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

Nutriente Reglamento mediante la alimentación continua de expansión a gran escala de células de mamífero en esferoides

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

Con el fin de generar un gran número de células humanas viables y funcionales para el trasplante, la regulación de las condiciones de cultivo es imprescindible. El agotamiento de nutrientes, junto con la acumulación de residuos metabólicos son los principales contribuyentes a los cambios de senescencia y metabólicos que reducen la calidad del producto de células 1-3. Este procedimiento demuestra un método para células de mamíferos de cultivo en esferoides utilizando un biorreactor agitado en combinación con un sistema de alimentación de perfusión tasa ajustada para regular la glucosa en un rango fisiológico 4 durante toda la duración del cultivo. Para los fines de estos estudios, el intervalo fisiológico se definió como entre 100 y 200 mg / dl. Los mismos métodos se pueden utilizar para regular otros nutrientes y residuos metabólicos, tales como lactato.

cultivos estáticos en pequeños volúmenes (1 – 30 ml) se utilizan normalmente en el entorno de laboratorio para mantener y diferenciar líneas celulares para EXPERIMENTAL propósitos. pases de células se lleva a cabo con los cambios de medio completo, según sea necesario, a intervalos regulares. La mayor parte de medio de cultivo "convencional" tiene una alta concentración de glucosa (450 mg / dl en DMEM utilizado en estos estudios) para permitir cambios de medio de menos frecuentes, sin el riesgo de limitaciones de nutrientes. Sin embargo, este método por lotes de alimentación de aún requiere una manipulación frecuente, introduce variabilidad en el medio ambiente celular, y aumenta el riesgo de contaminación 5-9. Biorreactores suspensión agitada (SSB) proporcionan una mejor mezcla y la disminución de gastos de envío de 3,10 – 20, pero al igual que cultivos estáticos, requieren cambios manuales medianas que contribuyen a las fluctuaciones en los niveles potencialmente dañinos de productos de nutrientes y desechos. alimentación de la perfusión de las culturas SSB reduce estos problemas mediante la infusión continua y la eliminación del medio, pero los grandes cambios en los niveles de nutrientes debido al crecimiento de las células siguen siendo un problema. El uso de un RA de alimentación ajustadate de los cálculos de utilización de nutrientes sobre la base de los requisitos de células estimado puede proporcionar el entorno celular estable requerido para optimizar la viabilidad celular y la función 21 – 24.

Hay una gran cantidad de literatura que describe métodos para cultivos escalables SSB de células de mamíferos específicamente para el cultivo y expansión de células pluripotentes 25 – 32, con otros se centraron en las células de los islotes 17,33,34 (beta), o la producción de productos biológicos 24, 35-38. Muchos de estos tipos de células investigadas pueden crecer en cultivos de esferoides, y procedimientos específicos para el tipo de célula que se utiliza debe ser optimizado antes de implementar un sistema de alimentación continua. En esta demostración, se utilizó un método de alimentación de perfusión para expandir una línea de células beta crecido como esferoides en un biorreactor agitado 39-43. El método descrito en este documento proporciona unala aplicación directa de la alimentación de los ajustes del tipo basado en las mediciones de glucosa fuera de línea para lograr condiciones de cultivo específicas. Ajuste de la velocidad de alimentación con este método para mantener un nivel de glucosa fisiológica se muestra a los rendimientos celulares aumenta. Las células de mamíferos son dependientes de un nutriente clave, glucosa, para la producción de energía, por lo que el uso de esta línea celular representa un modelo para muchas células de mamífero en cultivo 44. Además, esta línea es un ejemplo de la mayor complejidad de las células beta, que son sensibles a los niveles crónicos elevados de glucosa 45. Para este estudio, se dejó que las células ß-TC6 para formar esferoides en cultivo para aproximar el tamaño medio de los islotes de Langerhans in vivo. El sistema de biorreactor de perfusión 17 – 19,21,46 con una velocidad de alimentación ajustada a la glucosa consumo, se tradujo en el mantenimiento de condiciones fisiológicas y los rendimientos celulares más altas sin cambios en la viabilidad.

Protocol

1. Línea celular y el mantenimiento Obtener células ß-TC6 (u otra línea celular de mamíferos adherentes deseada). En preparación para el estudio, la cultura, el paso, y criopreservar las células de acuerdo con las instrucciones del proveedor. 2. Montar el sistema de alimentación continua NOTA: El diseño del sistema de alimentación continua en el método a continuación se basa en sistemas similares se describen en la literatura 17</s…

Representative Results

Niveles de glucosa del medio y las fluctuaciones de restringir la expansión de la célula en la norma SSB culturas Los niveles de glucosa fluctúan en cultivos estáticos y culturas SSB durante todo el periodo de cultivo de 3. Estas fluctuaciones se intensifican a medida que aumenta el número de células durante el periodo de cultivo de 21 días y fueron casi idénticos en ambos cultivos estáticos y SSB. Estas observaciones se presentan en nuestra publicación …

Discussion

Generación de productos de células de mamíferos para la producción de agentes biológicos y para las terapias con células requiere la cultura y el seguimiento de las células de mamífero en gran escala 55-58. Además, estas aplicaciones requieren condiciones de cultivo definidas y validadas. Simplemente aumentando el volumen de las células utilizando tecnologías de investigación no va a cumplir con todos estos requisitos. cambios de medio manuales que causan fluctuaciones en nut…

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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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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