窝形成enteroids的小肠和colonoids的结肠。 <a href="https://www.jove.com/files/ftp_upload/52483/52483fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52483/52483fig2highres.jpg" /> 图2.解剖过程地穴隔离。（A）小肠specimen被拉伸和钉扎平成聚硅氧烷涂覆的培养皿。 （B）中的粘膜被从底层粘膜下层分离。 （C）的该解剖粘膜被拉伸和钉扎平成聚硅氧烷涂覆的培养皿。 （D）中的EDTA螯合作用后，将隐窝从组织中分离。 （E）一种活检拉伸和钉扎平成聚硅氧烷涂覆的培养皿。 （F）EDTA螯合后，隐窝从活检隔离。 <a href="https://www.jove.com/files/ftp_upload/52483/52483fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52483/52483fig3highres.jpg" /> 图3.地穴文化和人力enteroid和colonoid代从整个组织。（A）隔离后镀在基底膜基质空肠隐窝。隐窝是后3至4小时闭起来，并开始球囊起来，形成超出这个时间enterospheres。在第7天，空肠enteroids形成。 （B）分离培养后，回肠隐窝表现得像空肠隐窝，形成回肠enteroids。 （C）结肠隐窝被隔离后镀在基底膜基质。 7天后（比例尺：100微米）隐窝密切和形式colonoids。 <a href="https://www.jove.com/files/ftp_upload/52483/52483fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52483/52483fig4highres.jpg" /> 图4.地穴文化和人力enteroid和colonoid一代从活检。（A）隔离后镀在基底膜基质十二指肠隐窝。隐窝是3收盘后至4小时，并开始了气球beyo次此时间形成enterospheres。在第7天，将enteroids形成。 （B）分离培养后，结肠隐窝铺在基底膜基质。隐窝收起来，形成colonospheres再经过7天 ​​（比例尺：100微米）colonoids。 <a href="https://www.jove.com/files/ftp_upload/52483/52483fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52483/52483fig5highres.jpg" /> 经过6天的文化图5人enteroids肠谱系。（A）人力enteroid。 （B）在（A）展示上皮衬里enteroids的苏木精-伊红节（比例尺：100微米）。 （ 光盘 ）后的EdU染色（品红色）enteroids的共聚焦成像显示增殖性细胞的存在。 （E </str翁&gt;）enteroids的共焦成像表明存在：E-cadherin的上皮细胞（ECAD，绿色）（比例尺：50微米）， <a href="https://www.jove.com/files/ftp_upload/52483/52483fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52483/52483fig6highres.jpg" /> 图6.地穴文化和人力enteroid一代的病变组织。（A）Enteroids从囊性纤维化标本建立的。 （B）Enteroids从先天性簇绒肠病标本建立的（比例尺：100微米）。 <a href="https://www.jove.com/files/ftp_upload/52483/52483fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 电影1 Enterosphere形成人类enteroid培养32小时时间圈e动画显示了从人体小肠回缩，形成文化的enteroid建立了enterosphere。 <a href="http://www.jove.com/files/ftp_upload/52483/Movie1_v2.mp4" target="_blank">请点击此处观看该视频。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 溶剂 </td><td> 股票集中度 </td><td> 终浓度 </td><td> 评论 </td></tr><tr><td> Dulbecco氏磷酸盐缓冲盐水的Ca 2+，Mg 2 +的自由（DPBS） </td><td> Life技术; GIBCO </td><td> 14190-144 </td><td> - </td><td> - </td><td> 1X </td><td></td></tr><tr><td>乙二胺 四乙酸（EDTA） </td><td> Sigma-Aldrich公司</td><td> 431788 </td><td>超纯的dh 2 O </td><tD&gt; 0.5M的</td><td> 2毫米</td><td></td></tr><tr><td>山梨糖醇</td><td>菲舍尔科学</td><td> BP439-500 </td><td> DPBS </td><td>粉</td><td> 2％ </td><td></td></tr><tr><td>蔗糖</td><td>菲舍尔科学</td><td> BP220-1 </td><td> DPBS </td><td>粉</td><td> 1％ </td><td></td></tr><tr><td>牛血清白蛋白（BSA）的V组分</td><td>菲舍尔科学</td><td> BP1600-100 </td><td> DPBS </td><td>粉</td><td> 1％ </td><td></td></tr><tr><td> Gzntamycin /两性霉素B的解决方案</td><td> Life技术; GIBCO </td><td> R-015-10 </td><td> - </td><td> 500X </td><td> 1X </td><td></td></tr><tr><td> Wnt信号-3A conditionned媒体</td><td>在内部</td><td> - </td><td> - </td><td> - </td><td> - </td><td></td></tr><tr><td>先进的DMEM / F12 </td><td> Life技术; GIBCO </td><td> 12634-028 </td><td> - </td><td> - </td><td> - </td><td></td></tr><tr><td> 1M HEPES </td><td> Life技术; GIBCO </td><td> 15630-080 </td><td> - </td><td> 1M的</td><td> 10毫</td><td></td></tr><tr><td>含有GlutaMAX（谷氨酰胺） </td><td> Life技术; GIBCO </td><td> 35050-061 </td><td> - </td><td> 100X </td><td> 1X </td><td></td></tr><tr><td>青霉素链霉素（10,000 U /毫升） </td><td> Life技术; GIBCO </td><td> 15140-148 </td><td> - </td><td> 100X </td><td> 1X </td><td></td></tr><tr><td> N2补充</td><td> Life技术; GIBCO </td><td> 17502-048 </td><td> - </td><td> 100X </td><td> 1X </td><td></td></tr><tr><td> B27补充</td><td> Life技术; GIBCO </td><td> 17504-044 </td><td> - </td><td> 50X </td><td> 1X </td><td></td></tr><tr><td> N-乙酰半胱氨酸</td><td> Sigma-Aldrich公司</td><td> A9165-5G </td><td> DPBS </td><td> 1M的</td><td> 1毫米</td><td> &lt;/ TD&gt; </tr><tr><td> Nicotidamide </td><td> Sigma-Aldrich公司</td><td> N0636 </td><td> DPBS </td><td> 1M的</td><td> 10毫</td><td></td></tr><tr><td>基底膜，肾小球滤过率，无苯酚（基底膜基质） </td><td>康宁</td><td> 356231 </td><td> - </td><td> - </td><td> - </td><td> REQUIRED </td></tr><tr><td>人重组头蛋白</td><td>研发</td><td> 6057-NG / CF </td><td> DPBS </td><td> 100微克/毫升</td><td> 100ng / ml的</td><td rowspan="3">其他供应商：R＆D组; Anaspec和Preprotech </td></tr><tr><td>重组人R-脊椎</td><td> Preprotech </td><td> 120-38 </td><td> DPBS </td><td> 1mg / ml的</td><td> 1微克/毫升</td></tr><tr><td>重组人表皮生长因子</td><td> Sigma-Aldrich公司</td><td> E9644-.2MG </td><td> DBPS </td><td> 500微克/毫升</td><td> 50ng / ml的</td></tr><tr><td> Y-27632 </td><td> Sigma-Aldrich公司</td><td> Y0503-1MG </td><td> DPBS </td><td> 10毫</td><td> 10μM </td><td></td></tr><tr><td> A-83-01 </td><td> Tocris </td><td> 2939 </td><td> DMSO </td><td> 500μM </td><td> 500纳米</td><td></td></tr><tr><td> SB202190 </td><td> Sigma-Aldrich公司</td><td> S7067-5MG </td><td> DMSO </td><td> 30毫米</td><td> 10μM </td><td></td></tr><tr><td>人〔亮] 15胃泌素1 </td><td> Sigma-Aldrich公司</td><td> G9145-.1MG </td><td> DPBS </td><td> 100μM </td><td> 10纳米</td><td></td></tr><tr><td> CHIR99021 </td><td> Stemgent建立</td><td> 04-0004 </td><td> DMSO </td><td> 10毫</td><td> 2.5μM </td><td></td></tr><tr><td> Thiazovivin </td><td> Stemgent建立</td><td> 04-0017 </td><td> DMSO </td><td> 10毫</td><td> 2.5μM </td><td></td></tr><tr><td>的TrypLE快递酶（1X），酚红（细胞解离酶） </td><td> Life技术; GIBCO </td><td> 12605-010 </td><td> - </td><td> - </td><td> - </td><td>传代</td></tr><tr><td>胎牛血清</td><td> Life技术; GIBCO </td><td> 10082-1​​47 </td><td> - </td><td> - </td><td> - </td><td>传代</td></tr><tr><td> CTS合成器-A-冻结介质（冷冻介质） </td><td> Life技术; GIBCO </td><td> A13713-01 </td><td> - </td><td> - </td><td> - </td><td>对于冻结</td></tr><tr><td> L的Wnt-3A细胞系</td><td> ATCC </td><td> CRL-2647 </td><td> - </td><td> - </td><td> - </td><td> Wnt信号-3A conditionned媒体制作</td></tr><tr><td>海肾荧光素酶检测</td><td> Promega公司</td><td> E2710 </td><td> - </td><td> - </td><td> - </td><td> Wnt信号-3A conditionned媒体活动</td></tr><tr><td>人重组的Wnt-3A </td><td>研发</td><td> 5036-WN / CF </td><td> DPBS </td><td> 100微克/毫升</td><td> 100ng / ml的</td><td&gt;的Wnt-3A conditionned媒体活动</td></tr><tr><td> HEK293 TOPFLASH细胞系</td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td><td> Wnt信号-3A conditionned媒体的活动;从汉斯Clevers实验室礼品</td></tr></tbody></table> 表1.详细试剂列表首选的制造商和目录编号。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 设备 </td><td> 耗材 </td><td> 工具 </td></tr><tr><td>层流罩</td><td> 15-和50毫升锥形管中</td><td>杜蒙＃5镊子标准（FST;＃11251-20） </td></tr><tr><td>的CO 2培养箱</td><td>离心管</td><td>杜蒙＃7，弯细镊子（FST;＃11274-20） </td></tr><tr><td>立体显微镜</td><td> 24孔板</td><td>精细剪刀（FST;＃14060-09） </td></tr><tR&gt; <td>离心分离机</td><td> 0.22微米的过滤器（赛多利斯） </td><td> Vannas弹簧剪刀（FST;＃15018-10） </td></tr><tr><td>轨道摇床</td><td>血清移液器</td><td>直径minutien0.2毫米引脚（FST;＃26002-20） </td></tr><tr><td>冷冻集装箱（的Nalgene） </td><td>微量提示</td><td>直径minutien0.1毫米引脚（FST;＃26002-10） </td></tr><tr><td> SYLGARD 184硅（道康宁） </td><td> 150微米网孔，尼龙筛（动态水族供给） </td><td></td></tr><tr><td>玻璃培养皿</td><td> 5毫升圆底聚丙烯管（Falcon）中</td><td></td></tr><tr><td>血清学移液器</td><td> 18G钝补针（BD） </td><td></td></tr><tr><td>微管</td><td> 3毫升lsyringes与鲁尔锁定提示（BD） </td><td></td></tr><tr><td></td><td>离心管</td><td></td></tr></tbody></table><s仲&gt;表2.详细的消耗品，工具，设备等所需隐窝分离培养。

Watch this Scientific Journal Video about Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy at JoVE.com

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

establishment-human-epithelial-enteroids-colonoids-from-whole-tissue

Research

JoVE Journal

Medicine

34.4K 次观看.  Cincinnati Children's Hospital Medical Center. We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

视频: Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D全心脏心肌组织分析

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

经非透法皮下腹脂肪组织活检技术

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

18F-fdg Petct 对活性人棕色脂肪组织的全身和区域定量

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

人鼻上皮类器官的培养和成像

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.