Monocytes and macrophages are being the subject of many studies that position them as a regenerative therapeutic tool of great interest. Nowadays, current isolation methods are not designed for its use in clinical facilities near the patient. Manual methods (Ficoll, Percoll or magnetic beads) consist in opened procedures that require working on a GMP compliant facility and require including steps to remove media to avoid adverse/toxic effects. Automated closed devices (SEPAX from BIOSAFE or PRODIGY from Miltenyi Biotec) still require GMP certified facility and the cost is expensive.

Three different methods have been compared: negative PBMC isolation based on MACS (Magnetic activated cell sorter) columns (Miltenyi), an automated PBMC isolation method and Hydroxyethyl starch (HES), that has been used as a potential substitute of the erythrocyte initial removal. A flow cytometry using CD45 and CD14 for PBMCs and monocytes, respectively, has been performed to analyse the purity (percentage of target cells within the total cell population) and recovery (cell count after the protocol compared with the initial cell number) of the methods.

The results show that a mixed protocol with HES isolation followed by PBMC MACS columns could perform with similar purity as the whole Miltenyi protocol. Moreover, automated PBMC isolation experiments had not achieved the expected purity and reproducibility.

HES and centrifugation are not enough to isolate monocytes, needing the addition of other technologies. However, the study shows the technological feasibility of isolating PBMCs with a potential automatable closed system based on proven techniques.