在培养细胞的功能行为可以通过在更体内样的三维培养环境16-21培养得到改善。该原稿描述了一种中空纤维生物反应器系统的用于体内式的哺乳动物组织培养的设置和操作。
Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.
组织培养是用于已经被用于超过100年1,2-细胞的生长和/或维持已建立的技术。研究细胞和响应体外的便利具有深远允许实验,否则将极具挑战性的,如果不是不可能的优点,例如遗传工程细胞系的产生,并在高通量筛选测定法3中使用报道细胞的。最近的组织培养,已经引起了组织工程领域, 在体外模型的生成和再生医学。与这些应用中,在动态的三维(3D)培养系统兴趣显著增长。
三维培养的方法(这里定义为3D培养基材和/或引入定向动态流动的)更好概括的体内细胞环境的体系结构,用于实现重要更多的生理样的功能。提取,生长,分化和移植细胞修复病变和损伤组织的目标的能力是研究领域有病人的利益和商业机会,潜力巨大。例如使用用于治疗烧伤的自体角质形成细胞(见4),并用于治疗中风的使用基于细胞的疗法(见5)。同样,市场对体外模型涵盖药物发现到药物分层应用。在组织培养中的约定是粘附或锚定依赖性细胞类型的组织培养烧瓶中的2维(2D)表面上的生长。虽然目前公认的在研究设置的金标准,近期组织工程应用的兴趣强调,当前2D组培环境不足以在电池生产6所需要的规模。
对于贴壁细胞类型scaffold被需要,这将取决于最终用途,在这两种化学成分和机械性能的方面。一些系统采用的支架作为由从高内相乳液模板形成高度多孔的基质的多孔板插入件(参见7)或电纺纤维(见第8)规定从常规的2D培养技术的最小的适应。细胞可以接种到不同成分的微载体和搅拌槽内的输送养分和信号分子长大,并通过动态以及混合环境9带走废物(大众交通)。然而,这些系统都在它们的体内样环境限制,并且进一步改进可以与问候作出按比例放大的成本。中空纤维生物反应器(HFBs)是由固定在与通常接种在通过纤维内腔递送的多孔纤维和介质的外部单元的模块的纤维的三维培养系统(1审查0)( 图1)。 HFBs提供与纤维模仿毛细血管以及从与动态媒体递送相关联的剪切应力屏蔽的细胞,同时使规定剪切经由流体流动通过侧口如果需要的话可以应用于细胞的体内样的环境。这将创建具有优越的物质传输中高细胞密度可以达到11一个多功能的文化体系。的HFB系统非常适合于维持锚地依赖性细胞类型,并已用于培养多种细胞,包括朗格汉斯12,小鼠β-TC-3胰岛细胞系12,原代人肝细胞13人骨的大 鼠胰岛骨髓单核细胞14的Madin Darby犬肾细胞(MDCK)15和Caco-2细胞16仅举几。
除了传质和放大的系统的优点,细胞生长在3D炎告培养系统往往更体内样形态,更响应于实验线索。例如大鼠原代肝细胞显示更立方形形态,增加活力,细胞色素P450酶的活性更大的诱导和敏感性增加扑热息痛毒性相比,在2D培养17生长的细胞中的市售聚苯乙烯脚手架生长时。使用肝癌细胞系HepG2也已显示出增加白蛋白生产18,并显示在体内的更样响应于甲氨蝶呤相比2D培养细胞19相同的支架。原代人肝证明延迟去分化,较高细胞色素P450酶活性的和用于在灌注培养系统20测试的4/5的化合物增加的间隙。人神经干细胞衍生的神经元和神经胶质细胞中的市售聚苯乙烯支架培养显示出兼具高(动作电位)和低frequeNCY(局部场电位)自发活动,而没有神经元的活动在2D培养细胞21进行检测。 Caco-2细胞相比通过增加的碱性磷酸酶,γ谷氨和P-糖蛋白的活性和F-肌动蛋白和卵occludens-1蛋白16高表达测量的2D培养中HFB证明增强的分化。尽管优点,细胞在比二维组织培养瓶表面的其它系统的常规培养仍然没有在许多实验室实施,虽然出版物引用的3D细胞培养物的数量在不断增长(在过去的10年增加8倍。源:考研“结果按年份”工具,“3D文化”)的侦测。
该原稿描述HFB系统的用于哺乳动物细胞培养物中的设置和操作条件,并演示了在培养人肝癌细胞系HepG2 / C3A其效用。该方法的目的是要培养的细胞更在体内样的文化,保留足够的简单系统,使它适合那些谁是新的三维培养系统。后面在应用程序中使用HFBs理描述在这里,这是改善肝模型的可预测性是它在理论上是可能的HFB环境22内模仿肝窦。这不是目前与其他文化系统是可行的。
该原稿描述了一种中空纤维生物反应器(HFB)系统,用于哺乳动物细胞培养的设置和操作,它的效用是表现在增殖的肝细胞系HepG2 / C3A。该系统的设计,以适应到一个标准培养箱及其组式的货架足以任何主管细胞生物学家熟悉无菌技术进行简单。
在这里描述的研究系统中使用的纤维是在房子由相位反转自旋使用非生物可降解的专有聚合物浇铸(纺丝制造)。有可能通过从各种适于细胞培养,包括可生物降解的和不可生物降解的,例如材料纺丝使纤维;聚己内酯(PCL)27,聚-L-丙交酯酸(PLLA)28,聚(乳酸-共-乙醇酸)(PLGA)29,聚砜(PSU)12和聚醚醚酮(PEEK)13。每个人都有不同的属性次应该基于系统的需要来选择。检查与在杀菌工序中使用的乙醇所采用光纤的兼容性。 PLGA是已知用乙醇迫使一种替代治疗,如抗生素/抗 真菌溶液25增塑。
这里使用的玻璃模块的尺寸是基于目前的研究的需要选择。不同的大小可通过任何有信誉的玻璃吹制公司进行。在模块尺寸的考虑是细胞的数目,这是与在模块中的纤维和可能的流速的数量。在更多的细胞有所述模块的较高的流率将需要以维持良好的培养条件在生物反应器的流出端英寸这将达到一些点,一些试验和错误可能会在模块渗透监测的媒体条件要求的限制。数学建模可以提供一些见解所需模块扪sions和流速22。
这里使用的设备的尺寸被设计为装配到培养箱货架。管道的长度由同时还允许部件的足够的运动,以允许设置和操作的连接器之间,以达到所需要的长度所决定的。如果需要的时间当然采样,例如在模块中的媒体状况监测渗透然后喷射口可以加入渗余物和渗透物线,以促进此。
一种用于任何细胞培养系统的先决条件是保持细胞存活并在大多数情况下生长。在研究证明体内样表型的更多,在3D培养系统中生长的细胞的光似乎也很重要,以提供密切模仿由细胞所遇到的体内环境的环境。最后这一点往往被忽视的2D细胞培养赞成这种文化系统提供了方便。 ŧ他通过纤维内腔递送营养物质到细胞HFB 体内毛细管网络模拟物。废物产品也从系统的动态流中去除。这产生用于细胞培养和一个密切模仿由肝细胞看到的在体内环境中的体内样系统,使得该系统更好的选择相比2D组织培养塑料用于培养这些细胞。这由以下事实证实了细胞分泌15倍相比,生长在二维组织培养塑料白蛋白的量,一个重要的肝功能,在HFB培养系统。
虽然HFB系统适合于大多数,如果不是全部贴壁依赖细胞类型,这里的例子为肝细胞,因为有一个真正的需要,以便能够培养官能,更体内样用于在药物开发使用肝细胞由制药工业并在体外支持生物人工肝设备的肝衰竭患者。需要更多的功能单元延伸超出这些例子中,特别是作为再生医学的领域进入的平移工作阶段。更在体内样的文化环境的优势不可小觑。
The authors have nothing to disclose.
This work was funded by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) CRACK IT funding.
Glass HFB Module | Soham Scientific | — | Custom Item. (Section 2) |
Sigmacote® | Sigma-Aldrich | SL2 | (Section 2.1) |
Silicoset 151 | Intertronics | ACCSS151 | Silicone Glue. (Section 2.3) |
PTFE tape | Sigma-Aldrich | Z104388 | (Section 2.5) |
Reservoir bottle | Fisher | 11972619 | PTFE the screw threads of the adapters and fitting nut. Attach to the Q-series cap. Attach an 8.5cm section of the supplied PTFE tubing under the 1mm adapter and a 4cm section under the 3mm adapter. (Section 3.2.1) |
Q-series cap | Kinesis | 00932Q-3V | PTFE the screw threads of the adapters and fitting nut. Attach to the Q-series cap. Attach an 8.5cm section of the supplied PTFE tubing under the 1mm adapter and a 4cm section under the 3mm adapter. (Section 3.2.1) |
Adatpter, Male, 1.0mm ID | Kinesis | 008NB10-KD5L | PTFE the screw threads of the adapters and fitting nut. Attach to the Q-series cap. Attach an 8.5cm section of the supplied PTFE tubing under the 1mm adapter and a 4cm section under the 3mm adapter. (Section 3.2.1) |
Adatpter, Male, 3.0mm ID | Kinesis | 008NB30-KD5L | PTFE the screw threads of the adapters and fitting nut. Attach to the Q-series cap. Attach an 8.5cm section of the supplied PTFE tubing under the 1mm adapter and a 4cm section under the 3mm adapter. (Section 3.2.1) |
Fitting Nut | Kinesis | U-350 | PTFE the screw threads of the adapters and fitting nut. Attach to the Q-series cap. Attach an 8.5cm section of the supplied PTFE tubing under the 1mm adapter and a 4cm section under the 3mm adapter. (Section 3.2.1) |
Neoprene tubing | Fisher | 10366344 | Attach the Hepa filter to 6cm of neoprene tubing and attach this to the 'fitting nut'. Attach a 2x 30mm sections of L/S14 tubing to the top two barbs of the Y-connector and a 3cm section of L/S16 tubing to the bottom. Attach this to the barb of the 3mm ID adapter. (Section 3.2.1) |
HEPA-vent | Fisher | 11374634 | Attach the Hepa filter to 6cm of neoprene tubing and attach this to the 'fitting nut'. Attach a 2x 30mm sections of L/S14 tubing to the top two barbs of the Y-connector and a 3cm section of L/S16 tubing to the bottom. Attach this to the barb of the 3mm ID adapter. (Section 3.2.1) |
Y-connector, barbed | Cole Parmer | OU-06295-10 | Attach the Hepa filter to 6cm of neoprene tubing and attach this to the 'fitting nut'. Attach a 2x 30mm sections of L/S14 tubing to the top two barbs of the Y-connector and a 3cm section of L/S16 tubing to the bottom. Attach this to the barb of the 3mm ID adapter. (Section 3.2.1) |
L/S16 Silicone tubing | Cole Parmer | OU-96410-16 | Attach the Hepa filter to 6cm of neoprene tubing and attach this to the 'fitting nut'. Attach a 2x 30mm sections of L/S14 tubing to the top two barbs of the Y-connector and a 3cm section of L/S16 tubing to the bottom. Attach this to the barb of the 3mm ID adapter. (Section 3.2.1) |
L/S14 Silicone tubing | Cole Parmer | WZ-96410-14 | Attach the Hepa filter to 6cm of neoprene tubing and attach this to the 'fitting nut'. Attach a 2x 30mm sections of L/S14 tubing to the top two barbs of the Y-connector and a 3cm section of L/S16 tubing to the bottom. Attach this to the barb of the 3mm ID adapter. (Section 3.2.1) |
L/S13 Silicone tubing | Cole Parmer | OU-96410-13 | 80cm to connect the 1.0mm barbed adapter on the Q-series cap to the pump tubing = Feed tube. (Section 3.2.1) |
WM 205U/CA pump | Fisher | 1248-6300 | (Section 3.2.1) |
WM pump tubing, PVC, blue-orange, 0.25mm bore | Fisher | 12416310 | PTFE the screw thred of the male adapter and connect the female adapter. Work the pump tubing over one of the barbs. Repeat this set-up at the other end of the tubing. (Section 3.2.1) |
Adatpter, Male, 1.0mm ID | Kinesis | 008NB10-KD5L | PTFE the screw thred of the male adapter and connect the female adapter. Work the pump tubing over one of the barbs. Repeat this set-up at the other end of the tubing. (Section 3.2.1) |
Adatpter, Female, 1.0mm ID | Kinesis | 008NB10-KD2L | PTFE the screw thred of the male adapter and connect the female adapter. Work the pump tubing over one of the barbs. Repeat this set-up at the other end of the tubing. (Section 3.2.1) |
Female luer cap | Cole Parmer | WZ-45508-64 | Side port end caps. (Section 3.2.2) |
L/S13 Silicone tubing | Cole Parmer | OU-96410-13 | 40mm section to connect the pump tubing to a module connector. (Section 3.2.2) |
L/S16 Silicone tubing | Cole Parmer | OU-96410-16 | 3x 30mm of L/S16 fitted to 3x reducers = module connectors. (Section 3.2.2) |
Barbed reducer 1/8"x1/16" | Cole Parmer | 30616-43 | 3x 30mm of L/S16 fitted to 3x reducers = module connectors. (Section 3.2.2) |
L/S13 Silicone tubing | Cole Parmer | OU-96410-13 | 55cm section to connect the retentate to the L/S14 of the Y-connector on the Q-series cap. (Section 3.2.4) |
L/S13 Silicone tubing | Cole Parmer | OU-96410-13 | 45cm section to connect the permeate to the L/S14 of the Y-connector on the Q-series cap. (Section 3.2.4) |
Straight barbed union | Cole Parmer | WZ-30612-43 | Attach to the end of the L/S13 that will connect with the L/S14 of the Y-connector. (Section 3.2.4) |
Clamp | VWR | 229-0609 | (Section 3.4.2) |
4mm Silicone tubing | Fisher | FB68858 | Fold over a 40mm section of tubing and secure with a cable tie = Module end cap. (Section 4.3) |
Cable tie | Fisher | 12326377 | Fold over a 40mm section of tubing and secure with a cable tie = Module end cap. (Section 4.3) |
MACSmix tube rotator | Miltenyi Biotech | 130-090-753 | An adaptation may be required to attach the modules. (Section 4.4) |
Leur Injection port | Thistle Scientific | IB-10820 | Attach the end cap to the injection port. (Section 4.5) |
Female luer cap | Cole Parmer | WZ-45508-64 | Attach the end cap to the injection port. (Section 4.5) |
L-lactic acid kit | Megazyme | K-LATE | (Section 5) |
D-glucose kit | Megazyme | K-GLUC | (Section 5) |
Scalpel / micro knife | InterFocus | 10315-12 | (Section 6.2) |
Albumin ELISA | Bethyl Labs | E80-129 | (Section 7.4.3) |