Contract Cell Culture

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Contract Cell Culture BSR offers contract cell culture for protein expression and purification in insect or mammalian cells. Insect cells (Sf9, Sf21) or High Five cells are cultured in stirred bioreactors with 1 to 36L.Mammalian cell culture and monoclonal antibody scale-up employ stirred tank and membrane-based systems.

 

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Insect Cell Culture:

Insect CultureThe baculovirus expression system (BVES) is an efficient method for expressing proteins in insect cell culture. Baculovirus is in the family Baculoviridae, a diverse group of large dsDNA viruses that infect insects, arachnids and crustaceans. They are extremely species-specific and neither infect neither vertebrate life forms nor propagates in mammalian cells in culture.

The most common Baculovirus used in laboratory protein expression, and in CBRL's contract insect cell culture, has been the Autographa californica nuclear polyhedrosis virus (AcNPV). This is in a sub-group of the Baculoviridae (Nuclear polyhedrosis virus) that has large double stranded DNA, up to 200 kb, packaged in rod-shaped nucleocapsids. The virus is shed from infected cells by budding of the cell membrane during early stages of infection (10 hr to 3 days).

In latter stages of infection (3 to 6 days), viruses are encased in large protein crystals, called Occlusion bodies, composed predominately of the Polyhedrin protein. Occlusion bodies are released from the nuclease when the infected cells lyse. AcNPV infects insects in two sub-families of Lipidotera (moths). Several tissue culture cell lines have been derived from susceptible hosts. The most commonly used laboratory lines for AcNPV propagation are Sf9 and Sf21, both were derived at the USDA Insect Pathology Laboratory from a cell line originating from pupal ovarian tissue of the fall army worm, Spodoptera frugiperda. Sf21 cells have greater range in size and have a doubling time of 24 hr compared to 48-72 hr for Sf9 cells.

A third cell line, High Five™ was developed from ovarian cells of the cabbage looper, Trichoplusia ni by Boyce Thompson Institute for Plant Research, Ithaca, New York, and is covered by U.S. patent no. 5,300,435. High Five cells do not propagate AcNPV well, but can increase the level of secreted protein, 5-10 fold, when compared with Sf9 or Sf21. CBRL uses a three cell lines in its contract insect cell culture services. AcNPV is the most thoroughly studied Baculovirus, its DNA has been fully sequenced and numerous transfer vectors and modified AcNPV DNAs are available, each with special advantages for selection or transfection efficiencies.

Advantages of the Baculovirus Expression Vector system and contract insect cell culture: q Recombinant Proteins are expressed in high yield, often up-to 30% of the total insect protein. q Large proteins can be expressed, no known maximum size q Multiple genes can be simultaneously expressed in the same cell q Signal peptides are cleaved q Intron junctions are properly spliced q Proteins are usually soluble q Expressed proteins are properly folded and biologically active q Post-translation modifications are effective, including Glycosylation, Phosphorylation and Acylation. q Safe, non-infective to humans or mammalian cell lines

  • Baculovirus Expression Protocols (Methods in Molecular Biology, Vol 39) by Christopher D. Richardson (Editor); Hardcover — 450 pages Spiral edition (March 1998) Humana Pr; ISBN: 0896032728
  • Baculovirus Expression Vectors : A Laboratory Manual by David R. O'Reilly, Lois Miller, Verne A. Luckow; Paperback Spiral edition (June 1994) Oxford Univ Press; ISBN: 0195091310
  • The Baculovirus Expression System : A Laboratory Guide by Linda A. King, R.D. Possee; Hardcover (May 1992) Chapman & Hall; ISBN: 0412371502
  • Pharmingen, BD Biosciences/ Pharmingen and Invitrogen, have extensive web-based and printed Manuals and technical articles.

Mammalian cell culture:

Mammalian cell culture BSR cell culture services include primary cultures of human or animal origin as well as established cell lines. Cells are cultured for production of natural or recombinant proteins or for use in bioassays such as cell migrations, proliferation, or cytokine response. BSR specializes in immunomodulation and wound healing bioassays.

 

Monoclonal Antibody Scale-up:

Monoclonal Antibody Scale-up Monoclonal Antibody Scale-up

 

BSR offers cost–effective Monoclonal Antibody production from mg to gram quantities using in membrane Bioreactors.

Since 1997 there has been an orchestrated effort, by NIH and Animal Care and Use Committees, to replace Mouse Ascites methods in vivo Monoclonal Antibody production with in vitro technologies. “There is evidence that the mouse Ascites method of monoclonal antibody production causes discomfort, distress, or pain. Practical in vitro methods exist which can replace the Ascites method in many experimental applications without compromising the aims of the study.” Office for Protection from Research Risks (Department of Public Health Services) Report, November, 1997, Gary B. Ellis, Ph.D. and Nelson L. Garnett, D.V.M.

Costs vary depending on expression and growth characteristics of your hybridoma and any special media requirements.

Please use our information request form for a quote on your project.

Protein Purification:

Protein Purification BSR provides protein purification services that are available through our insect cell recombinant protein service, monoclonal antibody production service, or as a stand alone service for plasma proteins or proteins from your feed stock.

BSR can also lend its expertise to the development and transfer of a protein purification protocol to your production team.