Effect of culture conditions on the phenotype of THP-1 monocyte cell line

Abstract

Problem: Macrophage function has many implications in a variety of diseases. Understanding their biology becomes imperative when trying to elucidate immune cell interactions with their environment, and in vitro cell lines allow researchers to manipulate these interactions. A common cell line used is THP-1, a promyeloid cell line suggestive to outside factors, and therefore sensitive to culture conditions. In this study, we describe how culture conditions can alter THP-1 morphology and in turn affect their response to differentiation stimuli.

Method of study: THP-1 cells were cultured in two conditions and treated with phorbol 12-myristate 13-acetate (PMA) or MCSF. CD14 surface expression was determined by flow cytometry and cytokine/chemokine production determined by multiplex analysis.

Results: Culture conditions of THP-1 affect their response to PMA. Highly confluent THP-1 cells differentiate into a heterogeneous population responsive to PMA as seen by an increase in CD14 expression. However, these cells, cultured in low confluence, remain as a homogenous population and do not gain CD14. Additionally, there are major differences in the constitutive cytokine profile.

Conclusion: We demonstrate that the culture conditions of THP-1 cells can alter their response PMA. This suggests that culture techniques may account for the discrepancy in the literature of both basal THP-1 phenotype and their response to PMA.

Figure 1

Differential response to PMA by THP-1. THP-1 cells were treated with 10ng/ml PMA or MCSF as described in materials and methods and CD14 expression determined by flow cytometry. In Condition I (A), CD14 expression increased after treatment with PMA as compared to NT, however, in Condition II (B) there was no change; MCSF had little effect on CD14; data are representative of at least three experiments.

Figure 2

THP-1 cells differentially change morphology in response to PMA. THP-1 cells were treated with 10ng/ml PMA or MCSF as described in materials and methods and size (FSC) and complexity (SSC) determined by flow cytometry. In Condition I (A), PMA treatment creates a more macrophage phenotype characterized by larger cells that gain complexity. In Condition II (B) PMA pushes THP-1 cells to a more granular phenotype as seen by an increase in complexity but not size; data are representative of at least three experiments.

Figure 3

THP-1 morphology under basal conditions. THP-1 cells were collected after 48hrs in culture, and analyzed for size (FSC) and complexity (SSC) determined by flow cytometry size. Condition I THP-1 cells, without any stimulant, are heterogeneous in size while the cells cultured in Condition II are a more compact and homogeneous population; data are representative of at least three experiments.

Figure 4

THP-1 cytokine secretion under basal conditions. THP-1 cells were collected after 48hrs in culture, and cell-free supernatants analyzed for cytokine and chemokine secretion by multiplex. Condition 1 THP-1 cells constitutively secrete higher levels of cytokines as compared to condition II, with exception of IL-12 and VEGF.