von Briesen: HIV Specimen Cryorepository
The goal of this project was to establish a state-of-the-art specimen repository, the HIV Specimen Cryorepository (HSC), where samples and reagents relevant for HIV vaccine development are stored under controlled low-temperature conditions. The second goal was to provide high quality specimens, tools and procedures, having a direct benefit to the CAVD community. In this context, the production of HIV-1 envpseudoviruses and Infectious Molecular Clones has turned out to be of highest interest.
Within the project phase 1 (Aug 06 – Jul 09), the HIV Specimen Cryorepository (HSC) as one of the Central Services Facilities (CSF) for the CAVD had successfully developed these processes, the cryoequipment and the production of bioreagents. The second project phase (Aug 09 – Jul 11) had been used to employ, improve and distribute the new cryotechnology platform, to develop new cryomedia and to establish new co¬operations in the field of R&D as well as for technology transfer. In the third project phase (Aug 11 - Jul 13), the focus was on integrating the HSC services into the new VDC projects and to evaluate the newly developed cryotechnologies and cryoprocedures. In the current project phase (Aug 13 – Jul 16), the assignation of HSC has been focused on the generation of bioreagents for the CAVD, especially the production of HIV-1 Env pseudoviruses and Infectious Molecular Clones for the CA-VIMC (Duke University).
The HSC responds to the needs of the CAVD network of collaborators through the following tasks:
· Bioreagent Generation: Fostering the production of large-scale standardized HIV-1 Env-pseudovirus stocks and Infectious Molecular Clones of HIV-1 in cooperation with the Antibody Vaccine Immune Monitoring Consortium (CA-VIMC) through an automated certified system.
· Extension of bioreagent production portfolio (e.g.virus like particles (VLP))
· Development of protein-free and xeno-free cryomedia for the preservation of PBMCs.
· Evaluation of newly developed cryotechnologies and cryoprocedures.
In the initial phase of the project, different designs and prototypes of smart cryosubstrates were developed. An integrated memory chip can store all identifiers, additional sample description, legal constraints, and the complete parameter set for operating the laboratory workflow. Injection molding production started in the second year and the HSC cooperates with the company PermaCryo Technology (PCT) to produce the substrates industrially. Since 2009, theses cryosubstrates are commercially available.
Likewise, new cryoequipment has been developed in collaboration with the company ASKION. The first company-made prototypes of the hermetic cryo hood were delivered in December 2007. Since 2013, two fully automated cryo hood systems for freezing and withdrawal of samples are available from the companies Askion and Sysmex.
New Procedures and production of biomaterials
In 2010, the HSC developed two chemically fully defined, endotoxin-free and xeno- and protein free cryomedia. The HSC has proven that the PBMC recovery and viability using these new cryomedia is statistically equal or better than a cell cryopreservation with the FBS-based cryomedium (Schulz, J. et al. (2012) J. Immunol. Methods
382, 24-31). Likewise, the PBMC cryopreservation results not only in high cell recovery and viability values, but also in maintenance of T-cell functionality (ELISpot).This new medium has proved its suitability in the daily routine, especially for freezing cells that need to be cultured FBS free. Some other labs have established the medium in theirsample handling.
In order to evaluate the advantages of our newly developed cryotechnologies, we started a study of handling and storage procedures of PBMCs which typically occur in clinical studies compared to handling conditions without any temperature up- and downturn. Germann, A. et al. (Cryobiology (2013)67(2), 193-200) could show that the maintenance of cell viability, recovery and T-cell functionality is strongly dependent on maintaining the samples in storage conditions without temperature fluctuations. Repeated temperature shifts up to -60°C (i.e. still deeply cold) led to a decrease in all measured parameters.
Another focus of the HSC is the manual and automated production of bioreagents. Since July 2011, an automated system to prepare HIV pseudo-viruses is validated successfully (Schultz, A. et al. (2012) PLoS ONE 7(12), e51715) and the automated method is utilized in addition to the standardized manual procedure. The validation for the automated production of Infectious Molecular Clones (IMC) is ongoing. Up to September 2013, more than 100 different HIVpv and 45 IMCs amounting to 130,000 ml in total were produced manually or automated and distributed globally to the collaboration partners under validated conditions (according to Good Clinical Laboratory Practice).
The outstanding expertise in the field of biobanking/cryostorage is also stressed by the fact that the cryorepository has been selected by the German Government to host the human samples of the German Federal Environmental Specimen Bank since January 2012.