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

Næringsstof forordning ved Kontinuerlig Fodring for Storstilet Udvidelse af pattedyrsceller in 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

For at skabe et stort antal af levedygtige og funktionelle humane celler til transplantation, regulering af dyrkningsbetingelser er bydende nødvendigt. Udtømning af næringsstoffer, sammen med opbygning af metaboliske affald er væsentlige bidragydere til ældning og metaboliske ændringer, som reducerer kvaliteten af cellen produkt 1 – 3 ud. Denne procedure belyser en fremgangsmåde til kultur pattedyrceller i sfæroider ved anvendelse af en omrørt bioreaktor kombineret med en justeret sats perfusion fodringssystem at regulere glucose i en fysiologisk område 4 under hele kulturen. Med henblik på disse undersøgelser blev det fysiologiske område defineret som mellem 100 og 200 mg / dl. De samme fremgangsmåder kan anvendes til at regulere andre næringsstoffer og metaboliske affald såsom lactat.

Statiske kulturer i små volumener (1 – 30 ml) anvendes typisk i indstillingen laboratorium for at opretholde og differentiere cellelinier til experimental formål. Cell passage udføres med komplet medium ændringer efter behov med jævne mellemrum. Mest "konventionelle" dyrkningsmedium har en høj glucosekoncentration (450 mg / dl for DMEM anvendt i disse undersøgelser) for at muliggøre mindre hyppige udskiftning af medium uden risiko for begrænsninger næringsstoffer. Men denne batch-fodring metode kræver stadig hyppige manipulation, introducerer variation i cellen miljø, og øger risikoen for forurening 5 – 9. Omrørt suspension bioreaktorer (SSB) give bedre blanding og faldt håndtering 3,10 – 20, men ligesom statiske kulturer, kræver manuelle medium ændringer, der bidrager til potentielt skadelige udsving i næringsstoffer og spildprodukt niveauer. Perfusion fodring af SSB kulturer reducerer disse problemer ved kontinuerlig infusion og fjernelse af medium, men store ændringer i næringsstoffer niveauer på grund af cellevækst fortsat være et problem. Anvendelsen af ​​en justeret fodring rate fra beregninger af forbrug af næringsstoffer baseret på estimerede celle krav kan give den stabile celle miljø kræves for at optimere cellernes levedygtighed og funktion 21 – 24.

Der er en stor mængde litteratur, der beskriver metoder til skalerbare SSB kulturer af pattedyrceller specielt til kultur og udvidelse af pluripotente celler 25-32, med andre fokuserede på holmen (beta) celler 17,33,34, eller produktion af biologiske produkter 24, 35-38. Mange af disse undersøgte celletyper kan dyrkes i kugleformede kulturer, og særlige procedurer for den celletype, der bruges bør optimeres før at gennemføre en kontinuerlig fodring system. I denne demonstration blev en perfusion fodring metode bruges til at udvide en beta-cellelinie dyrket som sfæroider i en omrørt bioreaktor 39-43. Den heri beskrevne fremgangsmåde tilvejebringer enligetil implementering af fodring sats justeringer baseret på off-line glucose målinger for at opnå målrettede dyrkningsbetingelser. Justering af tilførselshastigheden med denne metode til at opretholde en fysiologisk glucose niveau vises til stigninger celleudbytter. Mammale celler er afhængige af en nøgle næringsstof, glucose, til energiproduktion, så brugen af denne cellelinie er en model for mange dyrkede pattedyrceller 44. Derudover denne linje eksemplificerer yderligere kompleksitet af beta-celler, som er følsomme for kroniske høje niveauer af glucose 45. Til denne undersøgelse blev p-TC6 celler lov at dannes sfæroider i kultur at tilnærme den gennemsnitlige størrelse af Langerhanske øer in vivo. Den perfusionsbioreaktor systemet 17 – 19,21,46 med en fødehastighed justeret til glucose forbrug, i bevarelse fysiologiske betingelser og højere celleudbytter uden ændringer i levedygtighed.

Protocol

1. Cell Line og vedligeholdelse Opnå p-TC6 celler (eller anden ønsket adhærerende mammal cellelinie). Som forberedelse til undersøgelsen, kultur, passage, og Cryopreservering cellerne i henhold til udbyder anvisninger. 2. Saml Kontinuerlig Feeding System BEMÆRK: Den kontinuerlige fødesystem design i nedenstående metode var baseret på tilsvarende systemer, der er beskrevet i litteraturen 17 – 19,21,47 -<…

Representative Results

Medium blodsukkerniveauet og Udsving Begræns Cell Ekspansion i Standard SSB Cultures Glucose niveauer svinger i statiske kulturer og SSB kulturer i hele kulturen perioden 3.. Disse udsving intensiveres med stigende celletal under 21-dages kultur periode og var næsten identisk i både statiske og SSB kulturer. Disse observationer er præsenteret i vores tidligere publikation 3. Glucoseniveauerne kan være super-fysiologiske under hele dyrkningsperioden…

Discussion

Generering pattedyrcellelinjer produkter til fremstilling af biologiske agenser og for celleterapi kræver kulturen og overvågning af pattedyrceller i stor skala 55-58. Endvidere disse applikationer kræver definerede og validerede dyrkningsbetingelser. Blot øge mængden af ​​celler ved hjælp af forskning teknologier vil ikke opfylde alle disse krav. Manuelle mellemstore ændringer forårsager udsving i næringsstoffer og ophobning af affaldsstoffer reducere cellernes kvalitet, le…

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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