Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.
This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.
Standard stem cell culture techniques suffer from several environmental constraints that place undue stresses on the cells and expose the cells to unacceptable risks of contamination. Among the stresses that cells may endure under standard cell culture conditions are precipitous changes in the levels of carbon dioxide and oxygen concentrations3,4. This occurs when the cells are moved from the incubator to the biosafety cabinet and/or microscope which may not be optimal for the cells. Previous studies have confirmed the advantages of culturing both pluripotent and neural stem cells in hypoxic conditions4,11, and for best results, these conditions need to be continuous. Moreover, risks of cellular contamination are higher as the laboratory environment and personnel impinge upon the cells at almost every step of their culture and manipulation. Traditional clean rooms comprise one effective method to greatly decrease contamination risks but they are expensive, have a large footprint and fail to address stressors related to carbon dioxide and oxygen concentrations.
A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria9 provides high quality cells for basic science research as well as clinical applications1,6,7. Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules. Using this basic infrastructure, a wide variety of tasks can be performed, such as standard feeding and passaging of pluripotent stem cells and multipotent neural stem cells, as well as more specialized methods like Sendai virus-based reprogramming, in vitro migration studies, and differentiation of neural stem cells for electrophysiological characterization.
在CPF内生长的细胞看到在氧气或二氧化碳的浓度没有变化,因为它们从培养箱移动到处理室对显微镜室和背部。至关重要的是,在每一个腔室的条件匹配的特定培养箱,其中从培养箱除去细胞之前将细胞保持。该装置内的气氛是连续HEPA过滤并是可定制对于氧气和二氧化碳的浓度。细胞可以在标准浓度的PSCs或神经干细胞,5%和9%,中生长;或替代浓度可以选择为不同的细胞类型或特定的实验。因此,该装置与医疗级氧气,二氧化碳,和氮( 图4)的常数源供给。所有这三种气体是由特定气体歧管系统来确保恒定的供应提供。该装置还与一校准气体混合物包括供应10%(±0.01%)二氧化碳的氧气。歧管系统被安置在电池生产设施外和气体管道输送到通过天花板设施。校准气体被容纳在设施内。该装置还与房子的真空供给,也通过在天花板上。使用电子监控系统和无线发送装置,全部歧管的输出压力不断监测。在任何压力下降超出范围的情况下,电池生产工厂运营商都将自动打电话并通知等适当的行为采取行动。
该装置的功率要求是由六个专用120伏的电路从天花板降会见并连接到医院的备用发电机,以确保恒定的供应。该设备的操作是通过软件来通过不间断电源供电的基于PC的计算机上进行控制。这些电源和电脑安排确保系统的功能连续地甚至在一个公共电力系统故障的情况下。该软件控制装置具有用户友好的图形界面( 图1),它允许对氧气和二氧化碳浓度的控制以及温度,湿度,以及腔室压力。所有这些参数的值连续记录提供的所有设备参数的运行记录。每天晚上这个数据备份到远程服务器上,以保护其完整性。计算机和软件可以远程管理通过用户访问,以评估和/或更改任何参数。此外,计算机和软件可以远程访问,允许设备参数互动的评估,并与当地用户的故障诊断。一个附加的警报发送单元被连接到该装置,使得细胞生产设施人员收到通知的装置的任何超出范围状态。远程访问Çapabilities允许登录并超出范围的条件的具体的评估。
该装置被设计成一个模块化系统无论在宏观和微观意义。个别的细胞培养的模块,如孵化器和处理室,可以在考虑到它们的尺寸和要求,以及在它们的布局相对于彼此进行定制。此外,大多数的单个模块的控制功能本身模块化使得个体气氛气体控制器,例如,可以容易地不显著中断系统取代。
专门处理室,例如一个用于显微镜可视化和细胞培养物的处理,很容易适应系统。两者相差和荧光显微镜是系统( 图6),使得细胞可被活染色内部,菌落可以在相同的大气条件被解剖作为内部吨他的孵化器。通过在处理室的侧壁密封垫圈电缆的路由允许设备如电源和计算机要保持该装置的外面,通常在一个购物( 图6)。
在细胞生产设施处理室具有比常规的BSC不同气流模式。在常规的BSC,气流从中央排气口向下流动并分裂成两个单独的流,然后将其由两个不同的进风口在机壳的底板的前端和后部吸收。相比之下,CPF在顶棚的前部的单个排气口。空气流向下和朝向腔室,在那里,然后向上抽吸到进气通风口的背面。虽然公积金本身是很干净,这种独特的气流模式意味着,技术人员需要稍微调整自己的技术,以减少污染的风险。与传统的BSC,实验室工人小号HOULD避免将自己的双手张开细胞培养板和媒体瓶的上游。然而,这是上游方向已经在CPF改变
该电池生产工厂实验室本身是相当标准,并配备了-20℃的冰柜,-80℃的冰柜,4℃冰箱,离心机和水浴。实验室还具有对方便免提操作脚控制一个接收器。为了使该实验室成为官能临床细胞生产设施,然而,几个另外的修饰仍必须进行。首先,该装置本身必须升级为具有监视挥发性有机化合物,颗粒,和二氧化氯是用于去污的浓度的能力。其次,含在FACS机器的处理腔室可以被容纳并且经由缓冲器模块连接到该设备的其余部分。这将允许细胞分选和TR的纯化适当的环境条件下ansplantable细胞群。最后,整个装置必须在软墙洁净室之内被安置。这提供了一个国际标准化组织(ISO)8级环境,为设备5。
森林合作伙伴关系的高度不育和计算机控制的特性使得它与细胞治疗和良好的制造工艺的未来应用的理想系统。污染的风险被大大减轻,但更重要的是,细胞扩增的条件,自动记录和由计算机系统归档。在气体浓度,温度,湿度,和接入到系统中的所有事件偏差严格记载。这可以调查产品出现质量问题时有很大帮助。然而,仍然有限制。任何和所有试剂和耗材的使用( 例如,媒体组件,移液管,板)必须单独记录。加itionally,也有可能出现,这是完全无关的CPF的监控系统记录的变量可能出现的问题(包括人为错误的多种形式),众说纷纭。因此,需要对训练有素的人员和任务的详细手册文档保持在原位。
The authors have nothing to disclose.
作者要感谢在Biospherix工作人员,他们在学习使用Xvivo封闭的细胞培养系统,尤其是马特·弗里曼帮助;的Miles&凯利建筑公司,公司为他们建立实验室基础设施建设,尤其是拉斯·休斯的工作人员;设施和支持服务工作的奥兰治县科儿童医院在协调实验室改造,尤其是亚当Lukhard和德文Hugie的人员;信息系统为他们建立的数据管理基础设施和远程访问,特别是越南陈德良帮助的奥兰治县科儿童医院的工作人员;奥兰治县执行管理团队的儿童医院为他们的长期支持该项目,特别是玛丽亚博士米农和布伦特Dethlefs的。这项工作是由橙县儿童医院和加州理工学院再生Medicin资助e到补助TR3-05476小灵通。所有作者同样促成了这一工作。
Equipment | |||
Xvivo System | Biospherix | custom made | |
Xvivo Software | Biospherix | version i.o.2.1.2.1 | |
O2 Manifold | Amico | P-M2H-C3-S-U-OXY | |
CO2 Manifold | Amico | M2H-C3-D-U-CO2 | |
N2 Manifold | Western Innovator | CTM75-7-2-2-BM | |
Microscope with DP21 camera and fluorescence | Olympus Corporation | CKX41 | |
Reagents | |||
DMEM/F12 Glutamax | Life Technologies | 10565-018 | |
StemPro hESC Supplement | Life Technologies | A100006-01 | |
Accutase | Millipore | SCR005 | |
Phosphate-Buffered Sodium | Hyclone | 9236 | |
Fibroblast Growth Factor 2 | R&D Systems | AFL233 | |
Dimethyl sulfoxide | Protide | PP1130 | |
Hank's-based Cell dissociation Buffer | Life Technologies | 13150-016 | |
2-Mercaptoethanol | Life Technologies | 21985-023 | |
Epidermal Growth Factor | R&D Systems | AFL236 | |
Oct-3/4 Antibody | Millipore | AB3209 | |
TRA-1-60 Antibody | Millipore | MAB4260 | |
SSEA4 Antibody | Millipore | MAB4304 | |
BIT-9500 Serum Supplement | Stemcell Technologies | 9500 | |
Consumable Supplies | |||
2mL Serological pipet | VWR | 89130-894 | |
5mL Serological pipet | Olympus Plastics | 12-102 | |
10mL Serological pipet | Olympus Plastics | 12-104 | |
25mL Serological pipet | Olympus Plastics | 12-106 | |
50mL Serological pipet | Olympus Plastics | 12-107 | |
6-well plate | Corning | 353046 | |
12-well plate | Corning | 353043 | |
T25 flask | TPP | 90026 | |
T-75 flask | TPP | 90076 | |
20uL pipet tips | Eppendorf | 22491130 | |
200uL pipet tips | Eppendorf | 22491148 | |
1000 pipet tips | Eppendorf | 22491156 | |
Cryovials | Thermo Scientific | 5000.102 | |
70% ethanol | BDH | BDH1164-4LP | |
Sanimaster 4 | Ecolab | 65332960 | |
Bleach | Clorox | A714239 |