Lentiviruses are a type of retrovirus used in scientific research and gene therapy. These modified viruses deliver new genetic material into cells, useful for studying gene function, developing disease models, and exploring potential treatments. Lentiviruses can integrate their genetic payload directly into the host cell’s DNA, allowing long-term gene expression in both dividing and non-dividing cells.
Why Lentiviruses Need Concentration
Lentiviruses are initially produced in dilute form. Many experimental and therapeutic applications require higher concentrations. In gene therapy, a high dose delivers therapeutic genes to target cells. For in vivo studies, limited injection volumes necessitate a potent, compact viral stock.
In laboratory settings, efficient genetic modification (in vitro transduction) relies on a high multiplicity of infection (MOI), meaning many viral particles per cell. A concentrated lentivirus stock allows researchers to achieve the desired MOI without adding excessive liquid to cell cultures. Without concentration, the volume of dilute virus would be impractical for most uses.
Common Methods for Concentration
Several methods concentrate lentiviruses.
Ultracentrifugation
Ultracentrifugation separates viral particles by density and size. It involves spinning the supernatant at high speeds (e.g., 100,000 x g for two hours at 4°C), often through a sucrose cushion, to pellet viral particles. It offers high purity but requires specialized equipment and can be time-consuming. However, high shearing forces can damage the viral envelope, reducing infectivity.
PEG Precipitation
PEG precipitation aggregates viral particles, causing them to precipitate. PEG solutions (often with NaCl) are added to filtered supernatant and incubated (e.g., overnight at 4°C) before lower-speed centrifugation to collect the pellet. This method is simpler and more accessible, as it does not require specialized ultracentrifugation equipment. While effective for concentrating virus (up to 100-fold with up to 90% recovery), it may result in lower purity than ultracentrifugation due to co-precipitation of cellular debris and proteins.
Ultrafiltration (Tangential Flow Filtration – TFF)
Ultrafiltration, particularly Tangential Flow Filtration (TFF), gently concentrates lentiviruses by removing water and smaller molecules through a semi-permeable membrane. In TFF, the supernatant flows tangentially across the membrane, preventing particle buildup that can clog the filter. This continuous flow allows processing large volumes and can achieve high concentration factors (e.g., over 2000-fold) with high recovery (often above 90%). TFF systems can also incorporate diafiltration to exchange buffers and remove impurities, making it scalable for large-scale and clinical applications. While generally gentle, some viral particle loss can occur, and process time may increase if membrane pore blockage occurs.
Verifying Lentivirus Concentration
Accurately determining lentivirus concentration (titration) is necessary for reliable experimental results and safe therapeutic applications. Titration ensures a consistent amount of viral particles is used, important for predictable gene delivery and comparing results. There are two main categories of titration methods: physical and functional.
Physical Titration
Physical titration quantifies total viral components, irrespective of infectivity. Quantitative PCR (qPCR) measures viral genome copies. The p24 ELISA detects the p24 capsid protein, a lentivirus structural component. A challenge with p24 ELISA is that it measures all p24 protein, including from non-functional or incomplete particles, potentially overestimating infectious titer.
Functional Titration
Functional titration assesses infectious viral particles by measuring their ability to transduce target cells and express the delivered gene. This is often achieved by incorporating a reporter gene (e.g., GFP) into the vector. After transduction, reporter-expressing cells can be counted using flow cytometry (FACS) or by observing colony formation. While functional titers provide a more accurate measure of a vector’s biological activity, they can underestimate the true viral titer because assays may not account for cells infected by multiple particles. Both physical and functional titers provide valuable, complementary information. Functional titers are generally considered more indicative of a vector’s effectiveness.
Storing and Handling Concentrated Lentiviruses
Proper storage and handling of concentrated lentiviral stocks is necessary to maintain stability and infectivity. Lentiviruses are delicate and susceptible to degradation, so immediate storage after concentration is recommended. For long-term preservation, concentrated lentiviruses should be stored at ultralow temperatures (e.g., -80°C), remaining stable for at least one year.
To avoid significant reduction in viral titer, aliquot the concentrated lentivirus into smaller, working volumes before initial freezing. Repeated freeze-thaw cycles can cause a substantial decrease in viral infectivity, with reports indicating a 10-fold drop in titer per cycle. When thawing, place vials on ice for gradual temperature change and keep them on ice during experiments to preserve viral integrity. General precautions, such as avoiding vortexing and other harsh manipulations, also contribute to maintaining stability.