Large‐Scale Cell Culture
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.
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
- Literature Cited
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
Figure a0.1U.2 Modes of operation. The basic principles for each mode of operation are schematically shown.
Figure a0.1U.3 Cell separation devices. Schematic representations of four types of cell separation devices are shown.
Figure a0.1U.4 A schematic image of a spinner flask.
Figure a0.1U.5 A schematic image of a stirred tank reactor.
Figure a0.1U.6 A schematic image of a roller bottle.
Figure a0.1U.7 A schematic image of a stacked plate system.
Figure a0.1U.8 A schematic image of a packed bed bioreactor.
Figure a0.1U.9 A schematic image of microcarriers with cells attached to the surface.
Figure a0.1U.10 A schematic image of a fluidized bed bioreactor.
Figure a0.1U.11 A schematic image of a hollow fiber bioreactor.
Figure a0.1U.12 A schematic image of a wave bioreactor.
|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.|
|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.|
|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.|
|A supplier of the roller apparatus and spinner flasks.|
|New Brunswick Scientific is a supplier of the packed bed bioreactor.|
|Amersham Biosciences, Nunc, Percell Biolytica, and SoloHill Engineering are all suppliers of microcarriers.|
|Mettler‐Toledo, Broadley James, and Hamilton are suppliers of DO and pH probes.|
|B. Braun Biotech International, New Brunswick Scientific, and Applikon Biotechnology are suppliers of stirred tank reactors.|
|Amersham Biosciences and Vogelbusch are suppliers of fluidized bed bioreactors.|
|FiberCell Systems and Biovest International are suppliers of the hollow fiber bioreactor.|
|Wave Biotech is the supplier of the Wave bioreactor.|
|Cambrex, Hyclone, Invitrogen Life Technologies, Irvine Scientific, and JRH Biosciences are suppliers of serum‐free media.|
|The cell separation device, ATF System, is available from Refine Technology.|
|The hollow fiber system is available from Amersham Biosciences.|
|The spin filter system is available from B.Braun Biotech International.|
|The acoustic system is available from Applikon Biotechnology.|
|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.|
|Cole Parmer is a supplier of tubing and sterile connectors.|