Dataset Information

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

ABSTRACT: Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNF? or IL-1?. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

SUBMITTER: Colombo I

PROVIDER: S-EPMC5748104 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

Colombo Irma I Sangiovanni Enrico E Maggio Roberta R Mattozzi Carlo C Zava Stefania S Corbett Yolanda Y Fumagalli Marco M Carlino Claudia C Corsetto Paola Antonia PA Scaccabarozzi Diletta D Calvieri Stefano S Gismondi Angela A Taramelli Donatella D Dell'Agli Mario M

Mediators of inflammation 20171217

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the <i>in vitro</i> studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate <i>in vitro</i>, a ...[more]

PMID: 29391667

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Project description:For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32 °C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic stimulation by calcium, serum, confluence and lower incubation temperature amplified the differentiation rate.

Dataset Information

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

Publications

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

Similar Datasets

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