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 border="0" cellpadding="0" cellspacing="0" width="641">
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			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Collection Kit</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Stripping tray</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:119px;">13-361B</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:220px;">Liberty Dressing Forcep, pointed 13cm</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>S17329&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:220px;">Scissors - Sharp/Blunt Straight</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>NC0562592&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Protective Apparel (Gown)</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Protective Apparel</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Isolation gowns</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">General purpose face mask</td>
			<td style="width:119px;">Cardinal Health</td>
			<td style="width:119px;">AT7511</td>
			<td></td>
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			<td height="21" style="height:21px;">Bonnets</td>
			<td style="width:119px;">Medline</td>
			<td style="width:119px;">CRI1001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Shoe covers</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-7873W</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Media and Reagents</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td style="width:119px;">Life Technologies</td>
			<td align="right" style="width:119px;">14175095</td>
			<td>without calcium or magnesium</td>
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		<tr height="21">
			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T3449</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T6522</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cryostor CS5</td>
			<td style="width:119px;">BioLife Solutions</td>
			<td align="right" style="width:119px;">205102</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-EpCam-PE</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130 - 091-253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-isotype control</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD90-PeCy5</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">555597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PeCy5-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">557224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD105-APC</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">340754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">50mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm petri dishes</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#956;m filters</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
			<td style="width:119px;">Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">20ml Syringe</td>
			<td style="width:119px;">BD/Medical</td>
			<td align="right" style="width:119px;">309661</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic spatula</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>14-245-97&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>02-202-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cryo vials</td>
			<td>Nunc</td>
			<td>377267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:119px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></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, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

人类肾脏血管周围间质细胞新型临床分级分离方法

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

人鼻上皮类器官的培养和成像

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

使用创新的无异种方法进行人类治疗，分离和培养人脂肪来源的干细胞

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...