Cryopreservation of human monocytes for pharmacopeial monocyte activation test.

EU Directive 2010/63/EU regarding the protection of experimental animals came into force in November 2010 with an obligation for EU member states to incorporate its requirements into their respective national legislations by 1st of January 2013. The directive stipulates the application of in vitro methods to replace animal experiments whenever such an in vitro method exits and is recognized by EU legislation. The monocyte activation test (MAT) for the detection and quantification of pyrogenic contamination in medicines is recognized by the European Directorate for the Quality of Medicines & Health Care (EDQM) and was published in the European Pharmacopeia (Pharm. Eur.) in April 2010. The methodology described here facilitates the use of the MAT by making monocytes available, in the form of cryopreserved human peripheral blood mononuclear cells (PBMCs). We have developed and qualified a procedure to prepare functional monocytes in the form of PBMCs from the leukocyte filters that are used for the separation of blood in blood donation centers. Once used, these filters are normally treated as biological waste. Here we describe the procedures that are critical for the successful cryopreservation of PBMCs, demonstrate protection of PBMC functionality using various ligands for the toll-like receptors (TLRs) that mediate pyrogenic responses, report validation of the methodology for linearity, precision and robustness and show examples of the practical application of cryopreserved in MATs with samples of drugs and vaccines. Another application of cryopreserved PBMCs, only mentioned here, is to serve as an alternative to freshly isolated PBMCs in tests for unwanted intrinsic pro-inflammatory activities of new biological therapeutics. Such tests use PBMCs or PBMCs over a layer of endothelial cells to detect (unwanted) cytokine release, PBMCs being more suited to this purpose than tests using whole blood.