The authors thank the support of the Zell Family Foundation. We recognize support from the Northwestern Memorial Foundation Dixon Translational Research Grants Initiative, the American Society of Transplant Surgeon&#8217;s Faculty Development Grant, and a Research Grant for the Young Investigator from the National Kidney Foundation of Illinois. We acknowledge support from the Robert R. McCormick Foundation. This work was also supported by NIDDK K08 DK10175 to J.A.W. Imaging and histology cores used for this research is supported by the Mouse Histology and Phenotyping Laboratory, Electron Probe Instrumentation Center (EPIC), and Simpson Querrey Institute Equipment Core at Northwestern University, and a Cancer Center Support Grant (NCI CA060553). The authors would like to acknowledge the Northwestern University Microsurgery Core for rodent kidney procurements. Evaluation of renal tubular epithelia morphology following recellularization conducted in the Fluorescence Microscopy Shared Resource supported by P30 CA118100.

道德守则：所有涉及动物的程序是根据经西北大学的机构动物护理和使用委员会的指导方针进行。 1.肾脱细胞<ol><li>准备脱细胞的解决方案。准备试剂以下几卷对每个肾脏被脱细胞，加上一个额外的容量（ 例如 ，4肾脏，准备5000毫升的Triton X-100的步骤1.1.3。）： <ol><li>制成500毫升之反渗透纯水（ROH 2 O）。注：另外，去离子水可在ROH 2 O指示步骤中使用。 </li><li>在ROH 2 O制备千毫升的Triton X-100，1％（体积/体积）慢慢加入10 mL的Triton X-100到990毫升的水在一个搅拌盘下快速搅拌的大烧杯中。允许将试剂完全溶解后再使用（至少10分钟）。 </li><li>制备千毫升的Triton X-100，1％（体积/体积）中ROH 2 O（SEP独的体积）。 </li><li>制备千毫升十二烷基硫酸钠（SDS），0.1％（体积/体积）中ROH 2 O.下快速搅拌上的轰动板慢慢加入5毫升SDS（20％原液）为995毫升的水在一个大烧杯中。允许试剂的使用（至少10分钟）之前均匀混合。 </li><li>制成500毫升之ROH 2 O（另册）。 </li></ol></li><li>准备为肾脏脱细胞。 <ol><li>恢复从雄性Sprague Dawley大鼠（250-300克）上进行肾如前所述7。 <ol><li>通过腹膜内注射戊巴比妥（50毫克/千克体重）麻醉大鼠。经常检查麻醉深度（每10-15分钟）通过手术​​过程中监测呼吸功能，心脏速率，和脚趾捏响应。如果动物具有升高的呼吸率或阳性踏板反射，辖补充剂量（用1/3至初始剂量的1/4）戊巴比妥。 </li><li>进行纵向中线abdomina升切口以暴露肾脏，腹主动脉和下腔静脉。 </li><li>注入2000 USP肝素单位/ kg体重到阴茎静脉。 </li><li>动员双肾轻轻清扫。仔细分离肾周脂肪肾，同时保持周围的肾脏完好的肾小囊。灌注通过红外线肾腹主动脉冷盐水（10毫升）中的肾脏。 </li></ol></li><li>插入24号导管到肾动脉，紧紧结扎导管到动脉，并且（使用注射器）轻轻灌注用10ml冷的磷酸盐缓冲盐水（PBS）中的血液完全清除器官的肾。 </li><li>内培养皿并将其放置在-20℃冷冻至逐渐冻结器官（由此诱导细胞裂解），用于存储直到脱细胞浸入肾脏在25毫升PBS中。 注：如果需要的话，输尿管和/或肾静脉还可插管以逆行播种，虽然在THI未描述的方案。 </li><li>完全解冻冷冻肾（equilibriate在室温（RT）），并用10毫升的PBS轻轻灌注检查结扎肾动脉是否有泄漏。如果泄漏或显著性观察，再插入导管肾动脉。 </li></ol></li><li>准备设备脱细胞。组装脱细胞灌注系统如在图1B中所描绘。 <ol><li>连接一个8“蠕动泵管（ 图1D，F）的长度为两个充分的长度（&gt; 36”1/16“内径（ID）硅橡胶管（ 图1D，H）推荐）。使用两个阳鲁尔锁由一个阴鲁尔个插座适配器露儿加入管段（ 图1D，J）加入1/8“带刺的适配器。额外的凸形Luer锁插入到1/8“倒钩的适配器（ 图1D，三）进入灌注线的下游端用于连接到肾动脉导管。 </li><li>连接使用大泵筒每个蠕动泵管段的四辊泵头。 </li><li>将硅橡胶管的上游端的第一溶液槽（ROH 2 O）后，持“擎天柱”按钮，完全素每个灌注电路的解决方案。临界：确认没有气泡在管保持截留，而上游端（左开吸取流体）的硅橡胶管被固定的试剂容器的液体-空气界面的下方，如在图1B中所描绘。 </li></ol></li><li>在RT 7进行脱细胞协议。 <ol><li>各解冻肾的肾动脉导管连接到从所述泵的下游的灌注回路管的末端，以确保没有气泡被截留在导管。允许肾脏被沿一个空的5升烧杯（灌注液收集容器的内壁悬挂，参见图茜1B），使得肾动脉不在扭结或盘绕。 </li><li>调整泵驱动到5毫升/分钟，并按下“开始”按钮。确认每个肾脏由从器官的底部观察溶液滴灌注。 </li><li>如在图1A中描述的灌注每个肾脏用以下试剂<ol><li>用500ml ROH 2 O灌注在5毫升/分钟1小时，40分钟。 </li><li>用1,000ml 1％的Triton X-100的灌注在5毫升/分钟3小时，20分钟。 </li><li>用1,000ml 1％的Triton X-100的灌注以1毫升/分钟的16小时，40分钟。 </li><li>用1,000ml 0.1％SDS中灌注在5毫升/分钟3小时，20分钟。 </li><li>用500ml ROH 2 O灌注在5毫升/分钟1小时，40分钟。 注意：每个脱细胞的肾脏可以存储在PBS（不含添加剂）在50ml锥形管中，在4℃下最多两个星期后使用。 </li></ol></li></ol></li></ol> 2.每融合生物反应器组件，肾脏消毒，并准备Recellularization <ol><li>准备生物反应器的船只。 <ol><li>用温水洗净玻璃生物反应器水库（体成分）和生物反应器磁头，盘稀释洗涤剂溶液（ 如自来水中的1％洗洁精溶液），并用自来水，然后ROH 2 O.彻底冲洗允许组件完全干燥。 </li><li>适用硅化试剂到每个容器的底部的小体积（〜5ml）中。确保试剂润湿整个底面几秒钟，然后沥干多余的液体。 </li><li>允许储层干燥在通风橱中在室温O / N。可替换地，储存器可以在烘箱中干燥，在100℃下进行30分钟，以加快干燥和改善涂层，其目的是防止细胞附着到玻璃生物反应器容器的底部的耐用性。 </li></ol></li><li>准备灌注电路元件进行灭菌 ​​（ 图1D）。 <ol><li>切管长度如下（对于每个生物反应器）： <ol><li>削减ID硅橡胶管（ 图1D，H）两台25“的1/16长度”。 </li><li>切蠕动泵管一8“长（ 图1D，F）。 </li><li>削减ID硅橡胶管1 4.25“的1/16长度”。 </li><li>削减ID硅橡胶管（ 图1D，G）2个1“的厚壁1/16长度”。 </li><li>切割的1/4“内径×0.5”外径（OD）的硅橡胶管（ 图1D，e）一种2“的长度。 </li></ol></li><li>准备以下的Luer适配器（对于每个生物反应器）： <ol><li>准备10阳鲁尔锁1/8“刺适配器（ 图1D，C） </li><li>准备2阳鲁尔插头（ 图1D，一 ） </li><li>准备2阴鲁尔帽（ 图1D，B） </li><li>准备5阴鲁尔个插座鲁尔适配器（X5; 图1D，D） </li></ol></li><li>用温水清洗所有管路和露儿适配器，稀洗碗精的解决方案，在自来水彻底冲洗，然后ROH 2 O，并让他们干。 </li></ol></li><li>组装灌注回路。 <ol><li>滑1“的厚壁1/16”ID硅橡胶管长度超过附着在生物反应器的入口头部受露儿。将阳鲁尔锁1/8“ID倒钩适配器插入管开口端。重复其他入口露儿受体，并连接一个女鲁尔帽封锁端口（用于在本协议中未说明输尿管或静脉播种技术）。 </li><li> ID硅橡胶管使用阳鲁尔锁蠕动泵管的8“段各25”的1/16段“连接到1/8”ID倒钩和女性露儿个插座适配器露儿（ 图1D，J）。 </li><li>连接硅橡胶tubin一端开口g至在生物反应器头部的外侧上的媒体灌洗液插座。连接ID管的4.25“的1/16长度”到媒体灌洗液出口的相反（内）侧上的生物反应器头部的内部。 </li><li>阳鲁尔锁连接到1/8“带刺的ID适配器灌注电路管道的剩余开口端。连接阴鲁尔个插座适配器露儿的阳鲁尔锁。连接阴鲁尔个插座露儿适配器开阳鲁尔锁进领导的生物反应器的入口露儿受体。这将作为媒体灌流入口直接通往插入导管肾动脉。 </li><li>确保在生物反应器盖没有端口处于打开或暴露。紧紧贴着对身体的生物反应器真空阀。 </li><li>适合的生物反应器头部在贮液器本体，与O型环相同的槽衬的每个组件夹在中间。放置一个金属夹具通过接口来将两个部件连接在一起。 </li><li>适合于在生物反应器头部的排气口与一0.2微米的通气滤膜，使用1/4“内径×0.5”OD的硅橡胶管（ 图1D，E）一2“长度连接两个组件。打开排气阀。 </li><li>稍微拧松生物反应器头部红色螺丝帽。 </li><li>将剩余的露儿适配器和6“锯齿形标本镊子在高温高压消毒袋和密封。 </li></ol></li><li>高压灭菌器组装生物反应器和阳鲁尔插头。 </li><li>播种（卷每列脱细胞肾）前准备脚手架消毒和中灌注下列试剂： <ol><li>内通风橱中，加入在1L瓶2.56毫升过乙酸溶液（39％储备浓度）和40毫升200标准乙醇至〜957毫升ROH 2 O制备50ml的0.1％过乙酸，4％的乙醇溶液。通过反复颠倒瓶调匀，放入生物安全柜。 </li><li>准备150ml的1×PBS溶液，预先高压灭菌。 </li><li>制备补充有10％胎牛血清（FBS）的50毫升DMEM / F12培养基和1％青霉素 - 链霉素（青霉素 - 链霉素）。 </li></ol></li><li>收集灌注生物反应器组件，其余的项目。放置在生物安全柜中的所有项目。关键：确保生物安全柜配备有正常工作的电源插座，以数字泵的驱动功率。另外，使用延长线给水泵驱动器的外部电源插座柜外连接。 <ol><li>收集脱细胞肾脏。 </li><li>收集蒸压生物反应器与连接灌注回路。 </li><li>收集含露儿适配器和镊子高压灭菌袋。 </li><li>收集的数字泵驱动器连接四罗拉头，大泵筒（1元灌注回路）。 </li></ol></li><li>作为在无菌c在图1C的描述来完成灌注电路生物安全柜中onditions。 <ol><li>一个3路活塞（ 图1D，i）该阳鲁尔锁之间连接到附近的媒体灌洗液入口1/8“ID倒钩适配器和阴路厄个插座式路厄接头。留在水龙头打开的所有三个端口。 </li><li>除去在生物反应器头部和吸管将50ml 0.1％过乙酸，4％的乙醇溶液成通过开口介质容器红色螺丝帽。 </li><li>连接采用大泵筒的蠕动泵管段泵头。调节流速，以5毫升/分钟，按“开始”，并允许电路以素数。 <ol><li>当液体填充灌注线，并到达上述活栓的三通的其余开放口，采用阳鲁尔插头（图1D，一）插头的端口。 </li><li>允许该电路完全素，直到没有空气在灌注回路管或在入口鲁尔受体的内侧观察到的。如果necessaRy的，增加流速暂时从灌注线驱逐气泡。当完全底漆，停泵驱动器。 </li></ol></li><li>肾动脉导管的阴鲁尔端小心插入到生物反应器头部使用灭菌6“钳的内表面上的阳入口鲁尔受体。确保连接是紧，没有空气留在导管内。让肾脏轻轻地从肾动脉导管挂，使肾动脉没有扭曲或扭结。 </li><li>拧紧夹子的金属一起举行的生物反应器的头部和身体，使生物反应器水库密封。关闭红色螺丝帽上的生物反应器头部。 </li></ol></li><li>消毒肾脏在RT通过灌注用0.1％过，4％的乙醇溶液，在5毫升/分钟进行1小时，然后三个连续1小时灌注在RT用50ml PBS中以5毫升/分。 <ol><li>变化的解决方案： <ol><li> 1小时后，停泵，打开红色的螺丝帽。采用STE激怒（灭菌）巴斯德吸管，小心吸所有来自生物反应器的储液。离开灌注电路完全做好准备。 </li><li>吸取50毫升新鲜溶液到生物反应器蓄水池，关闭螺旋盖，并启动泵。 </li></ol></li></ol></li><li>最终的PBS冲洗，吸出PBS从在补充了10％FBS和1％青霉素 - 链霉素50毫升DMEM / F12培养基的储存器和吸移管后。关闭螺旋盖，并用附带的灌注回路传送的生物反应器，以保持在37℃和5％CO 2的大容量的孵化器。 </li><li>如果必要的话，泵驱动转移到孵化器。连接的生物反应器灌注回路的泵，和灌注的肾脏在4毫升/分钟，至少1小时之前播种。 </li></ol> 3.肾Recellularization与肾皮质肾小管上皮细胞<ol><li>暖足够体积的DMEM / F12的（补充有10％FBS和1％青霉素 - 链霉素）和cELL游离酶细胞升降至37℃。建议卷列举如下： <ol><li>准备好10 mL细胞游离酶和每175厘米2培养瓶中10毫升DMEM / F12的提升。 </li><li>准备5-10毫升的DMEM /每肾脏F12进行接种。 </li></ol></li><li>收集足够数量的培养烧瓶的所需接种浓度（4×10 7个在使用这种播种策略RCTE细胞的最大植入永生化人RCTE细胞18％肾结果）。 </li><li>电梯从细胞利用细胞解离酶瓶。从瓶中吸介质，然后移取10 mL细胞游离酶进入瓶中。在37℃培养箱地方烧瓶加速分解。 </li><li>检查烧瓶上相差显微镜每2分钟，直到从烧瓶表面的细胞充分解离是观察。注意：孵育时间将取决于细胞的汇合的水平不同，但RCTE细胞将řequire约15分钟，充分解离。 </li><li>取的解离的细胞悬液样品前用于制粒计数。稀释细胞悬液用等体积的预先温热的DMEM / F12培养基中，离心5分钟232×g离心相对离心力（RCF）。 </li><li>离心期间计数细胞。稀释所得到的样品用等体积的台盼蓝的，并且吸管10微升到血细胞计数器进行计数的每个端部。计算所需粒料稀释体积，以获得所需接种浓度（2×10 7细胞/ ml）。 </li><li>离心后，稀释沉淀用培养基的适当体积，以获得2×10 7个细胞/ ml的终浓度。绘制增长2毫升悬浮播种到灭菌的5毫升注射器。 </li><li>灌注生物反应器转移至生物安全柜中。转活栓阀关闭流向播种端口。取下注射器阳滑塞水龙头，并连接装有接种悬浮液的注射器。 </li><li>所述灌注回路迅速转移回到培养箱，并使用大泵筒在蠕动泵管段固定到泵头部。 </li><li>细胞接种：关闭活塞阀口指向泵。慢慢将细胞悬浮液注入到肾脏，保证整个悬浮液从注射器转移到上述活栓和灌注线。转动旋塞阀从注射器关闭流，并启动泵25毫升/分钟进行15分钟。 </li><li> 15分钟后，降低了泵的流速到4毫升/分钟。交换介质（100 ml，此为随后的变化）次日，此后每隔两天。 </li></ol> 4.评估细胞活力和增殖用刃天青灌注试验<ol><li>准备刃天青试剂。溶解在搅拌下百毫升的PBS 110.5毫克刃天青钠盐，并加入5毫升的稀1:10所得溶液至45毫升新鲜的PBS，以创建一个440微米的刃天青原液。过滤消毒（使用0.2微米的注射器过滤器），和原液中的光保护50ml锥形管中储存于4℃。 </li><li>准备刃天青补充的媒体控制。制备刃天青试剂的培养基中的10％溶液（ 例如 5毫升刃天青原液+ 45 ml培养介质）来创建刃天青工作溶液。高压釜于10ml体积中的光的保护容器中的刃天青工作溶液。注意：这将完全还原刃天青化合物试卤灵，和将作为阳性对照用于计算刃天青还原百分比。 </li><li>分装各1mL的刃天青工作溶液（氧化），蒸压刃天青单独（空白）工作液（减少），并培养成独立1.5毫升收集管。将打开的管道在同一孵化器作为生物反应器灌注。 </li><li>转移肾脏灌注电路从孵化至生物安全柜中。除去从生物反应器头部的螺丝帽，并从使用巴氏吸管的生物反应器储抽吸培养基。 </li><li>吸管10毫升刃天青工作液进入储存器，靠近螺丝帽，以及肾脏灌注回路转移回到培养箱。 </li><li>启动泵在4毫升/分钟，并允许试剂通过肾脏灌注整整1小时。 </li><li> 1小时后，停泵和肾脏灌注电路转移到生物安全柜。 </li><li>收集经调节的（部分还原的）刃天青溶液，并加入100毫升培养介质至生物反应器库。转印灌注回路到孵化器，并恢复流（4毫升/分钟）。 </li><li>吸管100微升（×3次重复）空调刃天青溶液，刃天青工作溶液中，高压灭菌的工作溶液，并培养介质空白成黑色（不透明）的96孔测定板。阅读的荧光强度（excitat离子：三十五分之五百四;发射：二十零分之五百九十）使用分光光度计。 </li><li>计算减少百分比如由氧化刃天青介质产生由荧光强度（FI）标准化荧光强度的比率（ORM，或刃天青工作溶液不暴露于细胞）或减小的刃天青介质（RRM，或高压灭菌工作液）：％减少= 100×{[FI（空调刃天青溶液） - FI（ORM）] / [FI（RRM） - FI（ORM）]}。正常化的结果，通过培养时间（1小时）循环体积（10毫升）和除法乘法减少％。 </li></ol>

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The authors have nothing to disclose.

所描述的脱细胞协议始终如一地产生完全无细胞肾细胞外基质充当3D模板人肾皮质小管上皮细胞（近端和远端小管衍生）的培养中，除了血管内皮细胞7,17。管状肾血管用作均匀输送试剂和细胞的整个支架内的生物反应器设置的关键特征，因此能够使灌注脱细胞，细胞接种，长期灌注培养，和刃天青灌注协议。如肾动脉之前器官灌注等，适当的插管是至关重要的，必须采取特别小心，以确?...

An erratum was issued for <a href="http://www.jove.com/video/53271/epithelial-cell-repopulation-preparation-rodent-extracellular-matrix">Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development</a>.
The human RCTEC/RCTE cell stocks used here and in Caralt et al., 2015, Uzarski et al., 2015 were originally provided by Dr. Loghman-Adham (then at St. Louis University) to co-author Dr. Wandinger-Ness (University of New Mexico) through an MTA in 2001. The SV40 immortalized human RCTEC/RCTE cells were characterized as being of distal tubule cell line as detailed in Loghman-Adham et al., 2003. On the basis of recent short tandem repeat (STR) DNA sequencing (Performed by IDEXX BioResearch) of the earliest passages of the RCTEC/RCTE cell stocks it became evident the cells were of mixed lineage. Further analyses of PCR products using QIAxcel capillary electrophoresis demonstrated the presence of a canine product. The product was sequenced and established to be of canine origin. A set of canine specific STR markers were compared to the sample and showed that the sample had a genetic profile with 92% identity to the MDCK cell line.&#160; Later cell stocks that were used in the present publication were STR profiled and showed drift to 100% MDCK lineage. Despite being of canine origin, rather than human as was previously thought, MDCK is similarly a distal tubule epithelial cell line. For this reason, previous interpretation and conclusions drawn using these cells here and in Caralt et al., 2015, Uzarski et al., 2015 remain sound, but for purposes of rigor, reproducibility and experimental validation by others we report on this misidentification. This information will also be reported for listing on the International Cell Line Authentication Committee (ICLAC) database (http://iclac.org).

Erratum: Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

由于患者终末期肾功能衰竭患人数继续增加，存在可供移植的供体肾脏的数量严重和日益短缺。不能以满足不断上升的一些候选人的需求等待上市的肾移植，促使研究肾器官工程与开发定制，植入的肾脏移植需求1,2的终极目标。构建从患者自身的细胞功能肾组织将消除需要终身免疫抑制，减少患者的时间花费在透析等待肾移植的数量，并延长救命移植到更多的患者有慢性肾脏疾病。 朝向生物工程使用源自患者的细胞中的肾组织的第一步是建立一个支架，作为一个支持基板肾实质（ 例如管状上皮的Lial），间质成纤维细胞和血管细胞生长。从天然器官的细胞外基质（ECM的）生物材料衍生支架有几个特点，使它们希望在组织工程，其中包括他们的天然生物成分的使用;适当的宏观和微观结构赋予的生理功能;和细胞生物相容性，促进细胞粘附，迁移和建设性的组织重构3。一个有前途的方法生产的支架为肾组织再生是通过同种异体或异种肾脏脱细胞，同时保留大部分的肾脏ECM 4复杂天然蛋白质组合物，保留器官的固有建筑复杂，并克服与自下而上关联的难度了厚厚的细胞化组织提供血管供应制定细胞支架recellularization 5后工程。 灌注脱细胞是一个过程其中去污剂，酶，或其它细胞破坏解决方案通过器官6的血管网均匀地输送。这个策略已被确立为一种有效的方法，以获得无细胞器官的基于ECM的支架为三维（3D），用于全器官工程6-8生物模板，就证明了无细胞肾模板从废弃人的肾脏9的开发从大的动物（ 如猪10，山羊11）和啮齿动物来源获得12异种肾脏。特别是，使用小动物模型如啮齿类需要更少的细胞和培养基，这对于器官recellularization研究中，细胞数目通常是有限的特别有益的，因为与干细胞衍生的组织的情况下。所描述的脱细胞协议的目标是生产无细胞的肾细胞外基质，可以用来作为3D脚手架系统为肾脏structur的再生上课，包括对重新填充本例与人肾皮质小管上皮（RCTE）细胞在肾小管。我们先前描述的一个最佳的，基于洗涤剂的大鼠肾脱细胞协议7，其较为迅速（约1天）比以前报道的其它方法的我们的严格的评价（Ross 等 .-5天12，Song 等人 .-4.5天13），并暴露了器官的变性剂十二烷基硫酸钠（SDS）的相当低的浓度（0.1％）脱细胞比以前的报告12-15中。 研究数量有限所描述的使用灭鼠肾脏的脱细胞和随后用作3D支架的细胞再增殖的12-16（1别处审查）。在这个协议中，我们提供了先前建立的，最佳的脱细胞的策略的详细说明用于制备无细胞肾支架从只SD大鼠肾脏7。使用自定义设计的双能播种和维护灌注培养17灌注生物反应器，我们recellularize脱细胞支架肾脏与人体RCTE细胞，重新填充一致管状部件在这些脱细胞基质，增殖，而在灌注培养存活了一个多星期。我们进一步证明我们使用刃天青灌注试验的-一种廉价的，无细胞毒性的，非侵入性的代谢评估先前用于毒性研究17 -随时间7提供的recellularized肾脏内细胞的活力和增殖的指示。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON X-100, Proteomics Grade, AMRESCO</td>
			<td>VWR</td>
			<td>M143-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>05030</td>
			<td></td>
		</tr>
		<tr>
			<td>Peracetic acid solution, purum, ~39% in acetic acid (RT)</td>
			<td>Sigma-Aldrich</td>
			<td>77240</td>
			<td>Peracetic is flammable and corrosive. Prepare within a fume hood using appropriate personal protective equipment (e.g. gloves, goggles).</td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR</td>
			<td>V1001TP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote, siliconizing reagent for glass and other surfaces</td>
			<td>Sigma-Aldrich</td>
			<td>SL2</td>
			<td>For treatment of bioreactor reservoirs. Referred to in text as siliconizing reagent.</td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cellgro Fetal Bovine Serum Premium, Mediatech</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X 10,000 I.U. Penicillin 10,000 &#181;g/mL Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>12605-028</td>
			<td>Referred to in text as cell dissociating enzyme.</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive, 600 RPM, 115/230 VAC</td>
			<td>Cole-Parmer</td>
			<td>EW-07522-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S large cartridges for 07519-05 and -06 pump heads.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-70</td>
			<td>Referred to in text as large pump cartridge.</td>
		</tr>
		<tr>
			<td>Masterflex L/S 8-channel, 4-roller cartridge pump head.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight 6&#34; specimen forceps, serrated</td>
			<td>VWR</td>
			<td>82027-438</td>
			<td></td>
		</tr>
		<tr>
			<td>*Kidney perfusion bioreactor</td>
			<td>WilMad Labglass</td>
			<td>*Custom designs</td>
			<td>Bioreactors are produced as described by WilMad Labglass. The designs have been described in depth in a previous publication.</td>
		</tr>
		<tr>
			<td>Perfusion Circuit Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 G x 0.75 in. BD Insyte Autoguard shielded IV catheter (0.7 mm x 19 mm) made of BD Vialon biomaterial. Has notched needle. (50/sp, 200/ca)</td>
			<td>BD Biosciences</td>
			<td>381412</td>
			<td>Referred to in text as 24 gauge catheter.</td>
		</tr>
		<tr>
			<td>Masterflex PharMed BPT Tubing, L/S #14, 25&#39;</td>
			<td>Cole-Parmer</td>
			<td>HV-06508-14</td>
			<td>Referred to in text as peristaltic pump tubing.</td>
		</tr>
		<tr>
			<td>Peroxide-Cured Silicone Tubing, 1/16&#34;id X 1/8&#34;OD, 25 Ft/pack</td>
			<td>Cole-Parmer</td>
			<td>HV-06411-62</td>
			<td>Referred to in text as 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing, L/S 14, 25 ft.</td>
			<td>Cole-Parmer</td>
			<td>HV-96410-14</td>
			<td>Referred to in text as thick-walled 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>VWR Silicone Tubing, 1/4&#34; ID x 0.5&#34; OD</td>
			<td>VWR</td>
			<td>89068-484</td>
			<td></td>
		</tr>
		<tr>
			<td>Acro 50 Vent Filters, Pall Life Sciences</td>
			<td>VWR</td>
			<td>28143-558</td>
			<td>Referred to in text as 0.2 micron vent filter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Adapters, Male Luer Lock to 1/8&#34; ID, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>T-45505-11</td>
			<td>Referred to in text as male Luer lock to 1/8&#34; barbed adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Male Luer Plug, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45505-58</td>
			<td></td>
		</tr>
		<tr>
			<td>Female luer x female luer adapter, Nylon, 25/pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-22</td>
			<td>Referred to in text as female Luer x female Luer adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Smiths Medical Large Bore Hi-Flo Stopcocks # MX4311L - 3-Way Hi-Flo Stopcock with Extended Male Luer Lock, Non-DEHP Formulation, Latex Free (LF), Lipid Resistant, Non-PVD, 50/cs</td>
			<td>Careforde Healthcare</td>
			<td>10254821</td>
			<td>Smiths Medical (vendor) catalogue number is #MX4311L.</td>
		</tr>
		<tr>
			<td>Other Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL BD Luer-Lok disposable syringe with BD Luer-Lok&#8482; tip.</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm x 10 mm Easy Grip Culture Dish</td>
			<td>BD Biosciences</td>
			<td>353001</td>
			<td>Used to draw cell suspension into syringe for cell seeding.</td>
		</tr>
	</tbody>
</table>

epithelial cell repopulation and preparation of rodent extracellular matrix scaffolds for renal tissue development

该协议细节无细胞，但生物功能，肾细胞外基质（ECM）的支架是小规模的模型底物器官规模组织发育有用的产生。只SD大鼠肾脏通过将导管插入肾动脉插管和灌注一系列低浓度去污剂剂（Triton X-100和十二烷基硫酸钠（SDS））超过26小时，以获得完整的，全肾支架与完整perfusable血管，肾小球和肾小管。以下脱细胞，肾支架放在定制设计的灌注生物反应器容器内，并且所述插入导管肾动脉被连接到包括一个灌注回路：蠕动泵;油管;和任选的探针，用于pH值，溶解氧，和压力。杀菌用过乙酸和乙醇，和平衡pH值（7.4）的支架后，将肾支架是一个LARG内通过培养基的灌注准备播种电子容量培养箱保持在37℃和5％的CO 2。通过肾动脉15分钟减少流至生理速率前四千万肾皮质小管上皮（RCTE）细胞注射后，迅速灌注通过下高流动，支架（25毫升/分钟）和压力（〜230毫米汞柱） （4毫升/分钟）。 RCTE细胞主要在肾皮质内填充管状ECM小生，增生，形成小管上皮结构在灌注培养七天。在培养基A 44μM的刃天青溶液通过肾脏中介质交流灌注1小时至小管中提供的细胞生存力和增殖荧光剂，氧化还原类代谢的评估。肾脏灌注生物反应器允许介质的非侵入性采样用于生化评估，和多个入口允许通过肾静脉或输尿管替代逆行播种。这些协议可以用于recellularize肾支架用多种类型的细胞，包括血管内皮细胞，肾小管上皮细胞，和基质成纤维细胞，对本系统内的快速评价。

肾脏依次灌注水和稀洗涤剂溶液中（1％的Triton X-100，0.1％SDS）中，根据先前建立的，最佳的脱细胞协议（ 见图1A，B）的7，逐渐变得更透明超过26小时的时间内，如图2A所示。由此产生的脱细胞支架肾脏是缺乏的细胞，并保留通过一个完整的肾小囊，这是完好无损以下灌注协议支持一个有凝聚力的肾ECM。通过最终的洗涤剂灌注（SDS）肾脏的血管网，特别是叶间血管...

Watch this Scientific Journal Video about Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development at JoVE.com

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

epithelial-cell-repopulation-preparation-rodent-extracellular-matrix

Research

JoVE Journal

Bioengineering

15.9K 次观看.  Northwestern University. This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

视频: Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

细胞外基质蛋白纤维的布里渊谱的制备

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...