Large‐Scale Cell Culture

Nicole A. Bleckwenn1, Joseph Shiloach1

1 NIDDK, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 1U
DOI:  10.1002/0471142735.ima01us59
Online Posting Date:  May, 2004
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Abstract

Mammalian cells offer a good production system for complex biologic products that require proper post‐translational processing and folding. These products can be endogenous proteins, recombinant proteins, or recombinant antibodies, which are often needed in large quantities for clinical evaluations and structural studies. Many cell lines are available for production purposes and are derived from various sources, therefore, exhibiting a wide variety of growth and production characteristics. In general, cell types can be divided into two major categories: those that are able to grow in suspension (anchorage‐independent) and those that require a physical support for growth (anchorage‐dependent). In either case, the products produced can be secreted into the production medium or can be accumulated inside the cells. The properties of the cells and the location of the produced product dictate the method used for growth and production, and consequently the selection of a suitable type of bioreactor. This unit focuses on the methods used to cultivate large amounts of cells for the purpose of obtaining an endogenous or recombinant product.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Large‐Scale Suspension Cell Culture in Spinner Flasks
  • Basic Protocol 2: Large‐Scale Suspension Cell Culture in a Stirred Tank Bioreactor
  • Basic Protocol 3: Large‐Scale Cell Culture Using Roller Bottles
  • Basic Protocol 4: Large‐Scale Cell Culture Using the Stacked Plate System
  • Basic Protocol 5: Large‐Scale Cell Culture Using the Packed Bed Bioreactor
  • Basic Protocol 6: Large‐Scale Microcarrier Cell Culture in a Spinner Flask
  • Basic Protocol 7: Large‐Scale Microcarrier Cell Culture in Stirred Tank Reactor
  • Basic Protocol 8: Large‐Scale Cell Culture Using a Fluidized Bed Bioreactor
  • Basic Protocol 9: Large‐Scale Cell Culture Using a Hollow Fiber Bioreactor
  • Basic Protocol 10: Large‐Scale Cell Culture Using a Wave Bioreactor
  • Support Protocol 1: Adaptation of Cell Cultures to Serum‐Free/Low‐Protein Medium
  • Support Protocol 2: Expansion of Attachment‐Dependent Cell Cultures in Tissue Culture Flasks
  • Support Protocol 3: Harvesting Attachment‐Dependent Cell Cultures from Tissue Culture Flasks
  • Support Protocol 4: Calibration of Bioreactor Dissolved Oxygen and pH Probes
  • Support Protocol 5: Monitor Nutrients and Metabolites in Cell Cultures
  • Support Protocol 6: Determining Cell Growth and Viability Using Large‐Scale Cell Cultures
  • Support Protocol 7: Bioreactor Tubing and Connector Considerations
  • Support Protocol 8: Adapting Cells to Suspension Culture
  • Support Protocol 9: Siliconizing Glassware
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Large‐Scale Suspension Cell Culture in Spinner Flasks

  Materials
  • Healthy suspension cells in late exponential growth phase
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Sterile spinner flasks (e.g., Bellco, Corning, or Nalgene)
  • Stir plate placed inside a 37°C, CO 2 humidified incubator (30 to 100 rpm)
  • Centrifuge with rotor to accommodate 50‐ and 250‐ml centrifuge tubes

Basic Protocol 2: Large‐Scale Suspension Cell Culture in a Stirred Tank Bioreactor

  Materials
  • PBS ( appendix 2A)
  • Gases such as air, nitrogen, carbon dioxide, and oxygen
  • Healthy suspension cells in late exponential growth phase
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Shear protective agents (e.g., 10% pluronic F‐68, Invitrogen)
  • Feed medium (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Stirred tank bioreactor vessel and control system (e.g., Braun Biotech, New Brunswick Scientific, or Applikon Biotechnology)
  • Probes to measure online parameters (e.g., pH, temperature, DO, level, and others; see protocol 14 for calibrating probes; pH and DO probes available from Mettler‐Toledo, Broadley James, Hamilton; temperature and level probes are generally available with the bioreactor units)
  • Cell separation device (e.g., ATF System, Refine Technology; hollow fiber, Amersham Biosciences; spin filter, B. Braun Biotech; acoustic filter, Applikon Biotechnology)
  • Sterile, vented medium transfer bottles in sizes in the range of 0.25 to 5 times the working volume of the reactor (Bellco Glass, Corning)
  • Silicone tubing and sterilizable connectors (Cole Parmer)
  • Centrifuge with rotor to accommodate 250‐ml centrifuge tubes

Basic Protocol 3: Large‐Scale Cell Culture Using Roller Bottles

  Materials
  • Harvested, healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Centrifuge with rotor to accommodate 50‐ and 250‐ml centrifuge tubes
  • Roller bottles (e.g., BD or Corning)
  • Roller apparatus (e.g., Bellco)
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 4: Large‐Scale Cell Culture Using the Stacked Plate System

  Materials
  • Harvested, healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Centrifuge with rotor to accommodate 50‐ and 250‐ml centrifuge tubes
  • Stacked plate unit with all components for type 1 (Nunc or Corning) or type 2 (Corning Costar) stacked plate system with control unit and all components
  • 37°C, 5% CO 2 humidified incubator
  • Sterile, vented medium transfer bottles with appropriate tubing and sterile connectors (Bellco Glass, Corning)
  • Silicone tubing and sterilizable connectors (Cole Parmer)
  • 75‐cm2 tissue culture flasks
  • Air pump

Basic Protocol 5: Large‐Scale Cell Culture Using the Packed Bed Bioreactor

  Materials
  • PBS ( appendix 2A)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Harvested, healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Packed bed bioreactor with all components and connectors
  • Packing material (e.g., Firba‐Cel disks, New Brunswick Scientific)
  • Probes to measure online parameters (pH, temperature, DO level, and others; see protocol 14 for calibration of probes; Mettler‐Toledo, Broadley James, Hamilton for pH and DO probes; temperature and level probes are usually available from bioreactor manufacturer)
  • Sterile, vented medium transfer bottles with appropriate tubing and sterile connectors (Corning, Bellco Glass)
  • Pump
  • Centrifuge with rotor to accommodate 250‐ml centrifuge tubes
  • Water bath at 4°C

Basic Protocol 6: Large‐Scale Microcarrier Cell Culture in a Spinner Flask

  Materials
  • PBS ( appendix 2A)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Harvested, healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Microcarriers (e.g., Amersham Biosciences, Nunc, Percell Biolytica, Sigma, or SoloHill Engineering)
  • 37°C, 5% CO 2 humidified incubator
  • Centrifuge with rotor to accommodate 50‐ and 250‐ml centrifuge tubes
  • Sterile, siliconized spinner flasks (e.g., Bellco, Corning, or Nalgene)
  • Siliconized bottle for preparation of some types of carriers ( protocol 19)
  • Stir plate placed inside a 37°C, 5% CO 2 incubator (30 to 100 rpm)

Basic Protocol 7: Large‐Scale Microcarrier Cell Culture in Stirred Tank Reactor

  Materials
  • PBS ( appendix 2A)
  • Harvested healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Gases: air, nitrogen, carbon dioxide, and oxygen (gas cylinders or building supply)
  • Shear protective agents (e.g., 10% pluronic F‐68, Invitrogen)
  • Siliconized stirred tank bioreactor vessel and control system (e.g., Braun Biotech, New Brunswick Scientific, or Applikon Biotechnology)
  • Probes to measure online parameters (pH, temp, DO, level, and others; see protocol 14 for calibration of probes), (Mettler‐Toledo, Broadley James, Hamilton for pH and DO probes; temperature and level probes are usually available from bioreactor manufacturer)
  • Cell separation device (e.g., ATF System, Refine Technology)
  • Sterile vented medium transfer bottles with appropriate tubing and sterile connectors (Corning, Bellco Glass for bottles, Cole Parmer for tubing and connectors)
  • Siliconized transfer bottle ( protocol 19; optional)
  • Microcarriers (nonporous or macroporous; Amersham Biosciences, Nunc, Percell Biolytica, Sigma, or SoloHill Engineering)
  • 37°C, 5% CO 2 humidified incubator
  • Pump
  • Centrifuge with rotor to accommodate 250‐ml centrifuge bottles

Basic Protocol 8: Large‐Scale Cell Culture Using a Fluidized Bed Bioreactor

  Materials
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Harvested, healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Gases: air, nitrogen, carbon dioxide, and oxygen (gas cylinders or building supply)
  • Fluidized bed bioreactor with all components and connectors (e.g., Amersham Biosciences, or Vogelbusch)
  • Probes to measure online parameters (pH, temp, DO, level, and others; see protocol 14 for calibration of probes; Mettler‐Toledo, Broadley James, Hamilton for pH and DO probes; temperature and level probes are usually available from bioreactor manufacturer)
  • Microcarriers appropriate for fluidized bed operation (e.g., Amersham Biosciences)
  • Sterile vented medium transfer bottles with appropriate tubing and sterile connectors (Bellco Glass, Corning for bottles and Cole Parmer for tubing and connectors)
  • Centrifuge with rotor to accommodate 250‐ml centrifuge bottles

Basic Protocol 9: Large‐Scale Cell Culture Using a Hollow Fiber Bioreactor

  Materials
  • Harvested, healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Hollow fiber bioreactor with all components and connectors (e.g., FiberCell Systems or Biovest International)
  • Centrifuge with rotor to accommodate 250‐ml centrifuge bottles
  • Sterile vented medium transfer bottles with appropriate tubing and sterile connectors (Bellco Glass, Corning)
  • Silicone tubing and sterilizable connectors (Cole Parmer)

Basic Protocol 10: Large‐Scale Cell Culture Using a Wave Bioreactor

  Materials
  • Gases: air, CO 2 (gas cylinders or building supply)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Harvested, healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Centrifuge with rotor to accommodate 250 ml centrifuge bottles
  • Wave bioreactor with all components and connectors including rocker platform, with temperature control or space in 37°C incubator (Wave Biotech)
  • Pre‐sterilized Cellbag (available from Wave Biotech; these can be prefilled by Wave Biotech with sterile microcarriers if using anchorage‐dependent cells)
  • Sterile, vented medium transfer bottles with appropriate tubing and sterile connectors, or medium supplied in bags (Bellco Glass, Corning, or medium suppliers for bags; see Internet Resources)
  • Silicone tubing and sterilizable connectors (Cole Parmer)
  • Pump, optional

Support Protocol 1: Adaptation of Cell Cultures to Serum‐Free/Low‐Protein Medium

  Materials
  • Healthy cells in late exponential growth phase (see Support Protocols protocol 122 and protocol 133)
  • Original medium at 37°C and any supplements such as serum
  • New complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Tissue culture flasks or sterile spinner flasks (e.g., BD, Corning, Costar, or Nunc for tissue culture flasks; or Bellco Glass, Corning Nalgene for spinner flasks)
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 2: Expansion of Attachment‐Dependent Cell Cultures in Tissue Culture Flasks

  Materials
  • Healthy cells in late exponential growth phase
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Tissue culture flasks (e.g., BD, Corning, Costar, or Nunc)
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 3: Harvesting Attachment‐Dependent Cell Cultures from Tissue Culture Flasks

  Materials
  • Healthy cells in late exponential growth phase
  • D‐PBS with calcium and magnesium (see recipe)
  • 0.05% to 0.25% trypsin solution with 1 mM EDTA or cell dissociation solution (Invitrogen)
  • Complete medium for cell line at 37°C (see Strategic Planning and Critical Parameters and Troubleshooting)
  • Tissue culture flasks (BD, Corning, Costar, or Nunc)
  • 37°C, 5% CO 2 humidified incubator
  • Centrifuge with rotor to accommodate 50‐ and 250‐ml centrifuge tubes
  • Additional reagents and equipment for trypan blue staining ( appendix 3B)

Support Protocol 4: Calibration of Bioreactor Dissolved Oxygen and pH Probes

  Materials
  • Nitrogen source
  • Air source
  • PBS ( appendix 2A)
  • pH buffers (7.00 and 4.00 or 10.00)
  • Dissolved oxygen (DO) probe, cable, and controller (Mettler‐Toledo, Broadley James, Hamilton)
  • Simulator testing device (Valley Instrument Company), optional
  • Bioreactor
  • pH probe, cable, and controller (Mettler‐Toledo, Broadley James, Hamilton)

Support Protocol 5: Monitor Nutrients and Metabolites in Cell Cultures

  Materials
  • Medium sample
  • Glucose/lactate analyzer (e.g., 2700, YSI; or equivalent) and buffers

Support Protocol 6: Determining Cell Growth and Viability Using Large‐Scale Cell Cultures

  Materials
  • Sample from reactor containing cells
  • D‐PBS containing calcium and magnesium (see recipe)
  • 1× trypsin‐EDTA solution
  • Medium containing serum
  • 1.5‐ml microcentrifuge and tubes
  • 37°C water bath
  • Additional reagents and equipment for trypan blue viability counting ( appendix 3B)

Support Protocol 7: Bioreactor Tubing and Connector Considerations

  Materials
  • Healthy cells in late exponential growth phase
  • Complete medium for growth in tissue culture flasks at 37°C
  • Complete medium for growth in suspension at 37°C (can be commercially available specialty medium or the same attachment growth medium with reduced or removed calcium concentration)
  • Centrifuge with rotor to accommodate 50‐ and 250‐ml centrifuge tubes
  • Sterile spinner flask (e.g., Bellco, Corning, or Nalgene)
  • Tissue culture flasks (e.g., BD, Corning, Costar, or Nunc)
  • 37°C, 5% CO 2 humidified incubator
  • Stir plate placed inside CO 2 incubator (30 to 100 rpm)

Support Protocol 8: Adapting Cells to Suspension Culture

  Materials
  • Siliconizing agent (e.g., Sigmacote, Sigma)
  • Glassware to be siliconized
  • Fume hood
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Figures

  •   FigureFigure a0.1U.1 Flowchart for choosing reactor type. The type of cell (suspension or anchorage‐dependent) and the location of the product (intracellular or secreted) will determine what types of reactors can be used and the modes in which they will operate.
  •   FigureFigure a0.1U.2 Modes of operation. The basic principles for each mode of operation are schematically shown.
  •   FigureFigure a0.1U.3 Cell separation devices. Schematic representations of four types of cell separation devices are shown.
  •   FigureFigure a0.1U.4 A schematic image of a spinner flask.
  •   FigureFigure a0.1U.5 A schematic image of a stirred tank reactor.
  •   FigureFigure a0.1U.6 A schematic image of a roller bottle.
  •   FigureFigure a0.1U.7 A schematic image of a stacked plate system.
  •   FigureFigure a0.1U.8 A schematic image of a packed bed bioreactor.
  •   FigureFigure a0.1U.9 A schematic image of microcarriers with cells attached to the surface.
  •   FigureFigure a0.1U.10 A schematic image of a fluidized bed bioreactor.
  •   FigureFigure a0.1U.11 A schematic image of a hollow fiber bioreactor.
  •   FigureFigure a0.1U.12 A schematic image of a wave bioreactor.

Videos

Literature Cited

   Chuppa, S., Tsai, Y.S., Yoon, S., Shackleford, S., Rozales, C., Bhat, R., Tsay, G., Matanguihan, C., Konstantinov, K., and Naveh, D. 1997. Fermenter temperature as a pool for control of high‐density perfusion cultures of mammalian cells. Biotechnol. Bioeng. 55:328‐338.
   Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. 1977. Characterization of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36:59‐74.
   Hayflick, L. and Moorhead, P.S. 1961. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25:585‐621.
   Jarvis, D.L. 2003. Developing baculovirus‐insect cell expression systems for humanized recombinant glycoprotein production. Virology 310:1‐7.
   Kaufman, J.B., Wang, G., Zhang, W., Valle, M.A., and Shiloach, J. 2000. Continuous production and recovery of recombinant Ca++ binding receptor from HEK 293 cells using perfusion through packed bed bioreactor. Cytotechnology 33:3‐11.
   Klima, G., Muller, D., Kreismayr, G., Lhota, G., Assadian, A.M., Schmatz, C., Wiederkum, S., Bluml, G., Coblhoff‐Dier, O., and Katinger, H. 1996. Process development for the large scale production of clinical grade human monoclonal antibody using a cytopilot fluidized bed bioreactor. In Animal Cell Technology: From Vaccines to Genetic Medicine (M.J.T. Carrondo, B. Griffiths, and J.L.P. Moreira, eds.) pp. 447‐450. Kluwer Academic Publishers, Dordrecht, The Netherlands.
   Kline, F., Ricketts, R.T., Jones, W.I., DeArmon, I.A., Temple, M.J., Soon, K.C., and Bridgen, P.J. 1979. Large‐scale production and concentration of human lymphoid interferon. Antimicrob. Agents Chemother. 15:420‐427.
   Lieber, M., Mazzetta, J., Nelson‐Rees, W., Kaplan, M., and Todaro, G. 1975. Establishment of continuous tumor‐cell line (panc‐1) from a human carcinoma of the exocrine pancreas. Int. J. Cancer 15:741‐747.
   Mowat, G.N. and Chapman, W.G. 1962. Growth of foot and mouth disease virus in a fibroblastic cell line derived from hamster kidney. Nature 194:253‐255.
   Puck, T.T., Marcus, P.I., and Cieciura, S.J. 1956. Clonal growth of mammalian cells in vitro: Growth characteristics of colonies from single HeLa cells with and without “feeder” layer. J. Exp. Med. 103:273‐284.
   Puck, T.T., Cieciura, S.J., and Robinson, A. 1958. Genetics of somatic mammalian cells III. Long‐term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 108945‐956.
   Schneider, I. 1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27:353‐365.
   Ubertini, B., Nardelli, L., Dal Prato, A., Panina, G., and Santero, G. 1963. Large scale cultivation of foot and mouth disease virus on calf kidney monolayers in rolling bottles. Sonderdruck Zentralblatt Veteeinarmedizin Reihe B Band 10 Heft 2:93‐101.
   Vaughn, J.L., Goodwin, R.H., Tompkins, G.J., and McCawley, P. 1977. The establishment of two cell lines from the insect Spondoptera frugiperda (Lepidoptera; Noctunidae). In Vitro 13:213‐217.
   Wickham, T.J. and Nemerow, G.R. 1993. Optimization of growth methods and recombinant protein production in BTI‐Tn‐5B1‐4 insect cells using the baculovirus expression system. Biotechnol. Prog. 9:25‐30.
   Yasumura, Y. and Kawakita, Y. 1962. American Public Health Association. Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health Association, Washington DC.
Key References
   Chu, L. and Robinson, D.K. 2001. Industrial choices for protein production by large‐scale cell culture. Curr. Opin. Biotechnol. 12:180‐187.
  Provides a good overview of commercial production processes and the products being produced.
   Freshney, R.I. 2000. Culture of Animal Cells: A Manual of Basic Technique, 4th ed. Wiley‐Liss, New York.
  This is a good general book on the culture of animal cells including a chapter on large‐scale culture.
   Lubiniecki, A.S. 1990. Large‐scale Mammalian Cell Culture Technology, Bioprocess Technology v. 10. Marcel Dekker, New York.
  Some perspectives on the elements of large‐scale cell culture, from recombinant technologies to manufacturing facilities.
Internet Resources
   http://www.bd.com/lifesciences/
  Suppliers of tissue culture flasks. Nunc is also the supplier for the Cell Factory. BD and Corning also supply roller bottles and spinner flasks. Corning is the supplier of CellCube and CellSTACK, two stack plate systems.
   http://www.corning.com/lifesciences/us‐canada/en/
  A supplier of the roller apparatus and spinner flasks.
   http://www.nuncbrand.com/
  New Brunswick Scientific is a supplier of the packed bed bioreactor.
   http://www.bellcoglass.com/
  Amersham Biosciences, Nunc, Percell Biolytica, and SoloHill Engineering are all suppliers of microcarriers.
   http://www.nbcs.com/
  Mettler‐Toledo, Broadley James, and Hamilton are suppliers of DO and pH probes.
   http://www.amershambiosciences.com
  B. Braun Biotech International, New Brunswick Scientific, and Applikon Biotechnology are suppliers of stirred tank reactors.
   http://www.nuncbrand.com/
  Amersham Biosciences and Vogelbusch are suppliers of fluidized bed bioreactors.
   http://www.percell.se/
  FiberCell Systems and Biovest International are suppliers of the hollow fiber bioreactor.
   http://www.sigmaaldrich.com/
  Wave Biotech is the supplier of the Wave bioreactor.
   http://www.solohill.com/
  Cambrex, Hyclone, Invitrogen Life Technologies, Irvine Scientific, and JRH Biosciences are suppliers of serum‐free media.
   http://www.mt.com
  The cell separation device, ATF System, is available from Refine Technology.
   http://www.broadleyjames.com
  The hollow fiber system is available from Amersham Biosciences.
   http://www.hamiltoncompany.com
  The spin filter system is available from B.Braun Biotech International.
   http://www.bbraunbiotech.com/
  The acoustic system is available from Applikon Biotechnology.
   http://www.nbsc.com/
  Bellco Glass Inc. and Corning are suppliers of transfer bottles. Caps must ordered separately with at least two openings for metal tubing to which silicone tubing for venting and liquid transfer can be attached.
   http://www.applikon.com
  Cole Parmer is a supplier of tubing and sterile connectors.
   http://www.amershambiosciences.com
   http://www.vogelbusch.at
   http://www.fibercellsystems.com/
   http://www.biovest.com/
   http://www.wavebiotech.com/
   http://www.cambrex.com/
   http://www.hyclone.com/
   http://www.invitrogen.com/
   http://www.irvinesci.com/
   http://www.jrhbiosciences.com/
   http://www.refinetech.com/
   http://www.amershambiosciences.com
   http://www.bbraunbiotech.com/
   http://www.applikon.com
   http://www.bellcoglass.com
   http://www.corning.com/lifesciences/us‐canada/en/
   http://www.coleparmer.com
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