JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Bioengineering

회전 타원체의 포유 동물 세포의 대규모 확장을위한 지속적인 공급함으로써 영양 규정

Published: September 25, 2016
doi:
10.3791/52224
, , , , , , , , ,
1Radiology,University of Minnesota, 2Medicine,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

이식 가능한 기능적 인간 세포의 다수를 생성하기 위해, 배양 조건의 조절이 필수적이다. 3 – 신진 대사 폐기물의 축적과 함께 영양분의 고갈은 셀 제품 1의 품질이 저하 노화 및 대사 변화에 중요한 기여한다. 이 절차는 배양 기간에 걸쳐 생리 학적 범위 4 글루코스를 조절하는 조절 된 속도로 관류 공급 시스템과 결합 교반 생물 반응기를 사용 타원체의 포유 동물 세포 배양하는 방법을 보여준다. 이 연구의 목적은 생리적 인 범위는 100 내지 200 밀리그램 / DL로 정의 하였다. 동일한 방법이 다른 영양소 및 락 테이트 대사 폐기물을 조절하기 위해 사용될 수있다.

작은 볼륨의 정적 문화 (1-30 ml)을 일반적으로 experime에 대한 세포 라인을 유지하고 차별화하는 실험실 환경에서 사용되는ntal 목적. 세포 계대는 정기적으로 필요한 완전 배지 변경과 함께 수행된다. 대부분의 "기존의"문화 매체는 영양 제한의 위험없이 덜 자주 매체 변경을 허용하는 높은 포도당 농도 (이 연구에 사용 된 DMEM 450 밀리그램 / DL)가 있습니다. 그러나,이 배치 급지에있어서 여전히 빈번한 조작을 필요로하는 세포의 환경에서 변화를 도입하고, 5 오염의 위험이 증가한다 – 9. 교반 정지 생물 반응기는 (SSB)는 더 나은 혼합을 제공하고 3,10 취급 감소 – 20 만, 정적 문화처럼, 영양 및 폐기물 제품 수준의 잠재적 인 손상 변동에 기여 설명서 매체의 변화가 필요합니다. SSB 문화의 관류 공급 연속 주입 및 매체의 제거,하지만 인해 세포 성장에 영양 수준에서 큰 변화로 이러한 문제가 문제가 남아 줄일 수 있습니다. 조정 된 공급 라의 사용예상 셀 요구 사항에 따라 영양소 사용량 계산에서 TE는 세포 생존을 최적화하는데 필요한 안정적인 셀 환경을 제공하고, 21 작용할 수 – 24.

25 구체적으로 만능 세포의 배양 및 확장을위한 포유 동물 세포의 확장 SSB 문화에 대한 방법을 설명하는 문학의 큰 몸이있다 – 다른 사람과 (32), 섬 (베타) 세포 17,33,34에 초점을 맞춘이, 또는 생물 의약품 24의 생산, 35-38. 이러한 연구 세포 종류의 많은 구형 배양에서 성장 될 수 있고, 사용되는 세포 유형에 대한 구체적인 절차는 연속 공급 시스템을 구현하기 이전에 최적화되어야한다. 43 -이 예제에서, 관류 공급 방법은 교반 생물 반응기 (39) 회전 타원체로 성장 베타 세포주를 확장 하였다. 본원에 기재된 방법을 제공한다타겟 배양 조건을 달성하기 위해 오프라인 혈당 측정에 기초하여 조정 레이트 공급의 간단한 구현. 이 방법으로 공급 속도를 조절하는 생리적 포도당 수준이 증가 세포 수율에 표시됩니다 유지합니다. 포유 동물 세포는 에너지 생산을 위해 중요한 영양소, 포도당에 의존하므로,이 세포주의 사용은 많은 포유류 배양 세포 (44)의 모델을 나타낸다. 또한,이 라인은 포도당 45 만성 높은 수준에 민감한 베타 세포의 추가의 복잡성을 예시한다. 본 연구를 위해, β-TC6 세포는 생체 내에서 랑게르한스섬의 평균 크기는 대략 배양 타원체를 형성시켰다. 관류 생물 반응기 시스템 (17) – 소비 포도당 조정 된 공급 속도와 19,21,46은, 생존의 변화없이 생리 학적 조건과 높은 셀 수율을 유지하는 결과.

Protocol

1. 세포주 및 유지 관리 구하는 β-TC6 세포 (또는 다른 원하는 부착 포유 동물 세포 라인). 연구를위한 준비, 문화, 통로, 그리고에서 제공 지침에 따라 세포를 cryopreserve. 2. 연속 급지 시스템을 조립 주 – 19,21,47 – (49) 아래의 방법에 연속 공급 시스템 설계 문헌 17에 기술 된 것과 유사한 시스템을 기반으로…

Representative Results

중간 포도당 수준 및 변동 표준 SSB 문화의 세포 확장을 제한 포도당 수준은 배양 기간 3를 통해 정적 문화와 SSB 문화에 변동. 이러한 변동은 21 일의 배양 기간 동안 세포 수의 증가와 함께 강화하고 정적 배양 SSB 모두에서 거의 동일 하였다. 이러한 관찰은 이전 발행 3에 제시되어있다. 포도당 수준은 두 가지 방법의 배양 기간의 기간 동안 슈퍼 생…

Discussion

(58) – 생물학적 제제와 세포 치료에 대한 생산을위한 포유 동물 세포의 제품을 생성하는 대규모 (55)의 문화와 포유 동물 세포의 모니터링이 필요합니다. 또한,이 응용 프로그램은 정의되고 검증 된 배양 조건을 요구. 단순히 이러한 요건을 모두 만족하지 조사 기술을 사용하여 세포의 양을 증가시킨다. 영양분과 노폐물의 축적에 변동을 일으키는 설명서 매체의 변화는 세?…

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)
DOWNLOAD MATERIALS LIST

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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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