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Effect of Monocyte Seeding Density on Dendritic Cell Generation in an Automated Perfusion-Based Culture System.

ABSTRACT: Dendritic cells (DCs) are increasingly important for research and clinical use but obtaining sufficient numbers of dendritic cells is a growing challenge. We systemically investigated the effect of monocyte (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) in MicroDEN, a perfusion-based culture system, as well as 6-well plates. Cell surface markers and the ability of the iDCs to induce proliferation of allogeneic T cells were examined. The data shows a strong relationship between iDC phenotype, specifically CD80/83/86 expression, and T cell proliferation. MicroDEN generated iDCs proved better than well plate generated iDCs at inducing T cell proliferation within the 200k-600k MO/cm2 seeding density range studied. We attribute this to perfusion in MicroDEN which supplies fresh differentiation medium continuously to the differentiating MOs while concurrently removing depleted medium and toxic byproducts of cellular respiration. MicroDEN generated fewer iDCs on a normalized basis than the well plates at lower MO seeding densities but generated equivalent numbers of iDCs at 600k MO seeding density. These results demonstrate that MicroDEN is capable of generating greater numbers of iDCs with less manual work than standard well plate culture and the MicroDEN generated iDCs have greater ability to induce T cell proliferation.

SUBMITTER: Kozbial A

PROVIDER: S-EPMC7441814 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Effect of Monocyte Seeding Density on Dendritic Cell Generation in an Automated Perfusion-Based Culture System.

Kozbial Andrew A Bhandary Lekhana L Murthy Shashi K SK

Biochemical engineering journal 20190705

Dendritic cells (DCs) are increasingly important for research and clinical use but obtaining sufficient numbers of dendritic cells is a growing challenge. We systemically investigated the effect of monocyte (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) in MicroDEN, a perfusion-based culture system, as well as 6-well plates. Cell surface markers and the ability of the iDCs to induce proliferation of allogeneic T cells were examined. The data shows a strong relatio ...[more]

PMID: 32831621

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Project description:The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.

Dataset Information

Effect of Monocyte Seeding Density on Dendritic Cell Generation in an Automated Perfusion-Based Culture System.

Publications

Effect of Monocyte Seeding Density on Dendritic Cell Generation in an Automated Perfusion-Based Culture System.

Similar Datasets

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