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Cell Culture Platforms with Controllable Stiffness for Chick Embryonic Cardiomyocytes.

ABSTRACT: For several years, cell culture techniques have been physiologically relevant to understand living organisms both structurally and functionally, aiming at preserving as carefully as possible the in vivo integrity and function of the cells. However, when studying cardiac cells, glass or plastic Petri dishes and culture-coated plates lack important cues that do not allow to maintain the desired phenotype, especially for primary cell culture. In this work, we show that microscaffolds made with polydimethylsiloxane (PDMS) enable modulating the stiffness of the surface of the culture substrate and this originates different patterns of adhesion, self-organization, and synchronized or propagated activity in the culture of chick embryonic cardiomyocytes. Thanks to the calcium imaging technique, we found that the substrate stiffness affected cardiomyocyte adhesion, as well as the calcium signal propagation in the formed tissue. The patterns of activity shown by the calcium fluorescence variations are reliable clues of the functional organization achieved by the cell layers. We found that PDMS substrates with a stiffness of 25 kPa did not allow the formation of cell layers and therefore the optimal propagation of the intracellular calcium signals, while softer PDMS substrates with Young's modulus within the physiological in vivo reported range did permit synchronized and coordinated contractility and intracellular calcium activity. This type of methodology allows us to study phenomena such as arrhythmias. For example, the occurrence of synchronized activity or rotors that can initiate or maintain cardiac arrhythmias can be reproduced on different substrates for study, so that replacement tissues or patches can be better designed.

SUBMITTER: Duran-Pasten ML

PROVIDER: S-EPMC6630216 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:Hemodynamic mechanical cues play a critical role in the early development and functional maturation of cardiomyocytes (CM). Therefore, tissue engineering approaches that incorporate immature CM into functional cardiac tissues capable of recovering or replacing damaged cardiac muscle require physiologically relevant environments to provide the appropriate mechanical cues. The goal of this work is to better understand the subcellular responses of immature cardiomyocytes using an in vitro cardiac cell culture model that realistically mimics in vivo mechanical conditions, including cyclical fluid flows, chamber pressures, and tissue strains that could be experienced by implanted cardiac tissues. Cardiomyocytes were cultured in a novel microfluidic cardiac cell culture model (CCCM) to achieve accurate replication of the mechanical cues experienced by ventricular CM. Day 10 chick embryonic ventricular CM (3.5 × 10(4) cell clusters per cell chamber) were cultured for 4 days in the CCCM under cyclic mechanical stimulation (10 mmHg, 8-15% stretch, 2 Hz frequency) and ventricular cells from the same embryo were cultured in a static condition for 4 days as controls. Additionally, ventricular cell suspensions and ventricular tissue from day 16 chick embryo were collected and analyzed for comparison with CCCM cultured CM. The gene expressions and protein synthesis of calcium handling proteins decreased significantly during the isolation process. Mechanical stimulation of the cultured CM using the CCCM resulted in an augmentation of gene expression and protein synthesis of calcium handling proteins compared to the 2D constructs cultured in the static conditions. Further, the CCCM conditioned 2D constructs have a higher beat rate and contractility response to isoproterenol. These results demonstrate that early mechanical stimulation of embryonic cardiac tissue is necessary for tissue proliferation and for protein synthesis of the calcium handling constituents required for tissue contractility. Thus, physiologic mechanical conditioning may be essential for generating functional cardiac patches for replacement of injured cardiac tissue.

Project description:The mechanisms involved in the development of skeletal muscle fibers have been studied in the last 70 years and yet many aspects of this process are still not completely understood. A myriad of in vivo and in vitro invertebrate and vertebrate animal models has been used for dissecting the molecular and cellular events involved in muscle formation. Among the most used animal models for the study of myogenesis are the rodents rat and mouse, the fruit fly Drosophila, and the birds chicken and quail. Here, we describe the robustness and advantages of the chick primary muscle culture model for the study of skeletal myogenesis. In the myoblast culture obtained from embryonic chick pectoralis muscle it is possible to analyze all the steps involved in skeletal myogenesis, such as myoblast proliferation, withdrawal from cell cycle, cell elongation and migration, myoblast alignment and fusion, the assembly of striated myofibrils, and the formation of multinucleated myotubes. The fact that in vitro chick myotubes can harbor hundreds of nuclei, whereas myotubes from cell lines have only a dozen nuclei demonstrates the high level of differentiation of the autonomous chick myogenic program. This striking differentiation is independent of serum withdrawal, which points to the power of the model. We also review the major pro-myogenic and anti-myogenic molecules and signaling pathways involved in chick myogenesis, in addition to providing a detailed protocol for the preparation of embryonic chick myogenic cultures. Moreover, we performed a bibliometric analysis of the articles that used this model to evaluate which were the main explored topics of interest and their contributors. We expect that by describing the major findings, and their advantages, of the studies using the embryonic chick myogenic model we will foster new studies on the molecular and cellular process involved in muscle proliferation and differentiation that are more similar to the actual in vivo condition than the muscle cell lines.

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Cell Culture Platforms with Controllable Stiffness for Chick Embryonic Cardiomyocytes.

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