| Currently,cardiovascular diseases are still the leading cause of human morbidity.Traditional drug discovery and development have been relied on animal models.Whereas,animal models could not fully recapitulate the native heart due to inter-species differences.Consequently,post-approval withdrawal of medicines could happen for the drugs may cause strong cardiotoxicity during preclinical and clinical development.Therefore,it is still a challenging road to develop new drugs for cardiovascular diseases.Over the past decades,researchers have been focusing on the construction of cardiac models in vitro using cardiomyocytes from human.Cardiac models incorporated human cardiomyocytes in vitro could better mimic the physiology and function of the native human heart,thus improving the safety and efficacy of the pre-clinical evaluation of drugs.In this study,we mainly investigated the source of the human cardiomyocytes,and introduction of new materials for the construction of the in vitro cardiac tissues,as well as study on the contractility of the engineered cardiac tissues.Because adult human cardiomyocytes are essentially postmitotic,they lack the ability to proliferate and regenerate.Therefore,the source of human cardiomyocytes mainly relies on the stem cell differentiation.Human pluripotent stem cells,including human embryonic stem cells and induced pluripotent stem cells,possess strong capacity to proliferate and they can differentiate into different cell lineages on special conditions.Herein,we used both small molecules and the commercial cardiomyocyte differentiation kit to induce the cardiogenesis from human induced pluripotent stem cells.The small molecule method was based on first activating the Wnt signaling pathway using the glycogen synthesis kinase 3(GSK3)inhibitor CHIR99021,and then inhibiting the Wnt signaling by the Wnt inhibitor IWR-1,thus inducing the induced pluripotent stem cells to differentiate into cardiomyocytes.As for the commercial cardiomyocyte differentiation kit,different agents were added at different time points according to its standard protocol.By comparing the two methods,we can find that the small molecule method could yield monolayer contracting cardiomyocytes with a lower cost,whereas the commercial kit could achieve much higher differentiation efficiency with a higher cost.Both methods were involved in the subsequent experiments.We chose human hair as scaffolds to construct the cardiac tissues in vitro.First,we used three short straight hair segments perpendicular to each other to construct the cardiac tissue(termed “cardiowire”)and cultured the cardiowire for a long period of time.We found that the arrangement of the hair segments facilitated the formation of the suspended cardiowire.The cardiowire could contract between the hair segments and maintained its contractility for as long as 118 days.Next,we fabricated hair springs,and assembled two different hair spring-based scaffolds to culture the engineered cardiac tissue.Results showed that the introduction of the hair spring induced a more three-dimensional contraction of the cardiac tissue,and significantly improved the contractility of the engineered cardiac tissue.Finally,to quantitatively measure the contractile force of the cardiac tissue in vitro,we fabricated a series of hair springs with different diameters and different length.Then we measured the spring constant of the hair springs and found that by adjusting the diameter and length,we could obtain hair springs with the spring constant as low as 0.019 N/m,which is approximately the spring constant of the probes of the atomic force microscope(AFM).Therefore,the hair spring was suitable to measure the contractile force of the engineered cardiac tissue.We developed a device through assembling one hair spring and two short hair segments for the measurement of the contractile force of the cardiac tissue.With this device,we could achieve a real-time measurement of the contractile force throughout the culture period. |
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