Evaluation of Retroviral Production Systems Using Quantitative Analysis

Abstract

Because of the low titer of retroviral supernatant, it is necessary to develop and optimize large-scale retroviral production systems. To quantitatively determine the effect of a given operating condition (e.g., temperature and serum content) on producer cells' retrovirus-producing capacity, a mathematical model was used to analyze the static retroviral production system described by three processes: viral diffusion, decay, and generation. The analytical solutions of the defined model equations were fitted with experimental data to determine the specific retroviral production rate constant, which represents the competence of a retroviral production system. Two different retroviral production systems, inducible production of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped retrovirus from 293GPG/EGFP cells and constant production of ecotropic retrovirus from GP+E86/LNCX cells, were employed to demonstrate the feasibility of the engineering analysis. Our results indicated that the time-variant specific retroviral production rate of 293GPG/EGFP cells reached its maximum value of 5.7 x 10(-)(3) CFU/cm(2).h.cell, and the constant specific retroviral production rate of GP+E86/LNCX cells was 1.49 x 10(-)(2) CFU/cm(2).h.cell for the whole period of production. Furthermore, the effects of serum concentration and temperature on the ecotropic retroviral production system were examined separately. Our results suggest that producing ecotropic retroviruses with 10% fetal bovine serum at 37 degrees C is the optimal operating conditions for the long-term production system used here.

Retroviral Transduction of Adherent Cells in Resonant Acoustic Fields

“…The increase of medium temperature will also give rise to thermal streaming that could disturb cell band formation and diminish retrovirus-cell encounters. Moreover, the infectivity of retrovirus will decrease because the half-life of retrovirus decreases along with the increment of culture medium temperature (24). Therefore, precise temperature stabilization is required for an efficient long-term operation.…”

Functional analysis of gammaretroviral vector transduction by quantitative PCR

“…Indirect methods have been developed to estimate the number of target cells successfully transduced by the retrovirus based on properties (reporter or selection markers) acquired after transduction 26–28. Direct quantitative methods, such as measurement of physical particles (electron microscopy) or indispensable components present in the supernatant of all retroviral particles (reverse transcriptase or genomic RNA), detect higher levels of vector particles in retroviral supernatants than those predicted by indirect methods (reporter protein expression or selection markers) 12–14, 29, 30, since direct methods cannot distinguish infectious from noninfectious viral particles 14, 15. In contrast, indirect methods could underestimate true titres because of a possible reduced capacity to detect all transduced cells 14, 30.…”