该项目得到了 nih K01OD0199、nih T32 OD011130、nih P30DK034987 和临床科学传播基金的支持。

在这些实验中, 所有动物研究都获得了北卡罗来纳州立大学机构动物保育和使用委员会 (IACUC) 的批准。
1. 文化的准备
注意: 所有试剂都列在材料表中。特定的增长因子浓度在表 1中列出。
<ol><li>在蒸馏去离子水 (ddH2O) 中制备0.5 米乙二胺乙酸 (EDTA) 溶液。使用氢氧化钠和/或 HCl 溶液, 充分混合并调整 pH 值为7.4。 注: 在每次试验前, 应先将 EDTA 库存溶液进行保鲜。</li>
	<li>准备离解试剂 #1 (DR #1) 如下和放置在冰上: 将磷酸盐缓冲生理盐水与无 Ca2 +和 Mg2 + (PBS)、0.5 米 EDTA、1M 14-Dithiothreitol (德勤)、10µM Y27632 和1X 抗生素防霉溶液 (抗反) 结合在一起。含青霉素、链霉素和两性霉素 B。 注: 在组织收集前立即添加 Y27632。</li>
	<li>准备离解试剂 #2 (DR #2) 如下和放置在37°c 水浴: 结合 PBS 没有 Ca2 +和镁2 +, 0.5 米 EDTA, 10 µM Y27632 和1X 抗反。注: 在组织收集前立即添加 Y27632。</li>
	<li>制备肠道上皮干细胞 (IESC) 培养基如下: 混合25毫升先进的 DMEM/F12 培养基与 1X N2 补充, 1X B-27 补充, 10mM HEPES, 2mM Glutamax 和1X 抗反。存储在4°c, 直到使用。</li>
	<li>准备一个主要的组合, 减少生长因子基底膜基质 (基质) 和补充生长因子如下: 在冰上解冻基质。在离心管中, 添加 100 ng/毫升重组人的脑袋, 500 ng/毫升重组人 R-Spondin 1, 50 ng/毫升重组人表皮生长因子 (EGF), 100 ng/毫升重组人 Wnt3a, 10 毫米烟酰胺, 10 nm 胃泌素, 500 nm A-83-01, 10 µM Y-27632, 10 毫米 SB202190, 500 nM LY2157299 和2.5 µM 糖原合成酶激酶3抑制剂 (GSK3i, CHIR99021)。当基质解冻时, 补充与生长因子混合 (使主混合) 和储存在冰上。 注意: 小心避免引入气泡, 因为这将在电镀过程中在基体帕蒂中产生工件。</li>
	<li>在开始细胞隔离程序之前, 在37°c 孵化器中预热一个24井组织培养皿。</li>
</ol>2. 缺血和组织收集的外科模型
<ol><li>使用八至十周大的约克郡杂交猪的任何性别 (推荐)。手术前取出所有饲料16-18 小时。让猪在任何时候都能接触到水。</li>
	<li>安排猪在实验前到达至少48小时, 以便进行环境驯化。 注: 经过培训的兽医和/或实验室技术员由有执照的兽医监督, 提供例行的动物护理和辅助麻醉程序。</li>
	<li>Premedicate 猪与甲苯噻嗪 (1.5 毫克/千克肌肉 (im)) 和氯胺酮 (11-20 毫克/千克 (im))15。一旦镇静, 将猪 orotracheally 插管。</li>
	<li>在全身麻醉下, 用异氟醚 (2-5%) 在 100% O2中汽化, 直至安乐死为止。</li>
	<li>放置静脉注射 (IV) 导管 (推荐的耳静脉), 并管理替代液体, 如乳酸枪手溶液, 维持率为15毫升/千克/小时。</li>
	<li>进行例行麻醉监测, 包括脉冲氧, 心电图和间接血压测量15。如果最终潮汐 CO2上升到 55-60 mmHg 以上, 则需要手动或机械地监测猪的呼吸速率和工作情况。 注: 通过持续监测心率 (范围80-130 节拍/分钟)、无创性血压 (平均动脉压 75-100 mmHg) 和呼吸率 (10-25 次呼吸/分钟)16和周期性检查来证实适当的麻醉。因为没有下巴和肛门的音调。麻醉深度也可以根据手术刺激的肌肉松弛程度和肌肉 fasciculation 来判断。眼睛反射通常没有值16。镇痛可以提供由机构 IACUC 政策指导。在这种情况下, 一种阿片类药物, 如丁丙诺啡, 可以在手术期间进行管理。</li>
	<li>将猪放在加热垫上, 并在背卧床上抑制手术。</li>
	<li>用手术磨砂 (洗必泰溶液) 和异丙醇来剃腹腹部和准备。</li>
	<li>使用以脐为中心的手术刀刀片进入腹部, 进行8-10 厘米腹中线切口。</li>
	<li>鉴别小肠 (空肠) 约40厘米口服到回盲交界。在创建随后的循环10厘米 (图 1) 之前, 用圆周将肠结扎两次, 在每条结扎之间形成一个厘米, 划定十厘米长的空肠环。</li>
	<li>创建两个循环, 每个时间点的缺血相邻, 一个为缺血, 一个为缺血, 如果需要额外的1h 再灌注。要创建完全缺血 (无论是动脉或静脉的流动), 钳或结扎肠系膜血管使用牛头犬血管钳, 弯曲霍尔斯特德蚊子止血剂, 或2-0 不可吸收的丝绸 1, 2, 3 和 4h, 然后删除夹具1h再灌注, 如果需要的话。 注: 作者创建了所有的缺血循环, 从4h 缺血性循环开始, 进展到下部 (以尽量减少总手术时间)。为解决相邻缺血性肠段可能损害相邻循环的可能性, 缺血性循环的顺序可以改变。这可能会改变总的手术时间。 注: 再灌注的时间范围可以根据感兴趣的研究问题或是否希望在再灌注期进行试验的方法来改变。然而, 随着缺血时间的增加, 组织损伤变得更加严重, 再灌注损伤可能不会导致进一步的上皮损伤。再灌注在轻度缺血性损伤后可能起到作用。</li>
	<li>在缺血时间点之间, 用无菌毛巾和/或毛巾钳保持腹部覆盖或闭合。 注: 在单个动物的实验中, 可以创建八个缺血性片段。确定一个额外的空肠段, 至少5-10 厘米近到最后的缺血性循环, 这将作为一个内部正常控制。</li>
	<li>每只牛头犬血管钳或弯曲霍尔斯特德蚊子止血大约三肠系膜血管阻塞。在止血的应用中必须注意, 因为血管容易受到创伤。试着堆叠在夹子的颚内的容器。</li>
	<li>在实验结束后, 与戊巴比妥85-100 毫克/千克 IV 的安乐死后, 收集所有循环的组织使用 metzenbaum 剪刀, 死后已确认没有心跳别人听诊和丧失角膜反射。首先收集一个正常空肠的控制块, 至少近 5-10 厘米到最后一个缺血性循环。</li>
	<li>将每个伤害时间点的循环分开并存储在冰冷 PBS 的小容器中, 直到准备好进行隐窝隔离。 注: 如果需要, 使缺血性循环足够长, 分成单独的标本, 用于组织学和肠道干细胞培养;然而, 作者发现, 超过约10厘米长的循环最有可能成为出血。</li>
</ol>3. 隐窝 (干细胞) 与缺血性和控制回路分离
<ol><li>用20口径的线和组织钳反转小肠的每个回路, 以暴露粘膜表面。使用缝合 (2-0 不可吸收的丝绸或同等物) 将环的顶部和底部牢固地连接到导线上。</li>
	<li>反转后, 在冰寒 PBS 中冲洗每个回路, 以去除腔内碎片。</li>
	<li>将样品立即放入50毫升锥形管中, 含 DR #1 在冰上30分钟。</li>
	<li>收集循环从控制开始, 并跟随循环递增增加的持续时间的缺血。从损伤较小的组织 (即控制和1小时缺血性组织) 的循环可以有力地动摇, 捕捉手腕, 以取得最佳效果。严重受损组织 (3 和4小时的缺血) 应轻轻摇晃或反转, 只有太多的震动会导致隐窝破坏和额外的组织破坏。在冰上每5分钟应摇动或倒管。</li>
	<li>在37°c 水浴中将样品转移到 DR #2 10 分钟。每5分钟摇动或反转管子。</li>
	<li>在转移组织后, 离心机中含有 DR #1 的圆锥管从2-4 小时损坏的循环中去除 EDTA 上清 (200 x g 5 分钟)。由于这些组织受到高度的破坏, 这是可能的墓穴已经成为离解。用小体积的 PBS (5 毫升) 除去上清和并用重悬颗粒, 然后进行步骤3.9。</li>
	<li>从 DR #2 取出样品, 并将组织样品直接放入冰上25毫升的冷 PBS (标签为洗涤 #1)。将样品放在 60 rpm 的冰上的轨道振动筛上。每次2至5分钟, 每管继续摇晃或反转。</li>
	<li>转移组织后, 将含有 DR #2 的圆锥管旋转2-4 小时, 以除去 EDTA 上清 (200 x g 5 分钟)。由于这些组织受到高度的破坏, 这是可能的墓穴已成为离解。用小体积的 PBS (5 毫升) 除去上清和并用重悬颗粒, 然后进行步骤3.9。</li>
	<li>去除50µL 整除从洗涤 #1 (或从 DR) 检查地窖离解的程度和碎片的数量。将组织放入一个新的50毫升圆锥管 (洗涤 #2, #3 等) 填充25毫升的冷 PBS 和动摇, 直到完整的墓穴被隔离, 最小的碎片和绒毛。 注: 目标是能够隔离干净的分数与完整的地窖和最小的碎片。每个额外的洗涤将清理细胞, 但太多的震动将开始导致二次组织/细胞损伤。此外, 如果将地窖从洗涤步骤中镀出, 而不是从离解试剂步骤中分离出来, 则最好的结果是最少污染。</li>
	<li>除去剩余的组织和离心机的50毫升圆锥管在 200 x g 5 分钟, 以清除上清, 然后并用重悬剩余的隐窝颗粒小体积的 PBS (5 毫升)。</li>
	<li>使用倒置显微镜, 检查每个样本的50µL 整除数, 以确定墓穴/分数的数量。目标是 50-100 crypts/50 µL, 因为这将是最终矩阵帕蒂的大小。 注: 如果样品过于集中, 继续添加冷 PBS, 直到达到预期浓度。如果样品太稀, 确定达到所需的隐窝产量和调整所需的整除数数量。</li>
	<li>整除适当容积 (由整除观察确定) 成离心管。例如, 如果样本包含 50 crypts/50 µL 然后整除50µL 每个帕蒂 X 3 复制 = 150 µL/离心管。如果样本只包含 25 crypts/50 µL 然后2整除数需要/帕蒂 X 3 复制 = 300 µL/管。</li>
	<li>200 x g 的球囊在 4°c 5 分钟。</li>
	<li>轻轻地并用重悬颗粒的地窖与适当的体积的主混合 (50 µL 每井)。在不产生气泡的情况下, 快速吹打15次混合。 注: 预冷却的吸管尖端与冷不育 PBS 之前, 吸主混合。这将有助于保持主混合不扩散。小贴士也可以放在冰箱里, 并放在引擎盖上立即使用。</li>
	<li>将50µL 主混合加上隐窝液滴进入预热前24井板的每个井的中心。</li>
	<li>在37摄氏度孵化培养板30分钟。</li>
	<li>覆盖每个矩阵帕蒂与500µL/井的 IESC 媒体 (没有额外的增长因素需要在0天, 因为它们是包含在主混合)。</li>
	<li>将500µL 无菌 PBS 添加到板上遗留的任何未使用的井中, 以保持湿度。</li>
	<li>计数被镀的地窖的数量在天0。 注意: 将每个细胞培养板预先栅格化, 有助于确保更准确的计数。</li>
	<li>enterospheres 每24小时计数, 并监测 enteroid 发展每日。</li>
	<li>每隔48小时将生长因子添加到每一个井中. 每96小时移除 IESC 介质, 并替换为500µL 新鲜介质 (必须将生长因子添加到介质中)。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+reperfusion+injury+an+important+cause+of+mucosal+damage+after+porcine+intestinal+ischemia.">Is reperfusion injury an important cause of mucosal damage after porcine intestinal ischemia.</a> Surgery. 121 (5), 526-534 (1997).</li><li>Shegarfi, H., 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=Regulation+of+CCN1+(Cyr61)+in+a+porcine+model+of+intestinal+ischemia/reperfusion.">Regulation of CCN1 (Cyr61) in a porcine model of intestinal ischemia/reperfusion.</a> Innate Immun. 21 (5), 453-462 (2015).</li><li>Gubernatorova, E. O., Perez-Chanona, E., Koroleva, E. P., Jobin, C., Tumanov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+intestinal+ischemia-reperfusion+injury.">Murine model of intestinal ischemia-reperfusion injury.</a> J Vis Exp. (111), (2016).</li></ol>

作者没有什么可透露的。

猪节段性肠缺血模型的建立扩大了以往的小鼠模型, 允许研究在同一动物体内组织损伤的多个时间点。该协议有几个关键的讨论点, 包括适当的血管结扎, 组织再灌注和成功的隐窝细胞培养。
正确的血管结扎是建立完全缺血模型的必要条件。如果缝合线不均匀或钳位不完全收紧, 厚壁动脉的血液可能继续进入组织, 由于薄壁静脉的倒塌而无法退出。这导致血液渗出到固有的层层, 造成额外的组织损伤。然而, 根据所研究的缺血性损伤类型, 可能需要完全或失血性缺血。例如, 在肠道移植过程中, 肠道在手术切除阶段完全与血管供应 (动脉和静脉) 分离, 导致完全的肠道缺血。然而, 当肠系膜在一个事件如肠扭转时被扭曲时, 静脉回流往往首先受到阻碍, 导致在动脉供应受阻之前, 组织内增加血液, 从而造成出血性缺血。
缺血性损伤会导致组织损伤, 从绒毛尖开始, 延伸到墓穴的底部3。在缺血期间, 以三磷酸腺苷为形式的能量继续被使用, 并产生代谢物嘌呤。当组织与氧气灌注时, 嘌呤会被黄嘌呤氧化酶代谢, 产生超氧化物自由基, 导致粘膜损伤, 并吸引组织损害的中性粒细胞17,18。在粘膜血管结构中的物种差异以及黄嘌呤氧化酶的变化表达, 导致不同程度的再灌注损伤3。猫和啮齿动物模型的缺血再灌注更容易受到活性氧代谢物的再灌注损伤19,20。相反, 猪被发现有较少的黄嘌呤氧化酶, 因此减少再灌注损伤, 使这个模型更类似于人类肠道缺血的21。此时, 使用挖空或转基因猪模型研究肠道损伤没有被描述, 这使得这一模式的一个主要的局限性。选择合适的动物模型取决于研究者希望研究的疾病过程或特定条件。例如, 6 小时的缺血性损伤的猪模型被描述为22, 而小鼠模型中大多数缺血性手术是45-60 分钟23。
成功地从正常和 ischemically 受损肠道中分离肠腺, 可以研究培养上皮的恢复。这个系统允许研究员单独地集中在上皮, 因为没有血管供应或免疫细胞组分考虑。这提供了一个机会来研究上皮细胞的相互作用和恢复后的伤害, 除了反应不同的生长因子, 或治疗过程中进行手术或继隐窝隔离通过补充培养基。这一步仍然是最困难的, 因为隔离从严重损坏的循环需要温和的震动和快速去除含 EDTA 的溶液, 以防墓穴过早离解。如果这些肠道的循环没有彻底冲洗, 地窖就有可能在文化中受到污染。因此, 除了 IESC 介质外, 还将抗生素防霉溶液添加到两种 DR 溶液中。另一个讨论点侧重于作为正常控制的肠道收集。当动物接受麻醉时, 可能会改变全身组织灌注, 同时可能会循环炎症介质继发于缺血, 即使 "正常" 控制组织也不能代表真正的控制。在这些实验中, 有一点值得注意的是, 在其他实验中, 控制组织出现了严重的和组织学上可比的动物组织 (冈萨雷斯, L.M., 未发表的数据, 2017)。
总之, 该方法描述了猪肠缺血的可再生模型, 并对人体缺血性损伤的发生进行了模型分析。此外, 还介绍了从缺血性循环中分离肠干细胞的方法, 为研究上皮修复和培养治疗的可能反应提供了方法。

肠道缺血性损伤, 由于减少或完全阻断血液流向肠道而导致的氧供应减少, 仍然是人类和动物患者发病率和死亡率的重要原因1,2。缺血性损伤, 随着随后的炎症和细胞浸润, 导致粘膜屏障的妥协。粘膜屏障是预防细菌易位和相关毒素进入系统循环的关键3,4。随后再灌注的缺血性组织可能导致破坏性的活性氧物种的形成, 从而加剧伤害5。由于肠道缺血是很少可预防的, 目前大多数的研究都集中在提高早期发现缺血的技术和开发新的治疗方法, 减少再灌注损伤或靶粘膜修复。
动物模型已被广泛地用于扩大我们的基本科学知识的缺血再灌注损伤, 并仍然是必要的翻译研究。啮齿类动物模型是最广泛使用的由于他们的能力被基因操纵6。然而, 最近, 使用大型动物模型, 特别是猪, 已被提倡未来的翻译研究由于一些优势, 包括猪解剖和生理相似的人类7,8。对缺血再灌注损伤进行了多种损伤模型的研究, 包括完整的血管闭塞、低流量缺血和节段性肠系膜血管闭塞。对这些模型的全面审查是在本文的领域外面然而作者直接读者到最近审查3。
除了体内模型外, 使用前体细胞培养系统为研究肠道稳态和修复损伤提供了一个有希望的工具。肠道干细胞是导致肠道上皮细胞增殖和流动的主要原因。当从正常或受损的肠道分离时, 肠道干细胞可以在培养中保持, 作为研究干细胞和上皮细胞生物学的工具或模型。方法分离和建立这些三维培养系统 (称为 enteroids 和 colonoids 时, 分别从小和大肠) 已经描述了各种种类和器官系统9,10 ,11,12,13。具体来说, 在胃肠道内, 这些培养系统被用来建模胃肠道疾病, 包括癌症, 病原体感染和炎症性肠病14。在这个时候, 没有报告描述从 ischemically 损伤小肠干细胞的分离和维护在任何种类。因此, 我们在这里描述了一个新的, 大型动物猪模型肠道缺血的过程, 导致可再生的伤害和隔离肠道干细胞从正常和 ischemically 受损肠道的能力, 进一步研究恢复以前的体内。

<table border="0" cellpadding="0" cellspacing="0" style="width:640px;" width="640">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Phosphate Buffered Saline, Ca2+, Mg 2+ free</td>
			<td style="width:107px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">BP-399</td>
			<td style="width:167px;">Dilute 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Distilled, deionized water (ddH2O)</td>
			<td style="width:107px;"></td>
			<td style="width:119px;"></td>
			<td>Used to prepare EDTA and PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">20688</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Ethylenediamene tetraacetic acid (EDTA)</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">ED45</td>
			<td>Make fresh before each experiment; pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">1,4-Dithiothreitol (DTT)</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">646563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Y-27632</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">Y0503</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Advanced DMEM/F12</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">12634-010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">N2 Supplement</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">B-27 Supplement</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">12587-010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">HEPES</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">15630-106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Glutamax</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Penicillin/Streptomycin/Amphotericin B</td>
			<td style="width:107px;">Gibco</td>
			<td style="width:119px;">15240-096</td>
			<td>Anti-Anti solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Recombinant human Wnt-3a</td>
			<td style="width:107px;">R &#38; D Systems</td>
			<td style="width:119px;">5036 WN/CF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Recombinant human Rspondin1</td>
			<td style="width:107px;">R &#38; D Systems</td>
			<td style="width:119px;">4645- RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Recombinant human Noggin</td>
			<td style="width:107px;">R &#38; D Systems</td>
			<td style="width:119px;">6057-NG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Recombinant human EGF</td>
			<td style="width:107px;">R &#38; D Systems</td>
			<td style="width:119px;">236-EG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">LY2157299</td>
			<td style="width:107px;">SelleckChem</td>
			<td align="right" style="width:119px;">52230</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">CHIR99021</td>
			<td style="width:107px;">Cayman Chemical</td>
			<td align="right" style="width:119px;">13122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Human [leu]15-Gastrin 1</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">G9145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">SB202190</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">57067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">A83-01</td>
			<td style="width:107px;">Tocris</td>
			<td align="right" style="width:119px;">2939</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Nicotinamide</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">N0636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Acetic Acid</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">695092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Water, WFI Quality</td>
			<td style="width:107px;">Corning, Inc.</td>
			<td style="width:119px;">25-055-CM</td>
			<td>Referred to as sterile water (SW); for growth factor stocks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Matrigel Matrix, GFR</td>
			<td style="width:107px;">Corning, Inc.</td>
			<td align="right" style="width:119px;">356231</td>
			<td>Phenol red free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">24 Well Culture Dish</td>
			<td style="width:107px;">Corning, Inc.</td>
			<td align="right" style="width:119px;">3524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Conical Tube, 50 ml&#160;</td>
			<td style="width:107px;">Corning, Inc.</td>
			<td align="right" style="width:119px;">430828</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Scalpel Handle</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td align="right">500236</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Carbon Steel Surgical Blade, No. 10</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td align="right">504169</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Tissue Forceps</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td align="right">15918</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Debakey Tissue Forceps</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td align="right">501239</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Mayo Scissors</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td align="right">501752</td>
			<td>Curved or straight</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Metzenbaum Scissors</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td align="right">501739</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Mosquito Forceps, Curved</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td>503724-12</td>
			<td>Curved or straight (503728-12)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Hopkins Bulldog Clamp</td>
			<td style="width:107px;">Stoelting Co.</td>
			<td>52120-40P</td>
			<td>Straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Silk, 2-0&#160;</td>
			<td style="width:107px;">Henry Schein</td>
			<td style="width:119px;">685S-BUT</td>
			<td>Any similar brand is acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Towel Clamps</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td align="right">501700</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Needle Holder</td>
			<td style="width:107px;">World Precision Instruments</td>
			<td style="width:119px;">V503382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Wire suture, 20 gauge</td>
			<td style="width:107px;">Henry Schein</td>
			<td align="right" style="width:119px;">19075</td>
			<td>Cut and straighten before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Surgical Towels</td>
			<td style="width:107px;">Henry Schein</td>
			<td style="width:119px;">ST1833</td>
			<td>Any similar product is acceptable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Lactated Ringers Solution</td>
			<td style="width:107px;">Henry Schein</td>
			<td align="right" style="width:119px;">9851</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Chlorhex antiseptic scrub (4%)</td>
			<td style="width:107px;">Henry Schein</td>
			<td style="width:119px;">VINV-CHMX-SCRB</td>
			<td>Any similar brand is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Isopropyl Alcohol 70%</td>
			<td style="width:107px;">Henry Schein</td>
			<td style="width:119px;">MS071HS</td>
			<td>Any similar brand is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">IV catheter, 22 gauge</td>
			<td style="width:107px;">Henry Schein</td>
			<td style="width:119px;">2225PUR</td>
			<td>May need 20g or 24 g depending on size of the vein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Xylazine (100 mg/ml)</td>
			<td style="width:107px;">Henry Schein</td>
			<td align="right" style="width:119px;">33198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Ketamine (100 mg/ml)</td>
			<td style="width:107px;">Henry Schein</td>
			<td style="width:119px;">11695-6835-1</td>
			<td>Controlled medication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Isoflurane solution</td>
			<td style="width:107px;">Henry Schein</td>
			<td align="right" style="width:119px;">10015516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Pentobarbital (Fatal Plus Euthanasia Solution (390 mg/ml))</td>
			<td style="width:107px;">Vortech Pharm.</td>
			<td style="width:119px;"></td>
			<td>Multiple brands of Pentobarbital Sodium available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Heating pad&#160;</td>
			<td style="width:107px;">Gaymar</td>
			<td style="width:119px;"></td>
			<td>Tpump Core Warming System; others are available.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Mindray Datascope Monitor</td>
			<td style="width:107px;">Mindray North America</td>
			<td style="width:119px;"></td>
			<td>Any equivalent piece of monitoring equipment acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Vaporizer&#160;</td>
			<td style="width:107px;">Vetland Medical</td>
			<td style="width:119px;"></td>
			<td>Recommended to use a Circle System w/ Y piece; multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Fluid Pump</td>
			<td style="width:107px;">Abbott Hospira</td>
			<td style="width:119px;"></td>
			<td>Plum A+; Any similar manufacturer is recommended.</td>
		</tr>
	</tbody>
</table>

intestinal stem cell isolation and culture in a porcine model of segmental small intestinal ischemia

肠道缺血仍然是人类和兽医患者发病率和死亡率的主要原因。许多疾病过程导致肠缺血, 当血液供应, 因此氧气减少到肚腑。这导致肠道屏障的损失和损害的基础组织。肠道干细胞位于 Lieberkühn 的窝底, 并负责在稳态和损伤后肠道的更新。"体外" 细胞培养技术通过建立支持三维上皮器官样系统生长的培养条件, 可以成功地研究上皮干细胞相互作用 (称为 "enteroids" 和 " colonoids "从小肠, 分别)。这些 enteroids 是由隐窝和绒毛状的领域和成熟, 以包含所有的细胞类型在上皮内发现。从历史上看, 小鼠模型被用来研究肠道损伤。然而, 猪模型提供了一些优势, 包括大小的相似性以及胃肠解剖学和生理学的人。通过利用猪模型, 我们建立了一种协议, 在这种情况下, 可以在单个动物体内建立肠缺血的节段循环, 从而研究缺血性损伤的不同时间点和修复体内。此外, 我们描述了一种方法, 以分离和培养肠道干细胞从缺血性循环, 允许继续研究上皮修复, 由干细胞调节,前体.

完全肠道缺血是在小肠循环中产生的, 利用血管闭塞缝合或钳, 如图 1所示。通过释放钳, 可以进行一个控制期的再灌注, 允许额外的研究后再灌注损伤如果需要的话。所有动物都在手术过程中幸存下来, 并发症极小, 直到安乐死。最常见的外科并发症是低血压, 它解决了补充一个积极的 inotrope, 如多巴酚丁胺。
如果正确执行, 缺血性损伤将开始在肠道绒毛的尖端, 并在墓穴内迁移下来, 因为缺血的持续时间增加 (图 2)。当血管没有结扎或夹紧时, 手术技术的一个常见错误就会发生。结果是失血性缺血 (图 3), 其中薄壁静脉在动脉前塌陷, 允许额外的血液渗入组织。在完全缺血 (图 3, 右) 的情况下, 与较浅的表面相比, 这被视为深紫色浆膜表面 (图 3, 左)。
在去除缺血性肠循环之后, 肠道墓穴在离解协议后成功隔离 (图 4)。正如预期的, 更严重损坏的 timepoints 的墓穴经常被打破 (f; 片段) 和墓穴分数包含更多的背景细胞碎片相比, 没有或轻微的损害。在该议定书中, 严重受损的肠道必须轻轻摇动, 以避免对底层组织造成额外伤害。摇晃得太粗略会导致进一步的隐窝损伤, 而大多数的墓穴最终会出现在含 EDTA 的 DR 溶液中, 从而造成额外的干扰。
一旦电镀, 所有的缺血时间点的地窖生存, 并能够成为建立在文化 (图 5)。当正常和轻度损伤的肠道墓穴被镀在培养, enterospheres 在24-48 小时内形成。由于严重缺血性损伤 (3 和4小时), 肠道墓穴存活, 但被破坏, 导致形成更小的球体最初。由72-120 小时, enteroids 变得更加复杂与明显的中央流明和萌芽结构。总体而言, enteroids 的生长效率下降, 以及从严重受损的肠道组织 (冈萨雷斯, L.M., 未发表的数据, 2017) 中提取的数量减少。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57647/57647fig1.jpg"> 图 1: 猪模型完全性肠缺血的手术模型.A) 正常的猪空肠形象化。B) 肠系膜血管已结扎, 缝合造成肠道缺血。C) 用牛头犬血管钳制造缺血, 如果需要, 可以组织再灌注。D) 切除血管钳后立即肠道血管。<a href="/files/ftp_upload/57647/57647fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57647/57647fig2.jpg"> 图 2: 组织学证据 (苏木精和伊红 (H & E) 染色) 在完全肠道缺血持续较长时间后上皮损伤增加.损伤开始于绒毛的尖端, 逐渐失去单细胞上皮层, 绒毛钝化和细胞损伤延伸到墓穴底部, 严重损伤 (最多4小时)。100µm 秤吧。I = 缺血。<a href="/files/ftp_upload/57647/57647fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57647/57647fig3.jpg"> 图 3: 出血性缺血的毛和组织学证据.A) 对出血性缺血 (左循环) 和完全缺血 (右环) 进行比较的总照片。当血管不被结扎或夹紧时, 血液会继续渗入组织, 导致额外的炎症和损伤。B) 1-4 小时内出血缺血的 h & E 图像。除了细胞损伤完全缺血, 有证据显示红细胞浸润在周围的固有层。100µm 秤吧。I = 缺血。<a href="/files/ftp_upload/57647/57647fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57647/57647fig4.jpg"> 图 4: 整除数从缺血性和正常肠道循环中分离出肠道墓穴。完整的, 完整的肠道墓穴 (星号) 成功地从每一个肠环分离。如预期, 较严重损坏的时间点的墓穴经常被打破 (f; 片段) 和墓穴分数包含更多的背景细胞碎片相比, 没有或轻微的损害。100µm 秤吧。I = 缺血。<a href="/files/ftp_upload/57647/57647fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57647/57647fig5.jpg"> 图 5: 从小肠缺血性循环分离后肠道干细胞生长的时间过程.当正常的肠道墓穴被镀在培养, enterospheres 形成24-48 小时内。由于严重缺血性损伤 (3 和 4 h), 墓穴存活, 但形成更小的球体最初。由72-120 小时, enteroids 变得更加复杂与明显的中央流明和萌芽结构。20µm 刻度条除非注明。<a href="/files/ftp_upload/57647/57647fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>生长因子</td>
			<td>稀释 剂</td>
			<td>库存浓度</td>
			<td>库存稀释</td>
			<td>工作稀释</td>
		</tr>
<tr>
<td>R-Spondin</td>
			<td>Pbs</td>
			<td>100X</td>
			<td>100µg/毫升</td>
			<td>1µg/毫升</td>
		</tr>
<tr>
<td>脑袋</td>
			<td>SW/0.1%BSA</td>
			<td>1000X</td>
			<td>100µg/毫升</td>
			<td>100 ng/毫升</td>
		</tr>
<tr>
<td>Egf</td>
			<td>10mM 乙酸</td>
			<td>10,000X</td>
			<td>500µg/毫升</td>
			<td>50 ng/毫升</td>
		</tr>
<tr>
<td>A-83-01</td>
			<td>Dmso</td>
			<td>1000X</td>
			<td>500µM</td>
			<td>500毫微米</td>
		</tr>
<tr>
<td>SB202190</td>
			<td>Dmso</td>
			<td>3000X</td>
			<td>30毫米</td>
			<td>10µM</td>
		</tr>
<tr>
<td>烟 酰 胺</td>
			<td>西 南部</td>
			<td>1000X</td>
			<td>1米</td>
			<td>1毫米</td>
		</tr>
<tr>
<td>胃 泌 素</td>
			<td>Pbs</td>
			<td>10,000X</td>
			<td>100µM</td>
			<td>10毫微米</td>
		</tr>
<tr>
<td>Y-27632</td>
			<td>Pbs</td>
			<td>1000X</td>
			<td>10毫米</td>
			<td>10µM</td>
		</tr>
<tr>
<td>LY2157299</td>
			<td>Dmso</td>
			<td>10,000X</td>
			<td>5毫米</td>
			<td>0.5 µM</td>
		</tr>
<tr>
<td>CHIR99021</td>
			<td>Pbs</td>
			<td>1000X</td>
			<td>2.5 毫米</td>
			<td>2.5 µM</td>
		</tr>
<tr>
<td>Wnt3a</td>
			<td>Pbs</td>
			<td>2000X</td>
			<td>200µg/毫升</td>
			<td>100 ng/毫升</td>
		</tr>
</tbody></table>表 1: 生长因子试剂表.本议定书使用的生长因子库存解决方案和工作解决方案摘要。

Watch this Scientific Journal Video about Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia at JoVE.com

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

该协议将使读者能够成功地建立猪节段性肠缺血模型, 随后分离和培养肠道干细胞, 用于研究损伤后的上皮修复。

猪节段性小肠缺血模型中肠道干细胞的分离与培养

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

intestinal-stem-cell-isolation-culture-porcine-model-segmental-small

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Medicine

Unit 1

Stem Cell Biology And Renewal in Epithelial Tissue

SCIENTISTS IN ACTION

10.7K 次观看.  North Carolina State University. 该协议将使读者能够成功地建立猪节段性肠缺血模型, 随后分离和培养肠道干细胞, 用于研究损伤后的上皮修复。

视频: Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

异位自体肾移植的猪模型:有步骤，分步协议

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀:一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

研究活体器官的肠道屏障分解

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

猪缺血/再灌注模型中脊髓微灌注的实时评估

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

透明质酸35 kDa对肠源单层早产小肠损伤体 外 模型及愈合的影响

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...

猪节段性小肠缺血模型中肠道干细胞的分离与培养

摘要

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此视频中的章节

一个小鼠模型在子宫内移植

在干细胞移植在体外模拟缺血/再灌注模型

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

异位自体肾移植的猪模型:有步骤，分步协议

肠缺血再灌注损伤的小鼠模型

毛喉素诱导在肠组织体溶胀:一个

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

研究活体器官的肠道屏障分解

猪缺血/再灌注模型中脊髓微灌注的实时评估

透明质酸35 kDa对肠源单层早产小肠损伤体外模型及愈合的影响

猪节段性小肠缺血模型中肠道干细胞的分离与培养

摘要

探索更多视频

此视频中的章节

一个小鼠模型在子宫内移植

在干细胞移植在体外模拟缺血/再灌注模型

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

异位自体肾移植的猪模型:有步骤，分步协议

肠缺血再灌注损伤的小鼠模型

毛喉素诱导在肠组织体溶胀:一个

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

研究活体器官的肠道屏障分解

猪缺血/再灌注模型中脊髓微灌注的实时评估

透明质酸35 kDa对肠源单层早产小肠损伤体 外 模型及愈合的影响

透明质酸35 kDa对肠源单层早产小肠损伤体外模型及愈合的影响