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

Nutrient regulamento, a alimentação contínua de Expansão em grande escala de células de mamíferos em Spheroids

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

A fim de gerar grandes números de células humanas viáveis ​​e funcionais para transplante, a regulação das condições de cultura é imperativo. O esgotamento de nutrientes, juntamente com a acumulação de resíduos metabólicos são importantes contribuintes para a senescência e alterações metabólicas que reduzem a qualidade do produto celular 1-3. Este procedimento demonstra um método para cultura de células de mamífero em esferóides utilizando um biorreactor agitado combinado com um sistema de alimentação de perfusão taxa ajustada para regular a glucose numa gama fisiológica 4 durante toda a duração da cultura. Para a finalidade destes estudos, a gama fisiológica foi definida como entre 100 e 200 mg / dl. Os mesmos métodos podem ser utilizados para regular outros nutrientes e resíduos metabólicos, tais como lactato.

culturas estáticas em pequenos volumes (1 – 30 ml) são normalmente utilizados no ambiente de laboratório para manter e diferenciar linhas celulares para experimefins ntal. Passaging celular é realizada com as mudanças de meio completo, conforme necessário, em intervalos regulares. Mais meio de cultura "convencional" tem uma concentração de glucose elevada (450 mg / dl para DMEM utilizado nestes estudos) para permitir as mudanças de meio menos frequentes, sem o risco de limitações nutricionais. No entanto, esse método de alimentação de lote ainda requer manipulação frequente, introduz variabilidade no ambiente celular, e aumenta o risco de contaminação 5-9. Biorreatores suspensão agitada (SSB) fornecer uma melhor mistura e diminuição da manipulação de 3,10 – 20, mas como culturas estáticas, exigir mudanças médias manuais que contribuem para flutuações potencialmente prejudiciais nos níveis de produtos nutricionais e de resíduos. alimentação perfusão de culturas SSB reduz esses problemas por infusão contínua e remoção do meio, mas grandes mudanças nos níveis de nutrientes devido ao crescimento celular continuam a ser um problema. A utilização de uma alimentação adequada rate a partir de cálculos de uso de nutrientes com base em requisitos de células estimados podem proporcionar o ambiente celular estável necessário para otimizar a viabilidade celular e funcionar 21-24.

Há um grande corpo de literatura métodos para culturas escaláveis SSB de células de mamíferos, especificamente para a cultura e expansão de células pluripotentes 25 descrevendo – 32, com os outros focados em (beta) células 17,33,34 ilhota, ou a produção de produtos biológicos 24, 35-38. Muitos destes tipos de células investigadas podem ser cultivadas em culturas de esferóides, e procedimentos específicos para o tipo de célula a ser utilizado deve ser optimizado antes da implementação de um sistema de alimentação contínuo. Nesta demonstração, um método de alimentação de perfusão foi usada para expandir uma linha celular cultivada como beta esferóides num biorreactor agitado 39-43. O método aqui descrito proporciona umaimplementação direta de alimentar ajustes de taxa com base em medições de glicose no off-line para alcançar condições de cultura alvo. Ajustando a taxa de alimentação com este método para manter um nível de glucose fisiológica é mostrado para colheitas de células aumenta. As células de mamíferos são dependentes de um nutriente essencial, glucose, para a produção de energia, de modo que o uso desta linha de células representa um modelo para muitas células de mamíferos em cultura 44. Além disso, esta linha exemplifica a complexidade adicional de células beta, que são sensíveis a níveis elevados de glicose 45 crónicas. Para este estudo, as células beta-TC6 permissão para formar esferóides em cultura para aproximar o tamanho médio das ilhotas de Langerhans in vivo. O sistema de bioreactor de perfusão 17 – 19,21,46 com uma taxa de alimentação ajustada ao consumo de glicose, resultou na manutenção de condições fisiológicas e rendimentos mais elevados de células, sem alterações na viabilidade.

Protocol

1. Linha de Células e manutenção Obter células-p TC6 (ou outra linha de células de mamífero aderente desejada). Em preparação para o estudo, a cultura, a passagem, e criopreservação de células de acordo com as instruções do provedor. 2. Montar o sistema de alimentação contínua NOTA: O design do sistema de alimentação contínua no método a seguir foi baseado em sistemas semelhantes aos descritos na literatura 17 -<…

Representative Results

Níveis de glicose médio e flutuações Restringir Expansão celular em culturas SSB Padrão Os níveis de glicose flutuar nas culturas estáticas e culturas SSB durante todo o período de cultura 3. Estas flutuações intensificar com o aumento do número de células durante o período de cultura de 21 dias e eram quase idênticas em ambas as culturas estáticas e SSB. Estas observações são apresentados na nossa publicação anterior 3. Os níveis …

Discussion

Geração de produtos de células de mamíferos para a produção de agentes biológicos e para terapias celulares requer a cultura e monitorização de células de mamífero em grande escala 55-58. Além disso, estas aplicações requerem condições de cultura definidas e validadas. Simplesmente aumentar o volume de células usando tecnologias de pesquisa não vai atender a todos esses requisitos. Manual de alterações a médio causando flutuações em nutrientes e acúmulo de resídu…

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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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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