Double Resonator Technique For Preliminary Measurements Of Mechanical Properties Of Primary Cardiomyocytes

The state of cardiomyocyte beating is closely related to cardiac function,so it is very important to establish an accurate and effective method to monitor the mechanical properties during cardiomyocytes' beating.In this thesis,based on the double resonator quartz crystal microbalance(DRQCM)technology,a non-destructive,real-time and dynamic method for simultaneous monitoring of cardiomyocytes' beating force and viscoelasticity was preliminarily established.The specific results are summarized as follows:1.The mixed enzymatic digestion method of 0.08% pancreatin and 0.08% type I collagenase was used to digest the neonatal rat cardiomyocytes and the differential adherence method was used to purify the cells.The results showed that the number of neonatal rat cardiomyocytes extracted by this method was relatively large and the survival rate was higher.2.Unmodified and fibronectin(FN)modified 40 nm and 100 nm gold deposited 9MHz AT and BT cut crystals were used as the substrates for the adhesion and beating responses of neonatal rat cardiomyocytes,the results showed that 40 nm gold electrodes had irregular beating responses,100 nm gold electrodes could measure more ideal beating responses.3.Unmodified 100 nm gold deposited 9 MHz AT and BT cut crystals were used to adhere neonatal rat cardiomyocytes,and the DRQCM showed periodic changes in frequencies and motional resistances,contractile force,relaxation force and viscoelasticity during cardiomyocytes' beatings with the following results.a)The frequency and resistance responses during the cardiomyocytes' beating cycles,demonstrated a rapid rise or fall process corresponding to the contraction,followed by a slower frequency decrease or resistance rise process accompanied by relaxation.b)Both positive and negative beating forces were observed,corresponding to the contraction and relaxation states of the cardiomyocytes,and the contraction force is slightly larger than the relaxation force,and the relaxation period is longer than the contraction period.c)The viscoelastic cycle is consistent with the beating cycle,indicating that force is the main factor causing the change of viscoelasticity.When the cells contract,the viscoelasticity becomes larger and the cells become harder;when the cells relax,the viscoelasticity becomes smaller and the cells become softer.4.Using unmodified and FN modified 9 MHz 100 nm gold electrodes,the desorption processes of the adhered cardiomyocytes in 10% serum medium,pH 7.2 PBS and 0.08%trypsin were monitored.The results showed that at the moments when the cells were desorbed from the electrodes,there had tendencies to "grip" the electrodes,resulting in a relatively short time of beating responses.In conclusion,different extraction methods for cardiomyocytes,the gold layer thickness deposited on the crystals and whether the gold electrodes are modified or not,all have different impacts on the beating dynamics of primary cardiomyocytes of neonatal rats.The preliminary exploration results of this study demonstrated the feasibility of using DRQCM technique to monitor the force and viscoelasticity of beating cardiomyocytes,which is of great importance to eventually develop the DRQCM technology into a real-time and quantitative measurement method for the mechanical properties of beating cardiomyocytes and its applications in assessing efficacy and toxicity of cardiovascular drugs.