A comparison of monocyte-derived macrophages with and without prior CD14+ magnetic sorting

For in vitro studies involving macrophages, an efficient protocol for isolation and cell culture of monocyte-derived macrophages (MDM) is essential. However, it remains unclear how far isolation steps improve purity or provoke cell changes. Here we compare the characteristics and functionality of macrophages isolated from leukocyte reduction (LRS) chambers by either CD14+ magnetic sorting (sMac) or by seeding PBMC with separation by plastic adhesion and subsequent washing steps (uMac) to evaluate the necessity of magnetic sorting.

PBMC were isolated from LRS chambers using density gradient centrifugation. sMac were enriched from PBMC by magnetic separation removing lymphocytes, uMac were seeded and separated from lymphocytes via plastic adhesion and washing steps. Both groups were plated under matched conditions to allow monocyte-to-macrophage differentiation after adhesion. Morphology, purity, cell viability, surface marker expression, and cytokine production upon stimulation were assessed using flow cytometry, qPCR, brightfield and confocal microscopy.

MDM were successfully cultured from both uMac and sMac in over 75%. Cell viability was similar between uMac (54%) and sMac (48%). CD14 expression was significantly higher in sMac (89.48%) compared to uMac (65.91%), the mean fluorescence intensity of CD14 did not differ significantly. No significant differences were found in CD80 and CD163 expression, nor in the production of pro-inflammatory cytokines upon stimulation.

LRS chambers serve as a valid source for culturing of MDM with high reliability and yield. Since both methods produced macrophages of comparable phenotype and function, our findings indicate that magnetic CD14+ sorting is not mandatory for most in vitro applications.