Name: conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation
Uploaded: 2017-03-22T13:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

We would like to acknowledge Connecticut Children's Medical Center Strategic Research Funding for supporting this work.

后从儿科患者的家长/监护人，并根据机构审查委员会（IRB＃13-094）获得知情同意，获得食管活检。 1.消毒仪器和明胶溶液<ol><li>高压灭菌镊子，刀片和前处理组织，以防止污染剪刀。 </li><li>使200毫升的0.1％明胶溶液，结合的蒸馏水200毫升含有0.2 1g明胶。高压灭菌并冷却后才能使用。 </li></ol> 2.镀层组织培养板<ol><li>前细胞分离约2小时，添加足够的0.1％明胶覆盖组织培养皿（100毫米），并在细胞培养培养箱中于37℃孵育。 注意:这些板也可以由提前时间和在使用前保持在培养箱中。 </li></ol> 3.使酶液解离<ol><li>大约前细胞分离30分钟，掂量出基于每毫克浓度的单位上的小瓶的平衡分散酶的10个单位。放置在一个15毫升锥形管的粉末与10的Dulbecco改良的Eagle培养基（DMEM）中的溶液，并发生在37℃水浴中。 </li></ol> 4.进行细胞培养基<ol><li>让50毫升的条件重新编程培养基中，将以下成分混合入无菌锥形管:35毫升F12的（咸的培养基），11.5毫升的DMEM，0.5毫升L-谷氨酰胺，0.5毫升100×青霉素/链霉素，2.5毫升的胎牛血清（FBS），人胰岛素的250微克，500纳克人表皮生长因子（EGF）的。 </li><li>让500毫升3T3培养基中，混合50毫升胎牛血清，5毫升100×青霉素/链霉素和5mL L-谷氨酰胺用500mL的DMEM中。 </li><li>让500毫升角质细胞无血清培养基的，解冻EGF和牛垂体提取物（BPE）补充剂（提供媒体）。补品加入到一瓶500毫升基础培养基中，加入5毫升100X青霉素/链霉素</li></ol> 5.培养及辐照3T3细胞作为馈线源<ol><li>制备3T3介质，包括DMEM，10％FBS，1×青霉素/链霉素和2mM L-谷氨酰胺。 </li><li> 文化3T3细胞在组织处理的T-75瓶患者样本到来前至少3天。 <ol><li>之前，为了患者样品的到来，trypsinize使用5的每烧瓶0.25％胰蛋白酶-EDTA溶液与3T3细胞孵育在37℃下5分钟，或直至大多数细胞是浮动</li></ol></li><li>添加5毫升3T3介质，使反应停止。吸出细胞悬浮液，并转移到15毫升锥形管中。旋在300×g下5分钟。 </li><li>吸出介质并悬浮细胞在台盼蓝的细胞计数一个限定的体积。结合细胞的10微升台盼蓝的10微升排除死细胞。放入10μL对细胞计数血球这一解决方案。 注:对于这个原山口，辐照3T3细胞接种密度为每厘米2，这等于每100mm板600,000个单元格10,900细胞。 </li><li>分装必要到板每名患者样品和重悬100mm的板在25mL 3T3介质的在50mL锥形管中，用3000拉德照射细胞的数目。 </li><li>照射之后，旋下来的细胞在300 XG在每100毫米的钢板5毫升5分钟，悬浮在有条件的重编程网上平台。 </li><li>抛开管，直到患者细胞镀在协议发生后（见步骤7.6）。 </li></ol> 6.获取，保存和运输小儿食管活检<ol><li>添加5毫升100微克/毫升primocin到15毫升锥形管中完整角质细胞无血清培养基中，并使其在冰上的医疗设施。 注意:介质的这些管子可被储存在4℃下长达2个月。 </li><li> 在TI获得约8食管活检我内窥镜单独的和，如下所示: <ol><li>放置在含有完整角化细胞的无血清培养基用100微克/毫升primocin和地点在湿冰上的15毫升锥形6活检。 </li><li>放置一个活检5毫升湿冰上福尔马林和地点。 </li><li>放置一个活检与最佳切削温度复合cryomold小活检。 <ol><li>这个模具立即放到置于液氮的异戊烷的烧杯中。一旦冷冻，将cryomold一个标本袋，并存储在一个聚苯乙烯泡沫塑料容器干冰。 </li></ol></li></ol></li><li>将含密封的袋子里，然后到含有湿冰包装箱在中等或福尔马林活检管。封并用生物危害标签标记框，并将其运送至实验室。 注:包含干冰的容器和冷冻活检也密封并用生物危害标签标记。 </li></ol> 7.处理病人组织对下游文化与分析<ol><li>一旦在实验室后，在干冰上转移冰冻活检，-80℃冷冻切片下游或RNA提取。放置在福尔马林样品。储存在4℃冰箱，并允许以固定过夜。 </li><li>旋转剩余活检下来300 XG，并吸出网上平台。加入10 mL分散酶溶液的锥形管，密封件，并允许它在37℃的水浴中孵育15分钟。 </li><li>孵育后，旋下活检以300×g的。吸中期和活检悬浮在5毫升0.05％胰蛋白酶EDTA的。 </li><li>转移5毫升含活检到100毫米的无菌板胰蛋白酶-EDTA，并剁碎使用2-灭菌刀片尽可能细的活检。 </li><li>传输含有剁碎活检胰蛋白酶-EDTA的5毫升到一个新的15毫升锥形。用另外5毫升0.05％胰蛋白酶-EDTA的冲洗所述板两次，并加入到锥形管。孵育管在37℃水浴中10分钟。 </li><li>继温育后，旋活检向下在300 XG（1.4 RPM）5分钟，并吸出培养基。加入5毫升的条件重新编程介质到锥形管以及包含经照射的饲养细胞的条件重新编程介质的5毫升（见步骤5.7）。 </li><li> ROCK抑制剂（1微升/毫升）添加到10毫升细胞悬浮液和板从组织培养皿抽吸明胶后。在37℃。 </li></ol> 8.培养和扩大细胞<ol><li>后约5天内培养的，吸含有切碎活检片起始培养基，用5ml无菌PBS冲洗培养皿2-3倍。新介质添加到板，包括在10μMROCK抑制剂。 </li><li>当达到70％汇合，展开的细胞。加入5毫升0.05％胰蛋白酶-EDTA的到板，并在37℃培养箱中孵育不超过2.5分钟，除去第Ë饲养细胞。以下该温育后，吸出胰蛋白酶-EDTA，并用5毫升的PBS冲洗板。 </li><li>吸出PBS中并加入5毫升0.25％胰蛋白酶-EDTA的到板并孵育5-10分钟，或直至所述细胞是从板松动。介质等量添加到板以停止反应，所有的流体转移到50毫升锥形管中并离心，在300×g离心5分钟。 </li><li>悬浮细胞在限定的条件重新编程介质的体积并用台盼蓝10μL的混合悬浮液的10微升。指望血球测定总细胞数。 </li><li>扩大小区100万人食管癌细胞添加至150mm的板沿与120万在15毫升的总体积照射3T3细胞。在10μM添加新鲜ROCK抑制剂和细胞悬浮液添加至明胶涂层150毫米菜。 </li></ol> 9.冷冻和保存人类食管上皮细胞<ol><li>为了使冷冻剂，加10％DMSO有条件重新编程网上平台。胰蛋白酶及数量后，分装细胞冻结到一个新的50毫升锥形管中并离心，在300×g离心5分钟。 </li><li>吸出培养基，并添加在每离心管2毫升体积冷冻介质。一旦进入细胞悬浮液是均匀的，分装10 6个细胞在预标记cyrovials。发生在在-80℃的冷冻容器之前移动所述小瓶长期液氮储存至少24小时。 </li></ol>

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The authors declare they have no competing financial interests.

为了从患者的活检隔离和扩大食管上皮细胞的最重要的步骤是:1）充分游离活检组织以最小的细胞死亡; 2）确保ROCK抑制剂加入到在每次更换培养基的细胞培养基; 3）不要使用更多的饲养层细胞比建议; 4）保持干净无菌文化; 5）传代细胞之前，为了达到汇合。 由于为条件重新编程得到的活检样品中的患者相关的差异，这是合理的预期生长动力学，表型和扩展到多个通道的能力?...

食管癌组织工程和嗜酸性粒细胞性食管炎（EOE）一直是研究在许多实验室在过去十年中的焦点。先天性缺陷，如食管闭锁，被认为在约1 4,000活产，这导致在食道导致无力的发育不完全吃1。 EOE的发病率和患病率一直在自从疾病实体于1993年鉴定EOE的发病率从0.7变化到每人10年/ 10万和患病率0.2范围到43/10万2的崛起。一种新的有吸引力的手术方法来治疗长差距食管闭锁在于生成的组织构建了利用患者自身的细胞植入。这些细胞在用合成脚手架结合将产生自体构造，不需要免疫抑制。一些团体已经开始调查美国食管组织工程3以及使用本地食管上皮细胞的干细胞样细胞电子重新填充粘膜4 - 7。疾病存在于儿童患者食道往往难以诊断或不干预研究。此外，利用动物模型或体外永生化细胞系模型的儿科疾病，如EOE不包括确切的发病机制或患者的具体差异8。因此，为了研究患者的疾病过程在体外的能力，以确定特定疾病触发抗原，评估潜在的机制和调查药物治疗将是新颖的和提供可在患者的治疗辅助信息临床医生。 已经有已被建议用于Tissu酒店使用许多自体或患者特定的细胞类型机电工程和研究人类疾病的发病机制。然而，这些类型的细胞在它们的能力是有限的，以产生一个特定的表型的足够的细胞接种大型支架或体外研究进行高吞吐量。使用多潜能或多能的干细胞已经大量的研究讨论的话题，但是，局限性和缺点对于使用这些细胞已被很好地描述9。利用人类胚胎干细胞的高度辩论，提出了许多伦理问题。最重要的是，这些细胞形成畸胎瘤，其类似于一个肿瘤，如果他们不从它们的多能性状态提供它们到生物主机10之前区分。此外，使用胚胎干细胞的不会是患者特异性的，并可能引起同种异体反应和需要免疫抑制10。诱导多能干细胞（iPS细胞）是多能干细胞，它可以从患者自身的细胞。体细胞，如皮肤细胞，可以使用各种一体化和非整合的技术来诱导多能状态。这些细胞然后作为用于组织工程或疾病调查患者特异性细胞来源。不需要的遗传物质整合到这些细胞是很多人所描述即使序列完全去除iPS细胞似乎节省了后生"记忆"对从它们所衍生11细胞类型值得关注。这些细胞还将形成畸胎瘤体内如果不是移植前11区分。许多分化的协议已经研究专注于上皮谱系12，13，14，但是，要注意的是在分化结束时所产生的细胞类型不均匀和O这是非常重要的唯一一句具有感兴趣的细胞类型的一小部分。这导致低收率和需要纯化所需的细胞类型。虽然iPS细胞是一个潜在的患者特定的细胞来源，该方法以获得对任何组织工程或疾病调查感兴趣的细胞类型是非常低效的。 肺15，乳房16，小肠17，结肠18，膀胱19和食道20:人上皮细胞已经从各种人体包括患病和非患病组织的被成功分离。要注意的是人的原代细胞具有其中表型被保持21,22通道的一个有限数量是很重要的。不幸的是，这意味着需要的疾病调查或播种的工程支架的细胞数用于植入可能无法达到。因此，需要新的技术来扩大患者的细胞，同时仍保持上皮表型。利用饲养细胞和ROCK抑制剂正常和癌性上皮细胞的有条件的重编程被刘等人在2012年描述。 2 3。这个技术被用于扩展从利用照射饲养细胞，ROCK抑制剂和有条件的重新编程介质前列腺癌和乳腺癌的活检获得的癌细胞的上皮细胞。我们的目标是产生足够的细胞用于在体外测定，如药物筛选。该技术是能够通过"重新编程"这些细胞干细胞或祖状态，这是高度增殖性无限期膨胀的上皮细胞的。已经证实，这些细胞是非致瘤性和不具有形成畸胎瘤23,24的能力。此外，没有染色体异常或遗传操作是使用这种技术23,24在培养传代这些细胞后存在。最重要的是，这些细胞仅能够分化成感兴趣的天然细胞类型。因此，这种技术提供了一个大的贮存器的患者特异性上皮细胞疾病调查或组织工程无需永生化。 从一个特定的器官，以研究疾病过程获得上皮组织往往有限，并不总是可能的，因为病人的风险。对于那些从食管疾病或缺陷的患者，内镜活检检索是用于获得可以解离，并有条件地重新编程，以提供特定于该病人的食管粘膜无限期细胞源的上皮组织微创方法。然后，这允许在体外研究上皮细胞，以评估疾病过程和屏幕为潜在治疗剂。一种疾病过程，可以极大地从该方法中受益是嗜酸性食管炎，已被描述为食道8的过敏性疾病。过敏试验以及治疗方法可以在体外使用患者自身的上皮细胞进行评估，然后这个数据可以被传递到治疗医师来开发个性化的治疗方案。条件重新编程的与来自儿科患者获得内窥镜活检一起使用时的技术提供了从任何患者无限膨胀正常食管上皮细胞的能力。此单元源因此可以用天然或合成的脚手架一起合作，以提供缺陷，疾病或创伤患者专用手术选择。有一个不确定的细胞数量将有助于工程师是拥有一个完全重新接种食道的构造为了与食管上皮细胞流明帮助促进剩余的细胞类型的再生。

<table><tbody><tr><td>Primocin</td><td>InVivogen</td><td>ant-pm2</td><td></td></tr><tr><td>Isopentane</td><td>Sigma Aldrich</td><td>277258-1L</td><td></td></tr><tr><td>Gelatin From Porcine Skin</td><td>Sigma Aldrich</td><td>G1890-100G</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher Scientific</td><td>11965092</td><td></td></tr><tr><td>Cryomold</td><td>TissueTek</td><td>4565</td><td></td></tr><tr><td>Cryomatrix OCT</td><td>Thermofisher Scientific</td><td>6769006</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Denville Scientific</td><td>C1017-p</td><td></td></tr><tr><td>Complete Keratinocyte Serum Free Medium</td><td>Thermofisher Scientific</td><td>10724011</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermofisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Glutamax</td><td>Thermofisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Insulin Solution</td><td>Sigma Aldrich</td><td>I9278-5ml</td><td></td></tr><tr><td>Human Epidermal Growth Factor (EGF)</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>ROCK Inhibitor (Y-27632)</td><td>Fisher Scientific</td><td>125410</td><td></td></tr><tr><td>F-12 Medium&amp;nbsp;</td><td>Thermofisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Denville Scientific</td><td>FB5001</td><td></td></tr><tr><td>Dispase</td><td>Thermofisher Scientific</td><td>17105041</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300062</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25200072</td><td></td></tr><tr><td>100 mm Dishes</td><td>Denville Scientific</td><td>T1110-20</td><td></td></tr><tr><td>150 mm Dishes</td><td>Denville Scientific</td><td>T1115</td><td></td></tr><tr><td>50 mL Conicals</td><td>Denville Scientific</td><td>&amp;nbsp; C1062-9&amp;nbsp;</td><td></td></tr><tr><td>Phosphate Buffered Saline Tablets</td><td>Fisher Scientific</td><td>BP2944-100</td><td></td></tr><tr><td>5 mL Pipettes</td><td>Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>10 mL Pipettes</td><td>Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>25 mL Pipettes</td><td>Fisher Scientific</td><td>1367811</td><td></td></tr><tr><td>9&quot; Pasteur Pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>NIH 3T3 Cells</td><td>ATCC</td><td>CRL1658</td><td></td></tr></tbody></table>

conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

在分离来自患者活检食管上皮细胞中的关键步骤的简要总结于图1。上皮细胞的集落将形成在约4-5天，将由成纤维细胞饲养细胞（ 图2A）所包围。作为这些菌落扩大他们将与其他的菌落合并，以形成较大的集落（ 图2B）。一旦培养物都成为70％汇合时，它们需要被膨胀（ 图2C）。以确保成纤维细胞从板中除去上皮细胞之前?...

Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

利用条件重新编程人类小儿食管上皮细胞的扩张提供了可用于工程食管构造为自体移植来治疗缺损或损伤，并作为治疗性筛选测定的储存器单元的特定病人群调查。

小儿食管上皮细胞重编程的条件在组织工程和疾病使用调查

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

conditional-reprogramming-pediatric-human-esophageal-epithelial-cells

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.9K 次观看.  UConn Health. 利用条件重新编程人类小儿食管上皮细胞的扩张提供了可用于工程食管构造为自体移植来治疗缺损或损伤，并作为治疗性筛选测定的储存器单元的特定病人群调查。 

视频: Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

科研

发育生物学

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

生物工程

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

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

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

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

从整体组织和活检建立人类上皮Enteroids和Colonoids的

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

医学

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

神经科学

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

成像引导的生物反应器，用于生成生物工程气道组织

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

在开顶器官芯片上重建人上皮组织的细胞结构和功能

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

人食管类器官和气液界面培养方案

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

作者聚焦:研究嗜酸性粒细胞性食管炎的病理生理学

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.