The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

所有涉及这个手稿中描述动物的程序进行了根据瑞士法律和瑞士苏黎世州的兽医行政管理部门批准。 1.缓冲液配制<ol><li>制备溶液1 - 4和抗利尿激素（ADH）溶液（ 表1）。 </li><li>制备透析缓冲液（ 表1）。 注意:这是用作灌注在透析缓冲的缓冲器。以后，红细胞将在这个缓冲液稀释以形成最终灌洗液。 </li><li>红细胞制剂。 <ol><li>稀释250毫升人红细胞浓缩至500毫升透析缓冲液（从当地血库获得测试材料）。离心在2000 xg离心8分钟。删除缓冲区，注意不要删除任何红细胞。重复3倍。 </li></ol></li><li>制备白蛋白（牛血清白蛋白; BSA）的缓冲液中。 <ol><li>在200毫升dialysi的的缓冲器中，用搅拌棒溶解44克BSA的。过滤用滤纸将溶液。 </li></ol></li><li>准备灌注液。 <ol><li>从步骤1.3.1通过滤纸进入BSA缓冲过滤红细胞。填充到800毫升的透析缓冲液的总体积。 注意:这是最后的灌注液。血细胞比容现在应该在8和12％之间。灌注液可以储存于4℃多达12小时下进行。 </li></ol></li></ol> 2.起爆透析和氧合<ol><li>打开周围较大缓冲储水，较小的缓冲液贮存和湿室（一个小的，双壁腔带到37℃和100％湿度稍后握住肾）至37℃的水浴（ 图2） 。 </li><li>填充透析缓冲液的较大的缓冲区贮存器和与灌注液较小贮存器。 </li><li> 2/95％O 2的气体流入转动5％CO到透析缓冲液。 </li><li>打开对透析缓冲灌注液的连续透析。小心使用低通量透析管。继续执行步骤3。 </li></ol><img alt="图2" src="/files/ftp_upload/54712/54712fig2.jpg" /> 图2:灌注回路的示意图。方案示出了灌注回路的主要组成部分和缓冲液流的方向。由深蓝色包围所有部件都保持在37℃的水浴/恒温器。 1:灌注缓冲液中的至少3倍体积的透析缓冲液连续地用95％O 2/5％CO 2鼓泡。 2:透析缓冲液和灌注缓冲液被连续地对在透析管彼此通过一个滚子泵透析。 3:由于这种透析，在灌注缓冲液用9％氧气富集2/5％CO 2和电解质水平保持恒定throughoUT灌注。 4:滚子泵推动朝肾脏灌注缓冲区。 5:删除的Windkessel蠕动波，并作为一个泡沫陷阱。 6:压力传感器（连接到4（滚子泵），以保持连续的压力，同时允许自由交替流）。 7:在整个灌注，肾保持在100％的空气湿度和37℃肾温度的潮湿室中。 <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.手术过程第1部分（适用于所有结扎的示意图，见图3） 注意:请用5-0外科缝线结扎所有。 <ol><li>麻醉通过腹膜内注射的小鼠（体重20毫克/毫升氯胺酮和1毫克/毫升甲苯噻嗪溶解在0.9％NaCl中的10微升/克）。确认anesth足够的深度ESIA通过测试后无足反射的。 </li><li>固定鼠标在湿室仰卧位置。保护眼睛与兽医药膏。放置脊柱低于1ml注射器来升高腰椎血管。 </li><li>从耻骨嵴到胸骨开幕先将患处，然后腹肌，用剪刀执行中位数剖腹探查术。 </li><li>除去肠并将其放置在从腹部鼠标侧的左侧。 </li><li>无结缔组织，膀胱，探索两侧输尿管和尿道。 </li><li>周围放置左侧输尿管（结扎I）的结扎。关闭它。 </li><li>将尿道周围（结扎II）连字。关闭它。 </li><li>将围绕整个膀胱（结扎III）一个"套索"连字。 </li><li>切开膀胱1毫米。 </li><li>导管插入与2厘米PE管材50开。 </li><li>关闭结扎III管道周围。 </li><li>剪切从结扎左侧输尿管和尿道远端。该BLAD德现在连接到右输尿管仅自由移动。 </li><li>清除结缔组织和脂肪的腹主动脉。 </li><li>将腹部中期主动脉结扎（结扎IV）。 </li><li>周围放置肠系膜上动脉和腹腔干（结扎Ⅴ）之间的隔膜下方的主动脉结扎。 </li><li>周围放置肠系膜动脉（结扎VI）的结扎。 </li><li>放置一个主动脉结扎正下方的右侧和左侧肾动脉（结扎Ⅶ）以上。 </li><li>周围放置尾静脉包（静脉）（结扎Ⅷ）结扎。继续执行步骤4。 </li></ol><img alt="图3" src="/files/ftp_upload/54712/54712fig3.jpg" /> 图3:手术时放置在连字的示意图。查看剖腹手术后开腹部。肠道移出到左侧。 L和R指示的左右肾。该黑线显示各个结扎的面积。连字放在前面，然后关闭，在文中给出的顺序排列。 X标记为主动脉插管切口的位置。 <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.灌注回路的引发<ol><li>启动回转泵和填充灌注液管路。小心从它清空所有气泡。 </li><li>该设备的Windkessel填写大约级中旬灌流。 </li><li>校准压力传感器为0毫米汞柱时所有管路被填充和流量为0保持在肾脏水平灌注针在这段时间。 </li><li>保持流动在一个恒定的最低水平（0.6ml /分钟），并继续执行步骤5。 </li></ol> 5.手术过程第2部分<ol><li>放置结扎IV和左RENA的分支之间的夹紧升动脉。 </li><li>使结扎IV的主动脉尾一小切口，注意不要切背墙。 </li><li>用扩张血管扩张主动脉开幕。 </li><li>导管插入用针主动脉（2厘米长，拉PE 50），推尖端正好夹。 </li><li>打开夹子。 </li><li>推针颅的前端，直到它到达右肾动脉和主动脉的连接处。 </li><li>关闭结扎七。 </li><li>关闭结扎IV。 </li><li>通过解剖隔膜打开用剪刀胸部。通过单一切口，分离主动脉，腔静脉，心脏和植物神经。与此步骤中，该动物是通过在连续深度麻醉迅速放血处死。 </li><li>启动灌注泵的压力控制。保持80至100毫米汞柱之间的平均压力。 </li><li>关闭结扎V. </li><li>关闭结扎VI。 </li><li>关闭结扎八。 </li><li>从结缔组织Tissu酒店免费右肾e和它嵌入到用剪刀脂肪囊。 </li><li>切主动脉近端至结扎线V. </li><li>切肠系膜上动脉远端，以结扎线VI。 </li><li>切肾血管配套捆绑了，小心不要切入血管自己。 </li><li>割肝在连接到肾脏。照顾到释放肾，但将肝粘附的一小部分到它，因此，腔静脉通过它保持打开。 </li><li>取肾捆绑了鼠标。从湿室中取出鼠标。 </li><li>周围放置肝脏和肾脏（结扎IX）的连接的"套索"连字。 </li><li>导管插入与静脉线路（2毫升的PE 50）的下腔静脉。 </li><li>关闭结扎九。通过静脉行静脉流出应立即启动。 </li><li>关闭湿室。 </li></ol> 6.下游分析<ol><li>在以下小时，连续监测血流量和intravasculAR压力15。收集静脉流出，它可以被用于分析，例如，肾肾素释放7。收集尿液的电解质浓度及肾小球滤过率14分析。灌注1小时后，肾脏可以管理单元冷冻western印迹或固定成像方法16。 </li></ol>

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The authors have no competing financial interests and nothing else to disclose.

离体灌注肾脏鼠标是用于在受控环境中的体外研究肾功能1小时，架桥在完整的动物体内实验，这可以通过许多全身性因素的影响，是有缺陷的之间的间隙的一个工具，并在体外实验中分离的肾段或培养细胞，这必然忽视完整器官结构对功能的影响。还有就是，以作者的知识，没有替代技术，用以执行这一特定的任务。对肾组织的生化研究，然而，可以在使用肾脏切片17</sup...

器官的隔离灌注一直生理学家中不断努力的主题几十年1。该技术使器官的功能，而没有全身的影响，如血压，激素，或神经，进行研究。卡尔·爱德华Loebell被认为是第一个所描述的一个孤立肾灌注成功，于1849年2。此后，灌注设备发生了显著细化。弗雷和格鲁伯引入了氧合搏动泵人工肺的连续灌流2。虽然早期的研究人员重点研究大型哺乳动物-即，猪2和狗3使用大鼠肾脏的-the第一份报告，魏斯等人的肾脏。 ，是小型哺乳动物器官灌注4研究的一个里程碑。 Schurek 等。报道添加哺乳动物红细胞到灌流如果有足够的肾小管的必要性氧合要达到5。临界长期实验是由相同的研究组6引进缓冲器的连续透析。在2003年，Schweda 等。是第一个报告功能的鼠标离体灌注肾（MIPK）7，后来被Rahgozar 等精制而成。 18和林德尔等。 14。 虽然在技术上不是孤立肾灌注大鼠更具挑战性，使用MIPK负有能够使用各种各样基因改造的小鼠中的优势。本文介绍了作者的方法的详细信息隔离灌注小鼠肾1小时。该方法允许对肾流量，血管阻力，激素释放，血气分析，尿液分析，以及药物的应用程序的连续评估。以下的方法，肾脏可以用于分子和生化分析进行处理，是固定的显微镜，或移植到受体小鼠（ 图1）。 <img alt="图1" src="/files/ftp_upload/54712/54712fig1.jpg" /> 图1:可能的输入/输出到离体肾的概述。 BGA:血气分析。 <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 这种技术可能会得到更多的关注，未来数年，多创新应用正在与长期常温肾脏血流灌注的曙光移植前的讨论（含或不含抗排斥或基因组编辑药物的应用）8，9，10 11，从脱细胞支架12整个肾脏的生物工程，剂量和高剂量荧光染料对多光子成像13应用</sup&gt;。这也是与研究急性肾损伤14中特定基因的作用的理想模型。 一个一步一步的协议，是考虑到让其他实验室成功地执行孤立的小鼠肾脏灌注。首先，组合物和制剂的缓冲液中被指定。然后，手术中详细描述和示出的关键步骤。第三，数据呈现的是，用于展示成功制备的:肾血流量，血管阻力，肾小球滤过率和分数电解质排泄-所有灌注肾脏的不同肾段的形态的生存能力和透射电子显微照片的功能性测量灌注后的1小时固定。

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Perfusion Circuit:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Moist chamber 834/8</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2901</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Cannular with basket and side port</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2947</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Thermostat TC120-ST5&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-4544</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">ISM 827/230V Roller Pump Reglo Analogue</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-0114</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 1L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3438</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 0.5L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3436</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Pressure Transducer APT300&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3862</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">TAM-D Plugsys Transducer</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-1793</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">SCP Plugsys servo controller</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2806</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Windkessel&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3717</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">HSE-USB data acquisition</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Low-Flux Dialysator Diacap Polysulfone</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">7203525</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:447px;">PE-Tubing for aorta cannulation 1.19mm I.D. x 1.70mm O.D.</td>
			<td style="width:173px;">Scientific Commodities Inc.</td>
			<td style="width:119px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:447px;">Name</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Buffer reagents:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Aminoplasmal 10%</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">134518064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium pyruvate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2256-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-Glutamic acid monosodium salt hydrate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1626-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-(-)-Malic acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1125-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium-L-Lactate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">L7022-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">alpha-Ketoglutaric acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">K1875-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaHCO3</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">60130-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">U5378-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatinine</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4255-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td style="width:173px;">Roche</td>
			<td align="right" style="width:119px;">10835242001</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2 * 6H2O</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2393-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">D-Glucose</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2 * 6H2O&#160;</td>
			<td style="width:173px;">Riedel-de-Ha&#235;n</td>
			<td align="right" style="width:119px;">12074</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Antidiuretic Hormone dDAVP</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">V2013-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">FITC-Inulin</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Filter used for erythrocyte filtration</td>
			<td>Macherey-Nagel</td>
			<td style="width:119px;">MN 615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BGA Analysis:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">ABL 80 flex</td>
			<td style="width:173px;">Radiometer Medical ApS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Electron Microscope:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Philips CM100 TEM</td>
			<td style="width:173px;">FEI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the mouse isolated perfused kidney technique

鼠标离体灌注肾（MIPK）是用于灌注和1小时官能体外条件下保持一个小鼠肾脏的技术。这是研究分离的器官，并为许多创新的应用程序，可能会在未来成为可能，包括灌注脱细胞肾生物工程或抗排斥或基因组编辑药物在高剂量黄金肾脏的管理生理学的一个先决条件用于移植。在灌注时，肾脏可以被操纵，肾功能可以评估，和给药的各种药物。手术后，肾脏可以移植或用于分子生物学，生物化学分析，或显微处理。 本文介绍了灌流，需要鼠标肾脏的体外灌注的手术技术。灌注装置的细节给出并且呈现表示V数据肾脏的制剂iability:肾血流量，血管阻力，和尿的数据作为不同肾段作为形态学读数，和不同肾段作为分子读出的转运蛋白的蛋白质印迹的官能，透射电子显微镜照片。

与所描述的方法，分离小鼠肾可为至少1小时保持存活。我们后官能（肾血流量和血管阻力，静脉流出，肾小球滤过率，尿分数Na +和K +排泄血气分析和尿液摩尔渗透压浓度）和形态（透射电子连续灌注的1小时测试的组织活力显微镜，TEM）的方法，在野生型C57BL / 6小鼠四个肾脏。此外，进行了不同的小管的根尖标记蛋白的蛋白质印迹，隔离灌注肾脏比较同一...

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The Mouse Isolated Perfused Kidney Technique

鼠标离体灌注肾（MIPK）是用于灌注和1小时官能体外条件下保持一个小鼠肾脏的技术。的缓冲液和外科技术进行详细说明。

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

the-mouse-isolated-perfused-kidney-technique

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

21.5K 次观看.  University of Zürich. 鼠标离体灌注肾（MIPK）是用于灌注和1小时官能体外条件下保持一个小鼠肾脏的技术。的缓冲液和外科技术进行详细说明。 

视频: The Mouse Isolated Perfused Kidney Technique

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

科研

生物学

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

表征隔离，通风，和仪表小鼠肺灌注与脉动流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...