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• W2200619637 abstract "While several reports focus on serum-containing media compared to serum-free culture conditions 1, 2 or on the influence of medium on the formation of 3D cultures 3, 4, only very few comparative studies exist on the influence of different serum-free culture media – even though widely used – on basic cell biological parameters in submerged cultures, and more particularly on parameters others than proliferation 5, 6. One aim of this study was to clarify whether the choice of medium may influence the outcome of an experiment in cultured keratinocytes. Additionally, we wanted to know to what extent donor variability influences the experiment. We chose two cell culture assays often used in experimental dermatology, that is barrier formation after Ca2+-induced stratification and scratch wound closure with and without manipulation of the cells by high glucose concentrations – mimicking diabetic conditions – and siRNA-mediated protein knock-down. All experiments were conducted on human keratinocytes isolated from foreskin of different donors from the age of 1 to 5 years as described previously 7. The keratinocytes were then expanded in submerged cultures for one to four passages (depending on the experiment) in the following serum-free growth media before they were used for the individual experiments: EpiLife (Cascade Biologics, Invitrogen, Darmstadt, Germany), KGM2 (Promocell, Heidelberg, Germany), DermaLife (Lifeline Cell Technology, Cellsystems, Troisdorf, Germany) and K-SFM (Gibco, Life Technologies, Darmstadt, Germany). For detailed descriptions, see Data S1. Barrier formation in keratinocytes cultured in DermaLife, EpiLife and KGM, respectively, was induced by elevation of the calcium level to 1.8 mm CaCl2 (t0). A gradual increase of transepithelial resistance (TER), which is a measure for ion barrier function, was observed in all three media after addition of calcium. However, at the beginning, TER increased significantly faster in EpiLife compared to the other two media, whereas from days 5 to 10 highest TER values were found for cells cultured in DermaLife (Fig. 1a). When comparing cells in DermaLife and EpiLife from different donors, we observed higher variances of TER values in DermaLife (maximum deviation of 117%) than in EpiLife (maximum deviation of 66%) (Fig. S1a,b). There is a general trend regarding the consistency of quantitative differences of the biological responses for keratinocyte strains from certain donors in different media, as it has been shown by repeated interindividual comparative studies. Interestingly, it was found that TER values of cells pooled from different donors mostly correspond to the donor cells with the leakiest barrier (Fig. S1c). Concerning barrier function to larger molecules, we observed time-dependent decreases in permeabilities in all three media for 4 and 40 kDa FITC dextrans (Figs 1b, S2). Lowest initial P values were constantly found for cells cultured in EpiLife indicating best barrier supporting properties at early time points, whereas after 4–9 days lowest permeabilities were measured in cells cultured with DermaLife (Figs 1b, S2). This was in line with the results of the TER experiments. Interestingly, the high interindividual differences in cells cultured with DermaLife seen in TER experiments cannot be seen in the tracer permeability experiments, also reflecting the different regulation of these two barrier properties 8. Because it is known that TJs are the main drivers of barrier formation in submerged cultures 9, we investigated the localization of the TJ-components Cldn-1, Cldn-4 and ZO-1 in the three media and observed pronounced differences: at early time points, staining intensity of TJ proteins at the cell–cell borders was more pronounced in EpiLife than in DermaLife in cells from most donors, reflecting the higher barrier function of keratinocytes in EpiLife (Fig. S3). After 10 days, Cldn-1 was found at the cell–cell borders in several cell layers of the meanwhile multilayered epithelium and it was strongly expressed in DermaLife and KGM, whereas only trace amounts were detected in EpiLife (Fig. 2). ZO-1 was found to be expressed in all media located in the uppermost layer of living cells, with stronger immunointensity in DermaLife than in EpiLife and KGM. When checking whether also a (rudimentary) stratum corneum may contribute to the differences in barrier function described above, we indeed observed in histology that keratinocytes grown in DermaLife built up 2–4 layers of cornified cells, cells grown in KGM developed 1–2 layers (which partly contained remnants of nuclei), whereas a cornified layer was either thinner or even absent in EpiLife (Fig. 2a–c). Thus, increased TJ protein expression and more cornified cells are likely to contribute to the increased barrier function of cells grown in DermaLife at later time points. Furthermore, we also investigated the impact of the medium on scratch wound assays (Fig. S4a,b), which are often used to investigate wound healing properties of keratinocytes. Influence of the medium was already found in normal scratch assay, but was especially obvious after manipulation of the cells by high glucose (Fig. S4a) or siRNA-mediated knock-down (Fig. S4b). Of note, as for barrier experiments, donor dependency was observed, and the influence of the donor was less pronounced in EpiLife than in DermaLife (Fig. S4c). We show here a major influence of medium on often used assays in human primary keratinocytes. During short-term incubation in barrier assay, cells grown in EpiLife show superior barrier function concerning ions (TER) and macromolecules (4 and 40 kDa FITC dextrans). After long-term incubation, cells grown in DermaLife show the best barrier function. This correlates with increased protein localization of certain tight junction proteins at the cell–cell borders and with the formation of cornified cells. Donor source also played a role, but was more pronounced in DermaLife than in EpiLife. Also wound closure rates in scratch wound assays differed between the media. Again, donor variability was more pronounced in DermaLife than in EpiLife. Interestingly, cultures in different media were also differently susceptible for the manipulation with high glucose and siRNA knock-down. Taken together, these data suggest that (i) the choice of medium influences the outcome of the experiment concerning barrier formation and scratch wound healing and (ii) donor variability is generally found, but is less pronounced in some media than others. Our data emphasize that unequivocal interpretation of experimental data based on cultures in different media may be rather complicated and that the choice of medium can reduce the influence of donor variability. J.M. Brandner, M. Zorn-Kruppa, T. Volksdorf and C. Ueck designed the research study. M. Zorn-Kruppa, T. Volksdorf, C. Ueck, E. Zöller and P. Houdek performed the research. K. Reinshagen, I. Ridderbusch and G. Bruning contributed essential reagents and tools. M. Zorn-Kruppa, T. Volksdorf, C. Ueck, E. Zöller and J.M. Brandner analysed the data. M. Zorn-Kruppa, T. Volksdorf, C. Ueck, I. Moll and J.M. Brandner wrote the manuscript. Ethics approval was obtained from the local medical ethics committee (WF061/12). None declared. Figure S1. TER values of keratinocytes from different donors cultured either in DermaLife (a) or EpiLife (b), n = 4, means ± SD. Note the higher variability – denoted by the higher SDs – between different donors in cells grown in DermaLife compared to Epilife (c) TER of keratinocytes from three donors compared to keratinocytes pooled from the same donors in one culture grown in DermaLife (means from triplicates; for better lucidity SDs are not shown). Figure S2. Paracellular tracer flux of 40 kDa FITC dextrans in DermaLife, EpiLife and KGM. Figure S3. TJ protein localisation in cells cultured in DermaLife, EpiLife and KGM 24 h after calcium switch. Figure S4. Scratch wound assays to study the influence of different serum-free media and donors on the wound closure rate of primary keratinocytes with and without manipulation of the cells by high-glucose conditions or siRNA mediated knock-down. Table S1. Antibodies used for immunofluorescence (IF) and Western blot analyses (WB). Table S2. Composition of some known additives (supplements) of the three culture media used. na, Data not available. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W2200619637 date "2016-02-10" @default.
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• W2200619637 title "Major cell biological parameters of keratinocytes are predetermined by culture medium and donor source" @default.
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• W2200619637 doi "https://doi.org/10.1111/exd.12922" @default.
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