Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV

Bennett, Samuel; Breit, Samuel N.

doi:10.1002/jlb.56.3.236

Cited by 65 publications

(11 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition to lymphocytes, the MCs of the spleen samples also consist of up to approximately 20% monocytes. Because the monocytes could interfere with additional experiments because of phagocytosis, they were removed by adhering to the plastic walls of the cell culture plates, as previously described [ 47 ]. Splenic MCs were cryopreserved with custodiol/10% DMSO and carefully thawed for stimulation experiments.…”

Section: Methodsmentioning

confidence: 99%

Effect of HLA-G5 Immune Checkpoint Molecule on the Expression of ILT-2, CD27, and CD38 in Splenic B cells

Rohn¹,

Lang

Schramm

et al. 2022

Journal of Immunology Research

View full text Add to dashboard Cite

The human leukocyte antigen G (HLA-G) is an immune checkpoint molecule with a complex network of interactions with several inhibitory receptors. Although the effect of HLA-G on T cells and NK cells is well studied, the effect of HLA-G on B cells is still largely elusive. B cells are of particular interest in the context of the HLA-G-ILT-2 interaction because the ILT-2 receptor is constitutively expressed on most B cells, whereas it is only present on some subsets of T and NK cells. To characterize the effect of HLA-G5 molecules on B cells, we studied splenic B cells derived from cytomegalovirus (CMV) sero-positive donors after CMV stimulation with antigens in the presence and absence of soluble HLA-G5. In the presence of HLA-G5, increased expression of the ITIM-bearing Ig-like transcript (ILT-2) was observed on B cells, but its expression was not affected by stimulation with CMV antigens. Moreover, it became evident that HLA-G5 exposure resulted in a decreased expression of CD27 and CD38 and, accordingly, in lower proportions of CD19+CD27+CD38+ and higher proportions of CD19+CD27-CD38- B cells. Taken together, our in vitro findings demonstrate that soluble HLA-G5 suppresses markers of B cell activation, suggesting that HLA-G5 has an impact on splenic B cell differentiation and activation. Based on these results, further investigation regarding the role of HLA-G as a prognostic factor and a potential therapeutic agent with respect to B cell function appears reasonable.

show abstract

Section: Methodsmentioning

confidence: 99%

Effect of HLA-G5 Immune Checkpoint Molecule on the Expression of ILT-2, CD27, and CD38 in Splenic B cells

Rohn¹,

Lang

Schramm

et al. 2022

Journal of Immunology Research

View full text Add to dashboard Cite

show abstract

“…However, the explant approach by mincing and culturing the pieces of tissues leads to highly heterogeneous cultures and has a low yield (9–11). The enzymatic digestion activates the expression of chemokines and adhesion molecules, leading to extensive leukocyte adhesion (12), affecting the yield and growth of cells derived from plaque (10,13).…”

Section: Introductionmentioning

confidence: 99%

“…Several groups previously isolated and cultured plaque smooth muscle cells by mincing or enzymatically digesting atherosclerotic tissues (9)(10)(11)(12)(13). According to those studies, the isolated cells resembled smooth muscle cells in culture.…”

Section: Introductionmentioning

confidence: 99%

Human primary plaque cell cultures to study mechanisms of atherosclerosis

Buono

Benavente

Slenders

et al. 2023

Preprint

View full text Add to dashboard Cite

Plaque smooth muscle cells are critical players in the initiation and advancement of atherosclerotic disease. They produce extracellular matrix (ECM) components, which play a role in lesion progression and stabilization. Despite clear phenotypic differences between plaque smooth muscle cells and vascular smooth muscle cells (VSMCs), VSMCs are still widely used as a model system in atherosclerotic research. Here we present a conditioned outgrowth method to isolate plaque smooth muscle cells. We obtained plaque cells from 27 donors (24 carotid and 3 femoral endarterectomies). We show that these cells keep their proliferative capacity for eight passages, are transcriptionally stable, retain donor-specific gene expression programs, and express extracellular matrix proteins (FN1, COL1A1, DCN) and smooth muscle cell markers (ACTA2, MYH11, CNN1). Single-cell transcriptomics of plaque tissue and cultured cells reveals that cultured plaque cells closely resemble the myofibroblast fraction of plaque smooth muscle cells. Chromatin immunoprecipitation sequencing (ChIP-seq) shows the presence of histone H3 lysine 4 dimethylation (H3K4me2) at the MYH11 promoter, pointing to their smooth muscle cell origin. Finally, we demonstrated that plaque cells can be efficiently transduced (>97%) and are capable to take up oxidized LDL (oxLDL) and undergo calcification. In conclusion, we present a method to isolate and culture primary human plaque cells that retain plaque myofibroblast-like cells' phenotypical and functional capabilities - making them a suitable in vitro model for studying selected mechanisms of atherosclerosis.

show abstract

“…In this context, the isolation procedure may have detrimental effects on the intended use as a cellular product. Besides monocyte isolation by adherence [ 13 ] or density centrifugation [ 14 , 15 , 16 , 17 ], magnetic sorting by positive or negative selection is a common way to isolate cells from multi-cell-suspension, i.e., buffy coats (BC). During positive selection, all monocytes are isolated with magnetic beads coupled with antibodies against the CD14 surface molecules of monocytes.…”

Section: Introductionmentioning

confidence: 99%

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

Hornschuh

Haas

Winkel

et al. 2022

Biology

View full text Add to dashboard Cite

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.

show abstract

Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV

Cited by 65 publications

References 32 publications

Effect of HLA-G5 Immune Checkpoint Molecule on the Expression of ILT-2, CD27, and CD38 in Splenic B cells

Effect of HLA-G5 Immune Checkpoint Molecule on the Expression of ILT-2, CD27, and CD38 in Splenic B cells

Human primary plaque cell cultures to study mechanisms of atherosclerosis

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

Contact Info

Product

Resources

About