Culture surface influence on T-cell phenotype and function.

When dealing with T lymphocyte culture there is currently very less information available about the interaction between T-cells and the culture system. In this study we look at the influence of the culture chamber on T-cell proliferation in two main aspects of the culture system, namely: culture chamber material and geometry. The study was carried out using unique polymeric closed cell culture inserts, which were processed via injection moulding from polystyrene (PS), polycarbonate (PC), polyetherurethane (PEU), polystyrene-co-acrylonitrile (PSAN) and polyetherimide (PEI). Furthermore culture chamber geometry was studied using commercially available 24, 12 and 6-well plates prepared from tissue culture plastic (TCP). For T lymphocyte stimulation two methods were used involving either EBV peptide pools or MACS iBead particles depending on the experiment performed. Culture was done with 1645 RPMI medium supplemented with foetal calf serum, penicillin, streptomycin and rhIL-2. We found four materials out of five we tested (PS, PC, PSAN and PEI) exhibited similar fold expansions with minimal influence on proportions of CD4 and CD8, while PEU had a negative influence on T cell growth along with adversely affected CD4/CD8 proportions. Changes in the geometry of TCP had no effect on T cell growth or maturation rather the size of geometry seems to have more influence on proliferation. T-cells appear to prefer smaller geometries during initial stages of culture while towards the end of the culture size becomes less significant to cell proliferation. The parameters tested in this study have significant influences on T-cell growth and are necessary to consider when designing and constructing expansion systems for antigen specific T lymphocytes. This is important when culturing T-cells for immunotherapeutic applications where antigen specificity, T-cell maturation and function should remain unaffected during culture.

Good Manufacturing Practices (GMP) manufacturing of advanced therapy medicinal products: a novel tailored model for optimizing performance and estimating costs.

BACKGROUND AIMS: Advanced therapy medicinal products (ATMP) have gained considerable attention in academia due to their therapeutic potential. Good Manufacturing Practice (GMP) principles ensure the quality and sterility of manufacturing these products. We developed a model for estimating the manufacturing costs of cell therapy products and optimizing the performance of academic GMP-facilities. METHODS: The "Clean-Room Technology Assessment Technique" (CTAT) was tested prospectively in the GMP facility of BCRT, Berlin, Germany, then retrospectively in the GMP facility of the University of California-Davis, California, USA. CTAT is a two-level model: level one identifies operational (core) processes and measures their fixed costs; level two identifies production (supporting) processes and measures their variable costs. The model comprises several tools to measure and optimize performance of these processes. Manufacturing costs were itemized using adjusted micro-costing system. RESULTS: CTAT identified GMP activities with strong correlation to the manufacturing process of cell-based products. Building best practice standards allowed for performance improvement and elimination of human errors. The model also demonstrated the unidirectional dependencies that may exist among the core GMP activities. When compared to traditional business models, the CTAT assessment resulted in a more accurate allocation of annual expenses. The estimated expenses were used to set a fee structure for both GMP facilities. A mathematical equation was also developed to provide the final product cost. CONCLUSIONS: CTAT can be a useful tool in estimating accurate costs for the ATMPs manufactured in an optimized GMP process. These estimates are useful when analyzing the cost-effectiveness of these novel interventions.