@misc{LutterEckeScholkaetal., author = {Lutter, Anne-Helen and Ecke, Annemarie and Scholka, Jenny and Waldeck, Peter and Tretter, Tim and Anderer, Ursula}, title = {Differentiation of human chondrocytes under the influence of microalgae-based exopolysaccharides}, series = {38. Jahrestagung der Deutschen Gesellschaft f{\"u}r Klinische Mikrozirkulation und H{\"a}morheologie : Programm, 22. und 23. November 2019, Braunschweig}, journal = {38. Jahrestagung der Deutschen Gesellschaft f{\"u}r Klinische Mikrozirkulation und H{\"a}morheologie : Programm, 22. und 23. November 2019, Braunschweig}, abstract = {BACKGROUND: Exopolysaccharides are formed by microalgae in response to adverse conditions and are found mainly as a coating around the cells. The protective function of EPS plays an important role in the survival of microalgae in polluted habitats. So far, the particular biological and physico-chemical properties of EPS have not been adequately investigated and their effect on cells such as human chondrocytes has not been tested. Microalgae in particular offer an ideal platform for the production of dietary supplements or drugs that enable new therapeutic approaches. METHODS: To investigate the influence of EPS on human chondrocytes, cytotoxicity and differentiation potential were analyzed in 3D cultures. RESULTS: We were able to demonstrate a positive, but donor-specific influence of EPS on the differentiation of human chondrocytes. Furthermore, the EPS showed no cytotoxic effects in a range of up to 250 µg EPS per ml medium. CONCLUSION: EPS have the potential to positively influence the differentiation of human chondrocytes. Further experiments are necessary to analyze the donor-specific effect.}, language = {en} } @misc{MartinNeubertLutteretal., author = {Martin, Frank and Neubert, Annemarie and Lutter, Anne-Helen and Scholka, Jenny and Hentschel, Erik and Richter, Heiko and Anderer, Ursula}, title = {MTS, WST-8, and ATP viability assays in 2D and 3D cultures: Comparison of methodologically different assays in primary human chondrocytes}, series = {Clinical Hemorheology and Microcirculation}, volume = {88}, journal = {Clinical Hemorheology and Microcirculation}, publisher = {IOS Press}, issn = {ISSN 1386-0291}, doi = {10.3233/CH-248101}, pages = {3 -- 19}, abstract = {BACKGROUND: Tissue engineering enables the production of three-dimensional microtissues which mimic naturally occurring conditions in special tissues. These 3D culture systems are particularly suitable for application in regenerative medicine or experimental pharmacology and toxicology. Therefore, it is important to analyse the cells in their 3D microenvironment with regard to viability and differentiation. Tetrazolium assays (WST-8 and MTS) are still the methods of choice for estimating the number of living, metabolically active cells, with WST-8 being cell-impermeable compared to MTS. In contrast to these methods, the ATP assay is an endpoint method based on the luciferase-induced reaction of ATP with luciferin after cell lysis. OBJECTIVE:We compared three methodologically different proliferation/toxicity assays (MTS, WST-8, ATP) in monolayer (2D) and 3D culture systems to improve the technically challenging determination of the number of viable cells. METHODS: Chondrocytes were isolated from human articular cartilage. Three different test systems (MTS, WST-8, ATP) were applied to monolayer cells (2D, varying cell numbers) and spheroids (3D, different sizes) in 96-well plates. The intracellular ATP concentration was determined by luciferase-induced reaction of ATP with luciferin using a luminometer. Formazan formation was measured spectrophotometrically after different incubation periods. Evaluation was performed by phase contrast microscopy (toxicity), correlation of cell count and ATP concentration or absorption signal (Gompertz function) and propidium iodide (PI) staining to proof the cell lysis of all cells in spheroids. RESULTS: In 2D culture, all three assays showed a good correlation between the number of seeded cells and the ATP concentration or absorption data, whereas the MTS-assay showed the lowest specificity. In 3D culture, the spheroid sizes were directly related to the number of cells seeded. The absorption data of the WST-8 and MTS assay correlated only for certain spheroid size ranges, whereas the MTS-assay showed again the lowest specificity. Only the measured intracellular ATP content showed a linear correlation with all spheroid sizes ranging from 100-1000 µm. The WST-8 assay revealed the second-best sensitivity which allows the measurement of spheroids larger than 240 µm. Phase contrast observation of monolayer cells showed toxic effects of MTS after 6 h incubation and no signs of toxicity of WST-8. Staining with propidium iodide showed complete lysis of all cells in a spheroid in the ATP assay. CONCLUSION: Among tetrazolium-based assays, WST-8 is preferable to MTS because of its non-toxicity and better sensitivity. When determining the number of viable cells in the 2D system, caution is advised when using the ATP assay because of its two-phase slope of the correlation graph concerning cell number and intracellular ATP. In 3D systems of human chondrocytes, the ATP-assay is superior to the other two test systems, as the correlation graph between cell number and intracellular ATP is biphasic. Since differentiation processes or other metabolic events can influence the results of proliferation and toxicity assays (determination of viable cells), this should be taken into account when using these test systems.}, language = {en} }