分离，冻存和文化的人羊膜上皮细胞的临床应用
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge financial support from the Victorian Government’s Operational Infrastructure Support Program.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Collection Kit | |||
| Stripping tray | Fisher Scientific | 13-361B | |
| Liberty Dressing Forcep, pointed 13cm | Fisher Scientific | S17329 | |
| Scissors – Sharp/Blunt Straight | Fisher Scientific | NC0562592 | |
| Sterile latex gloves | Fisher Scientific | 19-014-643 | |
| Protective Apparel (Gown) | U-line | S-15374-M | |
| Protective Apparel | |||
| Isolation gowns | U-line | S-15374-M | |
| Sterile latex gloves | Fisher Scientific | 19-014-643 | |
| General purpose face mask | Cardinal Health | AT7511 | |
| Bonnets | Medline | CRI1001 | |
| Shoe covers | U-line | S-7873W | |
| Media and Reagents | |||
| Hanks’ Balanced Salt Solution (HBSS) | Life Technologies | 14175095 | without calcium or magnesium |
| TrypZean(animal product–free recombinant trypsin) | Sigma Aldrich | T3449 | |
| Soybean Trypsin Inhibitor 1g/50mL | Sigma Aldrich | T6522 | |
| Cryostor CS5 | BioLife Solutions | 205102 | |
| Trypan blue reagent | Life Technologies | 15250-061 | |
| anti-EpCam-PE | Miltenyi Biotec | 130 – 091-253 | |
| PE-isotype control | Miltenyi Biotec | 130-098-845 | |
| anti-CD90-PeCy5 | BD Pharmingen | 555597 | |
| PeCy5-isotype control | BD Pharmingen | 557224 | |
| anti-CD105-APC | BD Pharmingen | 562408 | |
| APC-isotype control | BD Pharmingen | 340754 | |
| Collagen Type VI | Sigma Aldrich | C7521 | |
| Consumables | |||
| 50mL graduated pipette | BD/Falcon | 356550 | |
| 10mL graduated pipette | BD/Falcon | 356551 | |
| 5mL graduated pipette | BD/Falcon | 356543 | |
| 50mL falcon tubes | BD/Falcon | 352070 | |
| 15mL falcon tubes | BD/Falcon | 352096 | |
| 15-cm petri dishes | Corning | 351058 | |
| 70-μm filters | BD/Falcon | 352350 | |
| 0.22-μm filters | Millipore | SLGV033RS | |
| 1ml Pipette tips | Fisherbrand | 02-707-401 | |
| 200ul Pipette tips | Fisherbrand | 02-707-409 | |
| 20ml Syringe | BD/Medical | 309661 | |
| Plastic spatula | Fisher Scientific | 14-245-97 | |
| Plastic weighing boat | Fisher Scientific | 02-202-102 | |
| Cryo vials | Nunc | 377267 | |
| Equipment | |||
| Mr Frosty | Fisher Scientific | A451-4 | |
| Biohazard Cabinet |
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