Gradients Centrifugation

Recombinant adeno-associated virus (rAAV) is the most promising candidate for gene therapy due to its non-integrating nature and lack of pathogenicity in animal models. The production and purification of high titer rAAV vectors for experimental and therapeutic gene transfer are very important. Creative Biolabs has developed advanced purification methods, such as gradient centrifugation, to obtain high purity AAVs for better applications.

Background of AAV Vector Purification

In the past few decades, hundreds of different AAV serotypes have been isolated, which can be used for different cell groups, making the virus widely applicable as a gene therapy vector. In addition, rAAV can stably infect resting and replicating cells, stimulate the smallest immune response, and further expand its potential as a vector. Meanwhile, rAAV has many laboratory tools, for instance, it can be used to carry reporter genes, therapeutic genes or CRISPR (clustered regularly interspaced short palindromic repeat) associated protein 9 (CAS9) endonucleases for gene editing in animal models. Because of these numerous applications, it is becoming more and more important to obtain high titer rAAV vectors for experimental and therapeutic gene transfer.

Service

The purity and titer of the AAV vectors are important for achieving the stable and effective introduction of genes into individual animals and cultured cells. Creative Biolabs provides high-quality AAV purification services, making AAV an ideal raw material for in vivo experiments or for in vitro cell culture experiments. Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent AAV purification established to date.

CsCl Gradient Sedimentation

CsCl is widely used as a medium for isolating viral vectors by gradient centrifugation. This is called isomeric gradient ultra-centrifugation, and it is used to isolate and purify viral vectors based on their isodensity points (i.e., buoyancy density). Density gradients can be established by rotating viral preparations in solutions containing various CsCl concentrations in an ultra-centrifuge with high G-force, allowing the separation of empty, partially packaged and fully packaged viral carrier particles from cell fragments, proteins and nucleic acids.

Figure 1. The workflow for the production of AAV viruses.

Iodixanol Gradient Centrifugation

In medicine, iodixanol can be used as an equimolar contrast agent for intravenous injection. In research, iodixanol is also used as a picomolar density gradient medium for viral purification and isolation of cells, organelles, lipoproteins and macromolecules. Gradient purification of AAV with iodixanol was first proposed by researchers in 1999, and is still one of the most commonly used purification methods. Unlike other density gradient media, iodixanol is inert and non-toxic to mammalian cells when used in viral vector purification. Therefore, it is not necessary to remove iodixanol before using the purified virus.

Figure 2. The schema of AAV particles derived from a producer cell.

Features of Our Services

• Purification method is more economical, and has nothing to do with serotype, which minimizes the difference;
• Highest quality standards and upscaling options to fit your experimental needs;
• Each step of purification will communicate with our customers to ensure that the virus particles produced are fully suitable for the final application.

We're happy to answer any questions you have, or provide you with more info on the elements of Creative Biolabs. Just feel free to contact us or send us a short note.