Ching-An Peng

Determination of Infectious Retrovirus Concentration from Colony-Forming Assay with Quantitative Analysis

ABSTRACT The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 ± 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.

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

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.

Retroviral Transduction of Adherent Cells in Resonant Acoustic Fields

Ultrasound‐induced cavitation has been extensively used to enhance the efficiency of nonviral‐based gene delivery. Although such unique mechanical force could possibly augment the efficacy of retrovirus‐mediated gene transfer, we harnessed an alternative approach, a resonant acoustic field, to facilitate the retroviral transduction rate. NIH 3T3 fibroblast cells suspended in a culture well and mixed with ecotropic retroviruses were co‐treated with megahertz resonant acoustic fields (RAF). Suspended NIH 3T3 cells under RAF treatment agglomerated at acoustic nodal planes by primary radiation force within a short exposure time. These first arrived and agglomerated cells formed bands as nucleating sites for nanometer‐sized ecotropic retroviruses circulated between nodal planes to attach on and thereby increased cell‐virus encounters. According to the neomycin‐resistant colony assay, 2‐fold increment of retroviral transduction rate was obtained by exposing cells and retroviruses in the RAF for 6 min in the presence of 8 μg/mL Polybrene.

Impact of Cell Growth Morphology on Retroviral Transduction: Effect of Contact Inhibition

Retrovirus‐mediated gene transfer is one of the most commonly used methods to deliver, integrate, and express the gene of interest because the retrovirus can insert the desired gene into the chromosome of the target cells with high stability. However, to deliver the gene successfully, the retrovirus requires active division to integrate reversely transcribed DNA into the chromosome of target cells. In this study, we focused on the effect of cell‐cell contact inhibition on the efficiency of retroviral transduction with two anchorage‐dependent cell lines: NIH 3T3 and 293 cells. These two cell lines have very different cell morphologies and growth patterns on surfaces. Human embryonic kidney epithelial 293 cells tend to stick together after dividing, while NIH 3T3 cells migrate to occupy available surface and spread. Experimental data indicate that the abatement of the transduction rate of 293 cells was initiated in the early stage of the culture, whereas effect of contact inhibition of NIH 3T3 cells on the transduction rate became dominating at the end of the culture period. Experimental results were also quantitatively illustrated by plotting normalized multiplicity of infection (MOI) versus normalized cell density. According to the outcomes, cell inoculation density plays an important role in optimizing the retroviral transduction rate. The optimal time of retroviral transduction should be confined to the accelerating growth phase for 293 cells and at the exponential growth phase for NIH 3T3 cells. The implication drawn from this study is that contact inhibition effect on retroviral transduction should be taken into account for large‐scale gene transfer systems such as the microcarrier bioreactor.

Engineering analysis of ex vivo retroviral transduction system.

AbstractThe overall dynamics of the retrovirus-cell encounter under a static retroviral transduction system can be described in terms of the process of uptake (adsorption/internalization), decay, and diffusion. In this study, a mathematical model illustrating these processes was derived assuming a semi-infinite domain and solved analytically using the Laplace transform. The closed-form solutions for retroviral concentrations and time course profile of transduced cell colonies are presented to clarify the contributions of the processes involved in the retroviral transduction system. To manifest the usefulness of the closed-form solutions, the neomycin-resistant gene encoding retroviruses produced by two different packaging cells (human 293 cells and murine GP+E86/LNCX cells) were employed to transduce NIH 3T3 cells, which formed neomycin-resistant colonies after G418 selection. The experimental results were curve fitted with the model-derived analytical solutions to quantitatively determine transduction rate constant k and initial concentration of infectious retrovirus C0. Our study showed that the vesicular stomatitis virus G protein pseudotyped retrovirus produced from 293 packaging cells exhibited much higher transduction rate (k=0.0480cm/h} than the ecotropic retrovirus (k=0.0102 cm/h) produced from GP+E86/LNCX cells. The fitted values of C0 are approximately two orders of magnitude higher than the experimentally estimated titers for both retroviruses.

Differential interaction of retroviral vector with target cell: quantitative effect of cellular receptor, soluble proteoglycan, and cell type on gene delivery efficiency.

Retroviral vectors are powerful tools for gene therapy and stem cell engineering. To improve efficiency of retroviral gene delivery, quantitative understanding of interactions of a retroviral vector and a cell is crucial. Effects of nonspecific adsorption of retrovirus on a cell via proteoglycans and receptor-mediated binding of retrovirus to a cell on overall transduction efficiency were quantified by combining a mathematical model and experimental data. Results represented by transduction rate constant, a lumped parameter of overall transduction efficiency, delineated that chondroitin sulfate C (CSC) plays dual roles as either enhancer or inhibitor of retroviral transduction, depending on its concentrations in the retroviral supernatant. At the concentration of 20 microg/mL, CSC enhanced the transduction efficiency up to threefold but inhibited more than sevenfold at the concentration of 100 microg/mL. Transduction rate constants for amphotropic retroviral infection of NIH 3T3 cells under phosphate-depleted culture condition showed a proportional relationship between cellular receptor density on a cell and transduction efficiency. It was finally shown that amphotropic retrovirus transduced human fibroblast HT1080 cells more efficiently than NIH 3T3 cells. On the contrary, the transduction efficiency of NIH 3T3 cells by vesicular stomatitis virus G protein pseudotyped retroviruses was eightfold higher than that of HT1080 cells. This study implies usefulness of using quantitative analysis of retroviral transduction in understanding and optimizing retroviral gene delivery systems for therapeutic approaches to tissue engineering.