Enhanced retroviral transduction of 293 cells cultured on liquid-liquid interfaces

Abstract

In clinical research, retrovirus‐mediated gene therapy is one of the most commonly used methods to deliver and express the gene of interest due its ability to allow for stable gene integration into the chromosomes of target cells. To elevate the efficiency of viral transduction, several restrictions, such as low virus–cell encounters and the necessity for cell division, must be improved. In this study, we focused on the possibility of accelerating cell division and the ensuing increment of viral transduction on flexible substrata. Perfluorocarbon FC‐40 was harnessed to form a liquid–liquid interface with culture medium. Enhanced green fluorescence protein (EGFP) was employed as the marker gene to quickly illustrate the percentage of viral infection. The results indicate that the gene transfer efficiency to 293 cells cultured on protein‐precoated liquid–liquid interfaces was higher than in cells cultured on rigid polystyrene surfaces. This increased transduction rate on the liquid–liquid interface is consistent with the acceleration of division of 293 cells on a flexible interface, which exhibited less adhesiveness. The effect of cell–cell contact inhibition on the rate of gene transduction is also addressed in this study. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 331–338, 2001

Engineering considerations on the use of liquid/liquid two-phase systems as a cell culture platform

“…Flat surface no effect of perfluorocarbon on cells growth/adhesion, cells harvested without trypsin PFC/DMEM 12 human embryonic kidney cells 1 -PFC 1 -medium Flat surface cells attach and spread on coated FC-40 interface, gene transfer efficiency on coated interfaces higher than on polystyrene PFD/RPMI-1640 medium 13 dissipation rate) on drop size distribution and mean drop size was investigated at room temperature both during breakage (a step increase of the impeller speed from 530 rpm to 590 rpm and then to 650 rpm for D-PBS; from 450 rpm to 500 rpm and to 560 rpm for DMEM and from 530 rpm, to 590 rpm and then to 650 rpm for distilled water); and during coalescence (a step reduction of the impeller speed using the same speeds but in the reversed order). In all experiments, the flow was turbulent with Re number between: 19000 and 23100 in DMEM, 24700 and 29500 in D-PBS and in 23600 and 29000 in water.…”

Combined Multimodal Optical Imaging and Targeted Gene Silencing Using Stimuli-Transforming Nanotheragnostics

“…eGFP-expressing NIH 3T3 cells were obtained by retroviral transduction and subsequent G418 selection 30. To evaluate RNA interference, the eGFP-expressing NIH 3T3 cells were seeded in a 24-well plate at a density of 1.6 × 10 4 cells/well.…”

Evaluation of Retroviral Production Systems Using Quantitative Analysis

“…Retroviral Production. The procedure for producing VSV‐G MoMLVs from 293GPG/EGFP producer cells was reported previously (26). After switching from the tetracycline‐containing medium to the tetracycline‐free medium at 80% cell confluence, retrovirus‐containing supernatants (3 mL in a 6‐well plate) were harvested at different periods of time.…”