在球体哺乳动物细胞大规模扩张营养调控连续通
Summary
Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.
Abstract
In this demonstration, spheroids formed from the β-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.
Introduction
为了产生大量可行的和功能性人细胞移植，的培养条件调节是必要的。营养物质的消耗，与代谢废物堆积沿主要贡献者，以减少电池产品的质量1衰老和代谢改变– 3。此过程说明使用搅拌生物反应器具有调整速率灌注料系统相结合，以调节整个培养期间在生理范围4中的葡萄糖球体培养的哺乳动物细胞的方法。这些研究的目的，在生理范围被定义为100 200毫克/分升之间。相同的方法可用于调节其它营养物质和代谢废物如乳酸盐。
在小体积的静态培养物（1 – 30毫升）在实验室环境通常用于维持和分化为experime细胞系ntal目的。细胞传代与根据需要定期完全培养基变化进行的。最“常规”培养基具有高的葡萄糖浓度（在这些研究中使用的DMEM 450毫克/分升），以允许无养分限制的风险较不频繁的介质的变化。然而，这种分批输送方法仍然需要频繁操纵，在细胞环境中引入了可变性，并增加污染5的风险– 9。搅拌的生物反应器（SSB）提供更好的混合，降低处理3,10 – 20，但像静态的文化，需要有助于营养物质和废物产品级别潜在的破坏性波动手动媒介的变化。单边带培养物的灌注馈送减少这些问题通过连续输注和去除介质，但是在营养水平由于细胞生长大的变化仍是一个问题。使用调整后的喂养岭德从基于估计的细胞所需养分使用的计算可以提供优化细胞生存力所需的稳定的细胞环境和功能21 – 24。
有大量文献描述的哺乳动物细胞的可扩展SSB文化专为多能干细胞的培养和拓展方法25 – 32，与其他专注于胰岛（测试版）单元17,33,34，或生产生物制品24， 35 – 38。许多这些研究的细胞类型，可以在球体培养物中生长，并为所使用的细胞类型特异性的程序应之前实施的连续进料系统进行优化。在这个演示中，采用灌注饲养方法，扩大成长为在搅拌反应器39的球状体β细胞系– 43。本文所描述的方法提供了一种简单的实现饲养的基础上脱机葡萄糖测量来实现有针对性的培养条件下利率调整。用这种方法调节进料速率，以保持生理葡萄糖水平被示出为增加细胞产量。哺乳动物细胞是依赖于一个关键营养素，葡萄糖，用于能源生产，所以使用该细胞系的代表许多培养的哺乳动物细胞44的模型。此外，这条线例示的β细胞，这是慢性高含量葡萄糖45的敏感的进一步复杂性。在这项研究中，β-TC6允许细胞以形成培养球体近似体内胰岛的平均尺寸。灌注生物反应器系统17 – 19,21,46调整为葡萄糖消耗的进料速率，导致在保持生理条件和较高的细胞产量无生存力的改变。
Protocol
1.细胞系和维护得到βTC6细胞（或其他期望的粘附的哺乳动物细胞系）。在用于研究制备，培养，传代，并根据供应商的说明冷冻保存的细胞。 2.组装连续进料系统注：连续给料系统设计在下面的方法是根据文献中描述的17类似的系统- 19,21,47 – 49。这里使用的系统的组件的详细情况的先前出版物3…
Representative Results
中血糖水平和波动限制在标准SSB培养细胞扩增 血糖水平在静态的文化和文化SSB波动在整个培养周期3。这些波动加强与在21天的培养期增加细胞数量并分别在静态和SSB文化几乎相同。这些意见在我们以前的出版物3中。葡萄糖水平可以是超生理的培养期间这两种方法的持续时间。由于这种长期接触可能会抑制细胞生长54，连续供料系统的开?…
Discussion
生成哺乳动物细胞产品的生产生物战剂和细胞治疗需要大规模55文化和哺乳动物细胞的监测– 58。此外，这些应用要求定义和验证的培养条件。简单地增加使用研究技术不能满足所有这些要求细胞的体积。造成波动的营养物质和废物积聚产品中手动修改降低细胞的质量，生存能力和产量。利用生物反应器的是公认的微生物的生物制药应用的商业文化，和类似的策略已经应用?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Michael Garwood and Sam Stein for their helpful comments, and Kristen M. Maynard for assistance with manuscript preparation.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number / Link | Comments/Description |
| BTC-6 Cells | ATCC, Manassas, VA | CRL-11506 | Mouse Insulinoma cell line (adherent cell type) |
| DPBS No CA, No Mg | Invitrogen, Carlsbad, CA | 14190-144 | https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144 |
| Dulbecco's Modified Eagles Medium | Invitrogen, Carlsbad, CA | See below for product numbers | |
| DMEM High Glucose (500mM) | Invitrogen, Carlsbad, CA | 11965-092 | http://www.lifetechnologies.com/order/catalog/product/11965092 |
| DMEM Low Glucose (100mM) | Invitrogen, Carlsbad, CA | 11885-084 | http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate) |
| L-gultamine | Invitrogen, Carlsbad, CA | 25030081 | http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product |
| Sodium Pyruvate | Invitrogen, Carlsbad, CA | 11360070 | https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product |
| Heat Inactivated Porcine Serum | Gibco – Life Technologies | 10082147 | http://www.lifetechnologies.com/order/catalog/product/10082147 |
| Trypsin-EDTA | Invitrogen, Carlsbad, CA | 25200056 | https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product |
| T-150 Tissue Culture Treated Flasks | Corning, Corning, NY | 430825 | http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences) &categoryname= |
| NuAire Cell culture incubator | Princeton, MN | US Autoflow , Any water-jacketed CO2 regulating cell culture incubator could be used | |
| Centrifuge | Sorvall RT 7 (Any similar benchtop centrifuge may be used) | ||
| Refrigerator | Any laboratory refrigerator could be used (a small table-top version was used for these studies) | ||
| 1L Glass Bottle | Corning, Corning, NY | 1395-1L | Any vendor could be used http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences) &categoryname= |
| 2L Glass Bottle | Corning, Corning, NY | 1395-2L | Any vendor could be used |
| 250 ml stirred bioreactors | Corning, Corning, NY | 4500-250 | http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences) &categoryname= |
| Stir Plate | Fisher Scientific | 11-496-104A | Any incubator safe stir-plate can be used, any vendor |
| Tissue Culture Dishes 100mm Diameter | Nunc, Rochester, NY (Fisher Scientific) | 1256598 | Any vendor could be used (ordered through Fisher Sci) |
| FALCON 50 ml Conical Tubes | Falcon, San Jose, CA | 1256598 | Any vendor could be used |
| Delran Plastic Used for Custom Parts | McMaster Carr | Various | Any material of choice could be used, but Deran is chosen because it is autoclave safe, non-reactive, and easy to machine, http://www.mcmaster.com/#acetal-homopolymer-sheets/=rjrcac |
| Stainless Steel Pipe for custom lids | McMaster Carr | Various | Any vendor could be used, http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91 |
| Custom Modified Delran Bioreactor Lids for Continuous Feeding | Custom made | Not aware of any vendors producing a similar product | |
| Custom Modified Glass Bottle Lids for Continuous feeding | Custom made | Some vendors (eg. Fischer Sci, Corning) make similar products in the links below | |
| Masterflex Digital Peristaltic Pump | Cole Parmer, Vernon Hills, IL | EW-77919-25 | Any precision peristaltic pump could be used, http://www.coleparmer.com/Product/L_S_Eight_Channel_Four_Roller_ Cartridge_Pump_System_115_230 _VAC/EW-77919-25 |
| PVDF Tubing Connectors (various) | Cole Parmer, Vernon Hills, IL | see link | Any vendor could be used, http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium _Luer_Fittings/55889 |
| Pharmed BPT Tubing L/S 16 | Cole Parmer, Vernon Hills, IL | WU-06508-16 | Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing _L_S_13_25/WU-06508-16 |
| Pharmed BPT Tubing L/S 14 | Cole Parmer, Vernon Hills, IL | WU-06508-14 | Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing _L_S_13_25/WU-06508-14 |
| Pharmed BPT Tubing L/S 13 | Cole Parmer, Vernon Hills, IL | WU-06508-13 | Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing _L_S_13_25/WU-06508-13 |
| Millipore Millex GP PES membrane 0.22ul sterile syringe filter (used for venting, and medium filtration) | Fisher Scientific | SLGP033RS | Any vendor could be used |
| 25ml Graduated Pipette | Fisher Scientific | 13-678-11 | Any vendor could be used, and various sizes may be used |
| Pipetter | Fisher Scientific | 13-681-15E | Any vendor, or similar product could be used |
| Hemocytometer | Fisher Scientific | 02-671-6 | Any vendor, or similar product could be used |
| Trypan Blue | Gibco – Life Technologies | 15250-061 | Any vendor, or similar product could be used, https://www.lifetechnologies.com/order/catalog/product/15250061 |
| Inverted Light Microscope | Leica | Any vendor, or similar product could be used | |
| One Touch Ultra Blood Glucose Meter | Fisher Scientific | 22-029-293 | Any vendor, or similar product could be used (eg. Bayer) |
| One Touch Ultra-Strips | Fisher Scientific | 22-029-292 | Any vendor, or similar product could be used (eg. Bayer) |
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Cite This Article
Weegman, B. P., Essawy, A., Nash, P., Carlson, A. L., Voltzke, K. J., Geng, Z., Jahani, M., Becker, B. B., Papas, K. K., Firpo, M. T. Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids. J. Vis. Exp. (115), e52224, doi:10.3791/52224 (2016).