Effects of culture conditions on cell morphology and differentiation responses to PMA in THP-1 cells
Year: 2025
Type: article
Abstract: THP-1 monocyte cell line can be differentiated into macrophage-like cells upon stimulation with phorbol 12-myristate 13-acetate (PMA) and therefore widely used in the inflammatory research. However, there is a lack of evidence-based protocols for cell culture and differentiation. In this study, we highlight how culture conditions influence THP-1 cell and their response to PMA stimulation. After thawing, THP-1 cells were cultured for up to 4 weeks and monitored by phase-contrast microscopy. Stressed cells (>1×10⁶ cells/mL for 72 h without medium replenishment) were assessed for CD80 and MHC-II expression and the subset distribution by flow cytometry. For PMA stimulation (10 µg/mL), non-stressed cells at standard (5×10⁵ cells/mL), low (2 ×10⁵ cells/mL), and high (1×10⁶ cells/mL) cell densities, along with stressed cells at ×10⁶ cells/mL density, were seeded and observed at 24 and 48 h. Cells initially formed large aggregates during the first week in suspension but gradually transitioned to single cells with continued culture. Metabolically stressed cells developed protrusions, showed increased adherence, and expressed higher levels of MHC-II compared to standard cells (p = 0.02). These cells also displayed signs of partial differentiation, with fewer MHC-II⁻CD80⁻ cells (p < 0.001) and more MHC-II⁺CD80⁻ cells (p < 0.001) than non-stressed cells. Optimal and uniform differentiation was observed after 3-4 weeks of recovery, at seeding densities below 1×10⁶ cells/mL, and under non-stressed conditions. These findings show that THP-1 culture conditions, including recovery time post-thawing, stress status and seeding density, have a major impact on their responsiveness to PMA.
Source: Journal of Immunological Methods
Authors: Alexander Launa, Qiuyue Peng
Institution: Aalborg University
Language: English
Topic: Immune cells in cancer
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $2,970
OpenAlex ID: W4417177151