Negative Magnetic Sorting Preserves the Functionality of Ex Vivo Cultivated Non-Adherent Human Monocytes

Abstract

Background: Monocyte-derived macrophages or dendritic cells are of increasing interest for cellular therapeutic products to treat inflammation-related diseases and cancer. However, the isolation method and the culture conditions applied influence the functionality of cells. For some approaches, the adhesion-induced differentiation into macrophages must be prevented to maintain functions attributed to circulating monocytes. The effects of the isolation method on the functionality of non-adherent peripheral monocytes have not yet been investigated. Methods: The present study examines the impact of the isolation method on cell viability, growth, metabolism, inflammation-induced cytokine response, migratory capacity, and adherence of non-adherent human peripheral monocytes. The monocytes were isolated by magnetic sorting using either positive or negative selection and cultured in cell-repellent plates. Results: The purity and yield of monocytes were higher after positive selection. However, the adherence and migratory capacity, cytokine response, and metabolic activity were decreased compared to negatively selected monocytes. The impaired functionality presented in combination with cell shrinking, thus, indicates the start of cell viability loss. Negatively selected non-adherent monocytes showed no impairment in functionality, and the viability remained high. In conclusion, this approach is better suited for conducting ex vivo modifications of monocytes prior to the intended experimental setup or therapeutic application.

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

“…Although an enrichment for CD14 + monocytes by magnetic/fluorescence-activated cell sorting (MACS) might be reasonable to further enhance osteoclast yields, a priori positive selection for CD14 + precursors might affect cell differentiation due to the induction of different cellular phenotypes and the presence of residual beads in the culture system [ 32 ]. Indeed, Hornschuh and colleagues reported a significant decrease in monocyte functionality, cytokine release and viability after MACS-based CD14 sorting compared to negatively selected cells [ 33 ]. However, another study observed that negatively selected monocytes might be contaminated with high numbers of platelets and skewed towards a M2 polarization [ 34 ].…”

In vitro Production of Dendritic Cells as Cancer Immunotherapy: Highlights on Sample Source, Culture Period, Differentiation and Maturation Cytokines

“…For maturation, CD34 + cells take 7-12 days to differentiate using granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, and stronger pro-inflammatory cytokines such as stem cell factor (SCF), FMS-like tyrosine kinase 3 (Flt3L), thrombopoietin (TPO), TNF-α, IL-3, IL-4, and IL-6. 10,13,17,18 Meanwhile, monocytes only take 5-6 days for maturation. [19][20][21][22][23] There are approximately 5.28% and 7.91% monocytes that can be isolated from adult PB and UCB, respectively.…”

“…However, the potency to attach, migrate, and releasing cytokines are lower than negatively selected monocytes. 17,28 This may be because of interaction between antibody and CD14 indicating "danger signal", causing early maturation. In addition, by flowing through the magnetic field, the monocytes can shrink and lead to cell death.…”

“…In addition, by flowing through the magnetic field, the monocytes can shrink and lead to cell death. 17,28 Due to early maturation and dead cells, the monocyte had a lower adherence which is associated with a reduced migratory and metabolic potency compared to negatively sorted monocytes. 17,28 In addition, the beads from positive sorting still persist in patient, may cause hypersensitivity reaction.…”

“…17,28 Due to early maturation and dead cells, the monocyte had a lower adherence which is associated with a reduced migratory and metabolic potency compared to negatively sorted monocytes. 17,28 In addition, the beads from positive sorting still persist in patient, may cause hypersensitivity reaction. Even though there is no proof if the beads have also been injected to patient, but the beads still present after 6 days culture in laboratory.…”