Rapid Isolation of Mononuclear Cells with High Yield from Minimal Blood Volumes: A Simplified and Robust Approach for Immunotherapeutic Applications

Abstract

Objective: Currently, mononuclear cell (MNC) isolation is performed using density gradient methods, which are laborious and time-consuming, requiring a minimum of 10 ml of buffy coat or blood. This method is inadequate for isolating MNCs from small volumes, such as 100 µl. Therefore, we aimed to develop a straightforward, rapid, and cost-effective method for isolating MNCs from minute volumes of blood and buffy coat. Material and Methods: We utilized the MNC isolator to isolate MNCs from microvolumes, such as 100 µl and 500 µl of blood and buffy coat, by simple mixing and centrifugation. The isolated cells were cultured, and ligand-specific magnetic beads were employed to isolate CD4 and CD45 specific cells from the cultured cells. Results: In 15 experiments, the MNC isolator successfully isolated MNCs from small volumes of blood and buffy coat. The cell cultures were established and maintained for up to 3 years without contamination, with the cells remaining healthy. Successful isolations of CD4 and CD45 cells using magnetic beads were achieved, and the cells were successfully frozen and thawed. Conclusion: In this study, we have introduced a simple, cost-effective, and robust method for isolating MNCs from small volumes of buffy coat and blood, suitable for immunological applications and magnetic bead cell isolation. This method is user-friendly and can be adopted in cell culture laboratories worldwide, potentially opening new avenues for the development of novel antibodies, isolation of various cell populations using magnetic beads, and obtaining a large number of T-cells for immunotherapy development, such as CAR T-cell therapies. Keywords: mononuclear cell isolation, buffy coat, blood, CD4 positive cells, magnetic beads