Isolation of monocytes from whole blood by density gradient centrifugation and countercurrent elutriation followed by cryopreservation:: six years' experience

Background: Monocyte purification by means of counter-current elutriation and subsequent cryopreservation for future use was initiated in 1986 and has been established as a routine since 1993. Aim. To sum up and evaluate our method for the isolation and preservation of monocytes. Materials and methods: Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor blood by density gradient centrifugation, and monocytes were isolated from the PBMC by counter-current elutriation centrifugation using the Beekman J-6M/E centrifuge. The monocytes were then cryopreserved at -135 degrees C and thawed when required for experimental use. Results: Results are given for the last 6 years, including 59 elutriations and the fractions containing monocytes. The mean purity of monocytes was 93% (range 64-98%); mean recovery was 51% (range 22-55%). Studies of CD14 expression and Annexin V indicate that there are no differences between elutriated fractions immediately upon purification or after freezing and thawing. The studies also indicate that interdonor variations are much larger than intradonor variations. Discussion: Although it differs from other reports in certain respects, our procedure has nevertheless produced results in line with other findings. After extensive testing and use in different contexts we feel confident that we have established a method for producing a large number of purified and well-preserved monocytes. Conclusion: The goal of being able to perform a large number of experiments with monocytes of high purity and good functionality has been reached.