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Bioingegneria

Küresellerde Memeli Hücrelerinin Büyük ölçekli Genişleme için sürekli Beslenme ile Besin Yönetmeliği

Published: September 25, 2016
doi:
10.3791/52224
, , , , , , , , ,
1Radiology,University of Minnesota, 2Medicina,University of Minnesota, 3School of Public Health,University of Minnesota, 4Surgery,University of Arizona

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

Transplantasyon için uygulanabilir ve fonksiyonel insan hücrelerinin çok sayıda oluşturmak için, kültür koşullarının düzenlenmesi zorunludur. 3 – metabolik atıkların birikimi ile birlikte besinlerin tükenmesi, hücre ürününün 1 kalitesini azaltır yaşlanması ve metabolik değişikliklere önemli sebeplerindendir. Bu prosedür kültürünün süresi boyunca fizyolojik aralığında 4 glikoz düzenleyen ayarlanmış oran perfüzyon besleme sistemi ile birlikte karıştırılan bir biyoreaktör kullanarak küresellerde kültür memeli hücrelerinin bir yöntemi gösterir. Bu çalışmaların amacı için, fizyolojik aralığı 100 ila 200 mg / dl olarak tanımlandı. Aynı yöntemleri diğer besin ve laktat gibi metabolik atıkları regüle etmek için kullanılabilir.

küçük hacimlerde statik kültürler (1-30 mi), genellikle experime hücre hatları korumak ve ayırt laboratuar ortamında kullanılanntal amaçlar. Hücre pasajı düzenli aralıklarla gerektiğinde komple ortam değişiklikleri ile gerçekleştirilir. En "klasik" kültür ortamı, besin sınırlamalar riski olmadan daha az sık ve orta değişiklikleri sağlamak için, yüksek glukoz konsantrasyonuna (bu çalışmalarda kullanılan DMEM 450 mg / dL) sahiptir. Ancak, bu toplu beslenme yöntemi hala, sık manipülasyon gerektiren hücre ortamında değişkenlik tanıtır ve kontaminasyon 5 riskini artırır – 9. Karıştırılan süspansiyon biyoreaktörler (SSB) daha iyi karışmasını sağlamak ve 3,10 işleme azalmış – 20, ama statik kültürler gibi, besin ve atık ürün düzeylerinde potansiyel zarar verici dalgalanmalar katkıda manuel orta değişiklikler gerektirir. SSB kültürlerin perfüzyon besleme sürekli infüzyon ve orta kaldırılması, fakat hücre büyümesi besin seviyelerindeki büyük değişikliklere göre bu sorunların bir sorun olmaya devam azaltır. düzeltilmiş besleme ra kullanımıTahmini hücre gereksinimlerine göre besin kullanım hesaplamalarından te hücre canlılığını optimize etmek için gerekli kararlı hücre ortamı sağlamak ve 21 işlev görebilir – 24.

25 özel pluripotent hücrelerin kültür ve genişleme için memeli hücrelerinin ölçeklenebilir SSB kültürler için yöntemleri tarif literatür büyük bir gövde var – diğerleri ile 32, adacık (beta) hücreleri 17,33,34 odaklanmış, ya da biyolojik ürünlerin 24 üretim, 35-38. Bu araştırılmıştır hücre tiplerinin bir çok sferoid kültürlerde yetiştirilebilir ve kullanılan hücre tipi için özel prosedürler sürekli bir besleme sisteminin uygulanması öncesinde optimize edilmelidir. 43 – Bu gösteri, bir perfüzyon besleme yöntemi bir karıştıran bioreaktör 39 sferoidler olarak yetiştirilen beta hücre hattı genişletmek için kullanıldı. Bu tarifnamede tarif edilen metod sağlarhedeflenen kültür koşullarını elde etmek için off-line glukoz ölçümlerine dayanarak oranı ayarlamaları beslenme basit uygulama. Bu yöntem ile besleme oranı ayarlama fizyolojik glukoz seviyesi artar, hücre verimleri gösterilir korumak için. Memeli hücreleri, enerji üretimi için, önemli bir besin, glikoz bağlıdır, bu yüzden, bu hücre hattının kullanılması bir çok kültürlenmiş memeli hücrelerine 44 bir modeli temsil etmektedir. Buna ek olarak, bu hat glukoz 45, kronik yüksek düzeyde duyarlı olan beta hücreleri, daha karmaşık örnek teşkil etmektedir. Bu çalışma için, β-TC6 hücreler, in vivo olarak Langerhans adacıklarında ortalama büyüklüğü yaklaşık kültür içinde parçacıklarının oluşturulması için bırakılmıştır. Perfüzyon biyoreaktör sistemi 17 – tüketimi glikoz ayarlanmış bir besleme oranı ile 19,21,46, canlılığı değişiklik olmaksızın, fizyolojik koşullar ve yüksek hücre verimi muhafaza ile sonuçlanmıştır.

Protocol

1. Hücre Hattı ve Bakım Elde β-TC6 hücreleri (ya da diğer arzu edilen yapışkan memeli hücre çizgisi). Çalışmanın hazırlık, kültür, geçit ve sağlayıcı talimatlarına göre hücrelerin cryopreserve. 2. Sürekli Besleme Sistemi birleştirin NOT: – 19,21,47 – 49 aşağıda yöntemi sürekli besleme sistemi tasarımı literatürde 17'de açıklandığı benzer sistemler day…

Representative Results

Orta Glukoz Düzeyleri ve dalgalanmalar Standart SSB Kültürlerde Hücre Genişleme sınırla Glikoz düzeyleri kültür dönemi 3 boyunca statik kültürler ve SSB kültürlerde dalgalanma. Bu dalgalanmalar, 21 günlük kültür döneminde, hücre sayısı arttıkça yoğunlaştırmak ve statik ve SSB kültürleri hem de hemen hemen aynı idi. Bu gözlemler önceki yayında 3'te sunulmuştur. glikoz düzeyleri her iki yöntem için kültür dönem…

Discussion

58 – biyolojik maddelerin ve hücre terapileri için üretimi için memeli hücre üreten ürünlerin büyük ölçüde 55 kültür ve memeli hücreleri izlenmesini gerektirir. Dahası, bu uygulamalar tanımlanmış ve valide kültür koşulları için çağrı. Sadece bu gereksinimleri karşılamak olmaz araştırma teknolojilerini kullanarak hücrelerin hacminin artırılması. besin ve atık ürünlerin birikimi dalgalanmalara yol açması, Manuel ortam değişiklikleri hücre kalites…

Divulgazioni

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)
DOWNLOAD MATERIALS LIST

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Tags

Keywords: Nutrient RegulationContinuous FeedingLarge-scale Cell ExpansionSpheroidsBioreactorPeristaltic PumpMedium ReservoirWaste ReservoirPerfusion TubingCell CultureCellular TherapyNutrient ControlGas ExchangeCell DensityCell YieldCell Loss
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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).
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