A microfluidic bioreactor for increased active retrovirus output.

Retroviruses are one of the most commonly used vectors in ongoing gene therapy clinical trials. To evaluate and advance virus production on the microscale platform, we have created a novel microfluidic bioreactor for continuous retrovirus production. We investigated the growth kinetics of a retroviral packaging cell line in microfluidic bioreactors for several compartment sizes, and packaging cells perfused in the microdevices showed similar growth kinetics to those cultured in conventional static conditions. To evaluate the efficiency of retrovirus production, virus titers from the microdevices were compared to those obtained from static tissue culture. When retrovirus production and collection were maintained at 37 degrees C, virus production levels were comparable for the microdevices and static tissue culture conditions. However, immediate cold storage downstream of the packaging cells in the microdevices resulted in 1.4- to 3.7-fold greater active virus production levels with the microdevices compared to the conventional static conditions over a 5 day period. Lastly, the use of microfluidics for virus production provides a continuous supply of virus supernatant for immediate infection of target cells or for preservation and storage. Such devices will be valuable for the optimization of production and evaluation of retroviruses and other viral vectors for gene therapy applications.

[1]  G. Whitesides.  The origins and the future of microfluidics , 2006, Nature.

[2]  W. Stemmer,et al.  Breeding of retroviruses by DNA shuffling for improved stability and processing yields , 2000, Nature Biotechnology.

[3]  M L Yarmush,et al.  Large‐Scale Processing of Recombinant Retroviruses for Gene Therapy , 1999, Biotechnology progress.

[4]  D. Beebe,et al.  Microenvironment design considerations for cellular scale studies. , 2004, Lab on a chip.

[5]  D. Beebe,et al.  Cell infection within a microfluidic device using virus gradients , 2004 .

[6]  Darwin R. Reyes,et al.  Micro total analysis systems. 2. Analytical standard operations and applications. , 2002, Analytical chemistry.

[7]  J. L. Le Doux,et al.  Proteoglycans secreted by packaging cell lines inhibit retrovirus infection , 1996, Journal of virology.

[8]  K. Jensen,et al.  Cells on chips , 2006, Nature.

[9]  A. Goldstein,et al.  Effect of adsorbed fibronectin concentration on cell adhesion and deformation under shear on hydrophobic surfaces. , 2002, Journal of biomedical materials research.

[10]  A. Mortellaro,et al.  Correction of ADA-SCID by Stem Cell Gene Therapy Combined with Nonmyeloablative Conditioning , 2002, Science.

[11]  G. Truskey,et al.  The effect of fluid shear stress upon cell adhesion to fibronectin-treated surfaces. , 1990, Journal of biomedical materials research.

[12]  Rashid Bashir,et al.  Real-time virus trapping and fluorescent imaging in microfluidic devices , 2004 .

[13]  George M Whitesides,et al.  Propagation of viruses on micropatterned host cells. , 2003, Biotechnology and bioengineering.

[14]  B. Palsson,et al.  Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5.5 to 7.5 hours , 1997, Journal of virology.

[15]  P. Cruz,et al.  Comparison of Different Bioreactor Systems for the Production of High Titer Retroviral Vectors , 2001, Biotechnology progress.

[16]  F. Deist,et al.  Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. , 2000, Science.

[17]  Mehmet Toner,et al.  Cell handling using microstructured membranes. , 2006, Lab on a chip.

[18]  K. Cornetta,et al.  Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. , 1990, The New England journal of medicine.

[19]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[20]  O. Merten.  State‐of‐the‐art of the production of retroviral vectors , 2004, The journal of gene medicine.

[21]  J. L. Le Doux,et al.  Kinetics of retrovirus production and decay. , 1999, Biotechnology and bioengineering.

[22]  S. Chang,et al.  Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer. , 2000, Molecular therapy : the journal of the American Society of Gene Therapy.

[23]  W F Anderson,et al.  Improved methods of retroviral vector transduction and production for gene therapy. , 1994, Human gene therapy.

[24]  Yang Du,et al.  Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1 , 2006, Nature Medicine.