Cardiomyocyte syncytium combined with Atomic force microscopy, advanced setup of universal biosensor for phenotype screening
| Autoři | |
|---|---|
| Rok publikování | 2016 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in most cardiac diseases. However we routinely model such diseases using cardiomyocytes derived from human embryonic stem cells and especially from patient specific induced pluripotent stem cells; it is the biosensing of contraction and relaxation which makes the models a great tool for drug development. We employ atomic force microscopy to analyze these parameters. In this novel approach we employ the direct contact of the cantilever with standardized relaxation and stress conditions with the stem cells derived cardiac syncytium. We present advanced experimental setup for quantification of the cardiomyocyte dynamic changes induced by external stimuli such as extreme temperature, calcium level and stress conditions or administered various drugs. Further we show novel automatized data evaluation process. Advantages of feedback- gain parameter previously described (2) are discussed. Methods: Cardiomyocytes were generated as previously described (1) using aggregation in agarose micro-wells allowing homogeneous cluster formation in size and shape (size of the cluster as well as cardiomyocyte content was quantified). Atomic force microscopy analysis was performed in Tyrode’s solution, in Calcium concentration gradients, temperature gradients and ion channel- and adrenergic receptor-modulator gradients.. For data postprocessing and statistical evaluation was involved specific script allowing automatized evaluation. Results: The active AFM mode has no effect on contraction frequency, while providing for correct contraction force recording. We found the optimal value corresponds to average recorded value of EBs contraction force. It was set to 6 nN, in our case, which allowed for the measurement of 95% of maximum contraction force (4.6 nN vs. 4.9 nN, when SP was 6 and 18 nN, respectively). Use of noncoated AFM probes eliminates thermal instability and allows measurements for hours and days continuously. Calcium gradients allowed us to study the excitation-contraction (un)coupling as well as arrythmogenicity of calcium extreme concentrations. Beat to beat surface mapping has shown comparable values all over accessible syncytium rendering the method robust and unbiased by the cantilever position. |
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