Dataset Information

In vitro skin culture media influence the viability and inflammatory response of primary macrophages.

ABSTRACT: The replacement of animal models for investigation of inflammation and wound healing has been advancing by means of in vitro skin equivalents with increasing levels of complexity. However, the current in vitro skin models still have a limited pre-clinical relevance due to their lack of immune cells. So far, few steps have been made towards the incorporation of immune cells into in vitro skin and the requirements for immunocompetent co-cultures remain unexplored. To establish suitable conditions for incorporating macrophages into skin models, we evaluated the effects of different media on primary keratinocytes, fibroblasts and macrophages. Skin maturation was affected by culture in macrophage medium, while macrophages showed reduced viability, altered cell morphology and decreased response to pro- and anti-inflammatory stimuli in skin differentiation media, both in 2D and 3D. The results indicate that immunocompetent skin models have specific, complex requirements for supporting an accurate detection of immune responses, which point at the identification of a suitable culture medium as a crucial pre-requisite for the development of physiologically relevant models.

SUBMITTER: Griffoni C

PROVIDER: S-EPMC8007571 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

In vitro skin culture media influence the viability and inflammatory response of primary macrophages.

Griffoni Chiara C Neidhart Berna B Yang Ke K Groeber-Becker Florian F Maniura-Weber Katharina K Dandekar Thomas T Walles Heike H Rottmar Markus M

Scientific reports 20210329 1

The replacement of animal models for investigation of inflammation and wound healing has been advancing by means of in vitro skin equivalents with increasing levels of complexity. However, the current in vitro skin models still have a limited pre-clinical relevance due to their lack of immune cells. So far, few steps have been made towards the incorporation of immune cells into in vitro skin and the requirements for immunocompetent co-cultures remain unexplored. To establish suitable conditions ...[more]

PMID: 33782484

Similar Datasets

Project description:The isocyanate monomer 1,6-hexamethylene diisocyanate (HDI) and one of its trimers, HDI isocyanurate, are airway and skin sensitizers contained in polyurethane paint. The toxic response of cultured skin cells to these compounds was measured by evaluating the isocyanate concentrations at which 50% of the cells die (i.e., lethal concentration 50%, LC50) because the relative toxicity of each form of HDI should be considered when exposure limits of HDI-based paints are set. By using a luminescent ATP-viability assay, we compared the cytotoxic effects of HDI monomer and HDI isocyanurate on cultured human skin cells (keratinocytes, fibroblasts, and melanocytes) after 4-h isocyanate exposures using culture media with varying levels of nutrients in order to also determine the effects of media composition on isocyanate toxicity. Before analysis, experimental wells were normalized to controls containing cells that were cultured with the same vehicle and media. The measured mean LC50 values ranged from 5 to 200 µM across the experimental conditions, in which HDI isocyanurate in protein-devoid media was the most toxic to cells, producing the lowest LC50 values. For HDI monomer, keratinocytes were the most resistant to its toxicity and melanocytes were the most susceptible. However, when exposed to HDI isocyanurate, the opposite was observed, with melanocytes being the most resilient and the keratinocytes and fibroblasts were more susceptible. Depending on the type of skin cells, dose-response data indicated that HDI isocyanurate was 2-6 times more toxic than HDI monomer when using protein-devoid media whereas HDI isocyanurate was 4-13 times more toxic than HDI monomer when protein-rich media was used. Therefore, if the protein-devoid saline medium alone were used for these experiments, then a significant under-estimation of their relative toxicities in protein-rich environments would have resulted. This difference is because HDI monomer toxicity was more attenuated by the presence of protein in the culture media than HDI isocyanurate toxicity. Thus, conclusions based on comparative toxicity studies and consequent inference applied to potential human toxicity can be affected by in vitro culture media conditions. The physiochemical difference in reactivity of the two forms of HDI to biological molecules most likely explains the observed toxicity differences and may have implications for skin penetration, adverse effects like skin sensitization, and systemic responses like asthma. Future studies are warranted to investigate differences in the biological availability, cellular toxicity, and immunologic sensitization mechanisms for HDI monomer and HDI isocyanurate.

Project description:Macrophages are central to innate immunity and defining the mechanisms by which these cells mediate inflammation is critical to understanding in situ homeostatic and pathophysiologic roles. Human monocyte derived macrophages (hMDM) are widely used to examine these processes. Stimuli that influence macrophage function could influence these studies, including differences in culture environment that have been suggested significantly impact macrophage function. We examined baseline hMDM activity and the response to the TLR4 agonist, lipopolysaccharide (LPS) in different culture media in the presence and absence of serum. We focused on the most common macrophage medias, DMEM and RPMI 1640 Medium +/- fetal bovine serum (FBS), as well as Macrophage Serum Free Media (M-SFM), a media specifically formulated for serum free culture. Cells cultured in these five distinct media were treated with vehicle (H20) or LPS and evaluated for changes in morphology, transcriptomic profile, and alterations in inflammatory response. Culture in M-SFM resulted in the greatest morphological differences (area, length-to-width, perimeter-to-area ratio) and while there were few transcriptomic differences between DMEM and RPMI with FBS, both the absence of FBS supplementation and culture in M-SFM significantly altered the baseline transcriptome. Culture in M-SFM also significantly reduced macrophage sensitivity to LPS as measured by NF-kB activation. All hMDM conditions showed a dose dependent phagocytic response and elicited cytokine and chemokine secretion in response to LPS stimulation, but the magnitude of these effects varied between conditions, with different trends depending on the cytokine. These findings suggest that culture media and the presence of serum alters baseline activation and the response to stimulus in macrophages, potentially obscuring or driving artifactual results. These studies confirm and expand on previous findings showing that in vitro microenvironment is not a benign component of experimental design and demonstrate the need to optimize experimental conditions for such plastic cells.

Dataset Information

In vitro skin culture media influence the viability and inflammatory response of primary macrophages.

Publications

In vitro skin culture media influence the viability and inflammatory response of primary macrophages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets