Name: isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications
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The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

注:胎盘应该从健康的单胎妊娠进行收集，偏重短期选修剖腹产。书面知情同意应给予他们胎盘的收集。您有关人类研究伦理委员会的批准应该全部收集和使用人体组织。 1.分离羊膜上皮细胞<ol style="margin-left: 40px;"><li>将胎盘到无菌表面的II级生物安全柜中。 </li><li>使用无菌的Hanks平衡盐溶液（HBSS）中，从胎盘表面和膜洗尽可能多血越好。 </li><li>与胎盘放置脐带朝上，机械分离羊膜和绒毛膜膜的外边缘。 </li><li>一旦分离，手动从胎盘的绒毛膜膜剥离羊膜，围绕该膜的边缘的工作，向脐带。 注:carefUL，以消除任何碎片从羊膜绒毛膜污染膜。 </li><li>使用无菌剪刀，剪去羊膜约2cm从脐带并置于500毫升的基部密封含250毫升HBSS容器。 </li><li>通过摇动含羊膜的密封的瓶子，彻底冲洗。 </li><li>除去使用无菌镊子地方收集容器，以及羊膜成15cm的培养皿。 </li><li>切羊膜成片约5厘米长的（用血管钳垂直握持时），丢弃血性或撕成碎片。 </li><li>地方在500ml密闭容器中，并洗净，用250毫升的HBSS大约3-5次，或直到羊膜变成半透明，没有污染的血液。 注意:任何血液存在可能降低酶的消化效率的解决方案。 </li><li>转移羊膜的所有片放入含有50ml酶的消化溶液的容器中，同时注意尽量减少HBSS结转。 </li><li>孵育羊膜片在酶促消化在水浴或温室的溶液，在37℃下进行15分钟，温和搅拌，以除去任何剩余的血液。 </li><li>除去羊膜片从酶消化溶液并置于一个新的无菌容器中。 </li><li>加入100毫升新鲜的酶消化溶液的羊膜和孵化在37℃的水浴中或温暖的房间轻轻振荡（第一消化）60分钟。 </li><li>除去羊膜片，并放入新容器小心，以允许酶促消化溶液从每片膜的流失。 注意:这可以通过拖动羊膜片沿容器的内沿达到。 </li><li>加入100毫升新鲜的酶消化溶液的羊膜片和孵化在水浴或温室60分钟，在37℃轻微振荡（第二消化）。 </li><li>通过细胞suspensi从步骤1.14到1000 XG 10分钟70微米的过滤器和离心机。 </li><li>除去并弃去上清液，并重新悬浮含有1毫克/毫升大豆基酶抑制剂的细胞沉淀在4毫升HBSS中。轻轻地用1毫升移液管反复吸液管，以获得单细胞悬浮液。 注:在此阶段细胞可置于冰上直至从第二消化细胞是在处理的相同阶段。 </li><li>除去羊膜片，并放入新容器小心，以允许酶促消化溶液从每片膜的流失。 注意:这可以通过拖动羊膜片沿容器的内沿达到。 </li><li>通过一个70微米的过滤器和离心机通过从步骤1.18的细胞悬浮液在1000×g离心10分钟。 </li><li>除去并弃去上清液，并重新悬浮含有1毫克/毫升大豆基酶抑制剂的细胞沉淀在4毫升HBSS中。轻轻地用1毫升对反复吸取ipette以得到单细胞悬浮液。 </li><li>从两个摘要或备选池细胞保持单元概要分离直到细胞计数之后。 注意:这可以防止潜在的低存活率细胞具有高生存能力的细胞混合。 </li><li>添加HBSS至10-20毫升的最终体积，并使用台盼蓝测定存活进行细胞计数。 </li><li>确认上皮细胞表型通过流式细胞术。补使用抗EpCAM-PE适当抗体的鸡尾酒（1:2稀释）的抗CD90-PeCy5（1:250）和抗CD105-APC（1:100）在HBSS。 </li><li>重悬的细胞从一个或两个消化的抗体混合物的一个子集，在25×10 6细胞/ ml的浓度。 </li><li>对于流式细胞术，污点1×10 6个细胞用含有同种型对照抗体的鸡尾酒（例如IgG的1同种型对照-PE +抗CD90-PeCy5 +抗CD105-APC）。保持〜1×10 6个细胞未染色用于流式细胞仪建立。 </li><li>孵育细胞抗体鸡尾酒在4℃下60分钟。 </li><li>洗涤细胞3倍的HBSS重悬在5〜10万个细胞每毫升的流式细胞仪。 </li><li>执行流式细胞仪和笔记结果。细胞分离物（EpCAM的阳性和CD90 / CD105阴性细胞的百分比）的纯度，预期分别为90-95％和&lt;1％。 </li><li>预留的细胞的合适数量为测试对于适当的质量控制或释放标准由最终应用决定。 注意:这可以包括疾病/病毒筛查，或其他安全或生物标准。 </li><li>对于剩下的细胞，按照冷冻保存协议，或者直接放入培养（直接向培养步骤3.6）。 </li></ol> 2.冻存hAECs的<ol style="margin-left: 40px;"><li>确定使用流式细胞术数据或人工计数与台盼蓝排除细胞数量和存活力。 </li><li>离心在700×g离心5分钟。 </li><li>悬浮细胞秒每毫升1-10万细胞在动物产品无冷冻保存介质之间。 </li><li>吸取合适体积的细胞成O形圆环状冻存小瓶并放入一个受控速率冷冻装置，其中冷却在大约发生1℃/分钟，直至-80℃的实现。 </li><li>转移到小瓶液氮长期贮存。 </li></ol> 3.解冻Cyropreserved hAECs与文化<ol style="margin-left: 40px;"><li>从液氮中取出冻存小瓶，置于冰上。 注:对于只转移 - 迅速进行下一步。 </li><li>迅速解冻的冷冻保存的小瓶在37℃下，直到只有一小片（约5毫米×5毫米）的冰的存在。 注:该细胞溶液不暖，以减少在冷冻保存介质DMSO中的毒性作用是重要的。 </li><li>稀释冷冻保存介质10倍用冷（〜4℃）的无血清培养基，滴加在合适的尺寸卯管或容器。 </li><li>离心在700×g离心5分钟，在室温。 </li><li>重悬在无血清培养基中，以每毫升百万细胞的近似浓度。确定采用自动或手动计数与台盼蓝染色细胞数量和活力。预期存活率解冻后会比预先冷冻保存存活率少5-10％。 </li><li>板的细胞在标准细胞培养处理过的塑料制品，或可替代地使用I型胶原涂覆培养表面。 </li><li>对于I型胶原蛋白涂层遵循以下协议<ol><li>从人胎盘（15毫克的胶原蛋白用25毫升去离子水和50微升冰醋酸）准备酸溶I型胶原蛋白。 </li><li>覆盖烧杯用Parafilm搅拌适度，在37℃，直至胶原股被溶解。 </li><li>用0.22微米的过滤器无菌过滤的胶原溶液。 </li><li>稀释所述过滤的胶原股票1:10的去离子水。这种稀释股价的工作conce涂层塑料膜表面ntration解决方案。 </li><li>胶原涂层的塑料，玻璃和膜面进行2小时，在室温，在37℃，或O / N在2-8℃。 </li><li>从被涂覆表面除去多余的流体，并允许它以干燥O / N。 </li><li>引入细胞中之前用无菌组织培养级水或HBSS冲洗。 </li></ol></li><li>板hAECs以25,000细胞/ cm 2的无血清培养基中的密度，并允许附着到发生为72-96小时。 </li><li>以下附件，通过仔细地抽吸和与合适体积的无血清培养基中，每2-3天更换更换介质。 </li><li>使用以下协议代细胞<ol><li>吸细胞培养基洗净用无菌HBSS的1倍量</li><li>添加酶的1×体积消化溶液并孵育5-15分钟，或直至细胞被观察到从细胞培养表面分离。 </li><li>收集酶消化解决方案，包含电池，保证CELLS从细胞培养表面通过反复吹打酶促消化针对细胞培养表面分离的溶液</li><li>离心在700×g离心5分钟。 </li><li>悬浮在HBSS中的含有1mg / ml的大豆基酶抑制剂的合适体积的细胞沉淀。轻轻地用1毫升吸管反复吸取以获得单细胞悬浮液。 </li><li>加入无血清培养基，以100万个细胞/ ml的近似浓度。确定采用自动或手动计数与台盼蓝染色细胞数量和活力。 </li><li>板hAECs以25,000细胞/ cm 2的无血清培养基中的密度，并继续与标准培养方法。 </li></ol></li></ol>

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The authors have nothing to disclose.

有迹象表明，可以在该方法的成功显著影响几个关键参数。 hAECs分离之前胎盘或羊膜长达3小时的存储可能希望后勤或调度目的，然而，建议组织被尽快处理。如果组织是要被存储，则建议存储以下夹层的羊膜和洗涤来进行。羊膜可以在4℃储存在无菌HBSS中含有的抗生素，但是细胞生存力可与延长贮存时间减少。我们和其他人已经发现变异细胞产量和活力，并最大限度地减少这种变化，建议胎盘从?...

从围产期来源，如胎盘，胎盘胎膜，脐带和羊水来源的细胞已经吸引了研究人员和临床医生关注，因为细胞的再生医学1,2的潜在来源。这样做的原因兴趣的是，这些类型的细胞都具有一定程度的可塑性和免疫调节能力3，即是基本的，以它们的潜在的治疗应用的属性。 hAECs是可从术语或预术语羊膜4中可以得出，提供再生细胞材料的一种丰富的潜在来源的异质上皮人口。使hAECs吸引力作为细胞治疗的属性包括其多能，低免疫原性，和抗炎特性。 hAECs已被发现是在体外和体内的多能高度，能分化成中胚层谱系（cardiomyocytes，肌细胞，骨细胞，脂肪细胞），内胚层谱系（胰腺细胞，肝细胞，肺细胞）和外胚层谱系（头发，皮肤，神经细胞和星形胶质细胞）5-10。 令人放心，尽管它们的多能hAECs似乎没有任何形式的肿瘤或促进肿瘤的发展在体内 。此外，hAECs也是免疫特权，表达II类人类白细胞抗原（的HLA）8水平低。这个属性可能underlies他们逃避免疫排斥异体异种及移植后，这表现在使用免疫能力的猴子，兔子，豚鼠，大鼠和猪11-13研究能力。 hAECs显示强大的免疫调节和免疫特性，从而为自身免疫性疾病的治疗潜在的临床应用显著实用的优点。 hAECs被认为发挥免疫调节功能上都先天和适应性免疫系统。上Ë建议的机制，是通过免疫调节的分泌因子14。 hAECs在临床前的动物疾病模型中的电流的应用包括中风，多发性硬化，肝疾病，糖尿病和慢性和急性肺部疾病的治疗。研究人员已经证明在使用hAECs治疗中风后的脑炎症的兴趣，由于其独特的性质。有证据表明，hAECs能够穿越血脑屏障，他们可以嫁接，存活长达60天，分化为神经元，减少炎症和促进在神经系统疾病15的动物模型中损伤的中枢神经系统组织的再生。 hAECs提供目标和反向有助于多发性硬化的发生和发展的多个病理途径的能力。例如，从临床前动物研究结果表明，hAECs强烈的免疫抑制和有可能引起外周免疫耐受和反向持续炎症反应。 hAECs也已经显示出具有分化成神经细胞在体内并通过神经营养的繁多因子16的分泌增强内源性神经再生的能力。 人类和啮齿类羊膜上皮细胞已经表明它们的治疗功效肝病动物模型中的治疗。在肝脏疾病，HAEC导致在肝脏可行hAECs的移植移植，伴随着降低肝细胞凋亡的四氯化碳诱导损伤模型，降低肝脏炎症和纤维化17。 hAECs可以刺激表达胰腺因子包括胰岛素和葡萄糖转运。几项研究已经调查了潜力hAECs恢复的血糖水平在糖尿病小鼠18。在小鼠接受hAECs，无论动物体重和血糖水平在注射后的细胞下降到正常水平。这些研究提出了强烈的情况下使用hAECs对糖尿病的治疗。 hAECs具有预防和修复实验性急性和慢性肺损伤的成年和新生模型19在一个成熟的作用。这些研究发现，hAECs 在体外分化成表达多个肺相关蛋白质，包括囊性纤维化跨膜传导调节因子（CFTR），该突变在囊性纤维化患者20的离子通道功能的肺上皮细胞。此外，当hAECs被输送到受伤成人和新生儿肺部，它们通过宿主免疫细胞的调制发挥它们的修复效果，降低肺白细胞招募，包括中性粒细胞，巨噬细胞和淋巴细胞21-23。 鉴于其丰富，的安全记录，并用于多种疾病的公认的临床应用中，使用hAECs临床试验是不可避免的。以加快HAEC疗法进入临床试验的翻译的目标，我们开发的方法分离，冻存和文化hAECs适合于临床试验的方式，利用动物产品无试剂按照现行良好生产规范（cGMP）的指导方针。 我们根据这个协议，我们正在使用成功使用动物源性试剂6隔离hAECs先前公布的协议。我们改变了原来的协议与动物产品无试剂替代动物源性产品，以及随后的优化进行优化细胞产量，活力和纯度。我们的目标是开发一个协议，将符合法规标准的电池制造的人体临床试验。

<table><tbody><tr><td>Collection Kit</td><td></td><td></td><td></td></tr><tr><td>Stripping tray</td><td>Fisher Scientific</td><td>13-361B</td><td></td></tr><tr><td>Liberty Dressing Forcep, pointed 13 cm</td><td>Fisher Scientific</td><td>S17329&amp;nbsp;</td><td></td></tr><tr><td>Scissors - Sharp/Blunt Straight</td><td>Fisher Scientific</td><td>NC0562592&amp;nbsp;</td><td></td></tr><tr><td>Sterile latex gloves&amp;nbsp;</td><td>Fisher Scientific</td><td>19-014-643&amp;nbsp;</td><td></td></tr><tr><td>Protective Apparel</td><td></td><td></td><td></td></tr><tr><td>Protective Apparel (Gown)</td><td>U-line</td><td>S-15374-M</td><td></td></tr><tr><td>Isolation gowns</td><td>U-line</td><td>S-15374-M</td><td></td></tr><tr><td>Sterile latex gloves&amp;nbsp;</td><td>Fisher Scientific</td><td>19-014-643&amp;nbsp;</td><td></td></tr><tr><td>General purpose face mask</td><td>Cardinal Health</td><td>AT7511</td><td></td></tr><tr><td>Bonnets</td><td>Medline</td><td>CRI1001</td><td></td></tr><tr><td>Shoe covers</td><td>U-line</td><td>S-7873W</td><td></td></tr><tr><td>Media and Reagents</td><td></td><td></td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14175095</td><td>without calcium or magnesium</td></tr><tr><td>TrypZean (animal product&amp;ndash;free recombinant trypsin)</td><td>Sigma Aldrich</td><td>T3449</td><td></td></tr><tr><td>Soybean Trypsin Inhibitor&amp;nbsp; 1g/50mL</td><td>Sigma Aldrich</td><td>T6522</td><td></td></tr><tr><td>Cryostor CS5</td><td>BioLife Solutions</td><td>205102</td><td></td></tr><tr><td>Trypan blue reagent</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Anti-EpCam-PE</td><td>Miltenyi Biotec</td><td>130 - 091-253</td><td></td></tr><tr><td>PE-isotype control</td><td>Miltenyi Biotec</td><td>130-098-845</td><td></td></tr><tr><td>Anti-CD90-PeCy5</td><td>BD Pharmingen</td><td>555597</td><td></td></tr><tr><td>PeCy5-isotype control</td><td>BD Pharmingen</td><td>557224</td><td></td></tr><tr><td>Anti-CD105-APC</td><td>BD Pharmingen</td><td>562408</td><td></td></tr><tr><td>APC-isotype control</td><td>BD Pharmingen</td><td>340754</td><td></td></tr><tr><td>Collagen Type VI</td><td>Sigma Aldrich</td><td>C7521</td><td></td></tr><tr><td>Consumables</td><td></td><td></td><td></td></tr><tr><td>50 ml graduated pipette</td><td>BD/Falcon</td><td>356550</td><td></td></tr><tr><td>10 ml graduated pipette</td><td>BD/Falcon</td><td>356551</td><td></td></tr><tr><td>5 ml graduated pipette</td><td>BD/Falcon</td><td>356543</td><td></td></tr><tr><td>50 ml falcon tubes</td><td>BD/Falcon</td><td>352070</td><td></td></tr><tr><td>15 ml falcon tubes</td><td>BD/Falcon</td><td>352096</td><td></td></tr><tr><td>15 cm Petri dishes</td><td>Corning</td><td>351058</td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>BD/Falcon</td><td>352350</td><td></td></tr><tr><td>0.22 &amp;mu;m filters</td><td>Millipore</td><td>SLGV033RS</td><td></td></tr><tr><td>1 ml Pipette tips</td><td>Fisherbrand</td><td>02-707-401</td><td></td></tr><tr><td>200 &amp;mu;l Pipette tips</td><td>Fisherbrand</td><td>02-707-409</td><td></td></tr><tr><td>20 ml Syringe</td><td>BD/Medical</td><td>309661</td><td></td></tr><tr><td>Plastic spatula</td><td>Fisher Scientific</td><td>14-245-97&amp;nbsp;</td><td></td></tr><tr><td>Plastic weighing boat</td><td>Fisher Scientific</td><td>02-202-102&amp;nbsp;</td><td></td></tr><tr><td>Cryo vials</td><td>Nunc</td><td>377267</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Mr Frosty</td><td>Fisher Scientific</td><td>A451-4&amp;nbsp;</td><td></td></tr><tr><td>Biohazard Cabinet</td><td></td><td></td><td></td></tr></tbody></table>

isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

当此过程被正确执行，1.2亿hAECs平均产量应该预计，随着80-160百万细胞的典型范围。从这些产量，83±4％的平均存活率可以预期。增加的平均产量和稍低生存能力在临床方法可以由于缺乏血清蛋白是由于比动物衍生产物更高胰蛋白酶活性，或许也是。孤立hAECs有92％的EpCAM阳性细胞与&lt;1％CD90，C​​D105间充质标志物阳性细胞的平均细胞表面轮廓。这些标志物由于分别选择它们的特异性上皮24,25</s...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

isolation-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K 次观看.  Wake Forest University Health Sciences. We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. 

视频: Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

科研

发育生物学

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

生物学

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

人类羊膜不同解剖区域的皮下细胞体外培养

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

生物工程

推进人干细胞来源的视网膜色素上皮细胞的低温储存

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

作者聚焦:开发一种高效可行的视网膜色素上皮细胞冷冻方法

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...

分离，冻存和文化的人羊膜上皮细胞的临床应用

摘要

探索更多视频

此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多孔骨基质

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

无异条件下多原代羊水和膜细胞重新编程

人子宫内膜基质细胞的体外分离蜕膜化

人类胚胎干细胞冷冻和解冻

人类羊膜不同解剖区域的皮下细胞体外培养

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

分离，冻存和文化的人羊膜上皮细胞的临床应用

摘要

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此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

无异条件下多原代羊水和膜细胞重新编程

人子宫内膜基质细胞的体外分离蜕膜化

人类胚胎干细胞冷冻和解冻

人类羊膜不同解剖区域的皮下细胞体外培养

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

人类骨髓间充质干细胞的分离及其培养的多孔骨基质