An increase in mechanical load in the heart causes cardiac hypertrophy, either physiologically (heart development, exercise and pregnancy) or pathologically (high blood pressure and heart-valve regurgitation). Understanding cardiac hypertrophy is critical to comprehending the mechanisms of heart development and treatment of heart disease. However, the major molecular event that occurs during physiological or pathological hypertrophy is the dynamic process of sarcomeric addition, and it has not been observed. In this study, a custom-built second harmonic generation (SHG) confocal microscope was used to study dynamic sarcomeric addition in single neonatal CMs in a 3D culture system under acute, uniaxial, static, sustained stretch. Here we report, for the first time, live-cell observations of various modes of dynamic sarcomeric addition (and how these real-time images compare to static images from hypertrophic hearts reported in the literature): 1) Insertion in the mid-region or addition at the end of a myofibril; 2) Sequential addition with an existing myofibril as a template; and 3) Longitudinal splitting of an existing myofibril. The 3D cell culture system developed on a deformable substrate affixed to a stretcher and the SHG live-cell imaging technique are unique tools for real-time analysis of cultured models of hypertrophy.
The ability to place individual cells into an engineered microenvironment in a cell-culture model is critical for the study of in vivo-relevant cell-cell and cell-extracellular matrix interactions. Microfluidics provides a high-throughput modality to inject various cell types into a microenvironment. Laser guided systems provide the high spatial and temporal resolution necessary for single-cell micropatterning. Combining these two techniques, the authors designed, constructed, tested, and evaluated 1) a novel removable microfluidics-based cell-delivery biochip and 2) a combined system that uses the novel biochip coupled with a laser guided cell-micropatterning system to place individual cells into both 2D and 3D arrays. Cell-suspensions of chick forebrain neurons and glial cells were loaded into their respective inlet reservoirs and traversed the microfluidic channels until reaching the outlet ports. Individual cells were trapped and guided from the outlet of a microfluidic channel to a target site on the cell-culture substrate. At the target site, 2D and 3D pattern arrays were constructed with micron-level accuracy. Single-cell manipulation was accomplished at a rate of 150 μm/s in the radial plane and 50 μm/s in the axial direction of the laser beam. Results demonstrated that a single-cell can typically be patterned in 20-30 seconds, and that highly accurate and reproducible cellular arrays and systems can be achieved through coupling the microfluidics-based cell-delivery biochip with the laser guided system.
The basement membrane (BM), a network of laminin and collagen IV, mechanically supports individual cells and directly mediates cell-cell and cell-extracellular matrix (ECM) interactions. For example, the BM network that tightly encloses each cardiomyocyte (CM) mediates the alignment of CMs with collagen I in the ECM. Additionally, the BM-laminin is involved in the formation of gap junctions (GJs), which regulate electrical coupling between two CMs in the myocardium. The role of BM in GJ maturation remains unclear because of the complicated in vivo structures and lack of an ideal in vitro culturing mode. In this study, our laser cell-micropatterning system was used to place two neonatal CMs (NCMs) in contact on an aligned collagen gel (ACG) to study the relationship between GJ maturation and BM development. The results of double immunofluorescence staining and confocal imaging showed that BM-laminin was deposited earlier than the formation of GJs in the intercellular space and that newly expressed connexin 43 clusters were preferentially assembled near the deposited BM structures. Eventually the BM network surrounded the GJs.
Traditional cell-culture techniques lack the spatial control of single cells that is necessary to recreate the cell-cell contact arrangement found in tissue. Since Ashkins developed optical force-based cellmanipulation techniques [1], the laser microbeam has been used in a microscopic system to explore various biological interactions at molecular and cellular levels. These explorations of a single cell or subcellular organelle involve trapping (using a laser-tweezers microscope [2]) and guidance (using a laser-guided direct writing microscope [3,4]). When laser tweezers are used, a high numerical aperture (NA) microscope objective generates a strongly focused laser beam, which 3D traps a particle, such as a biological cell, in the beam's focal point. In laser-guided direct writing, a low NA microscope objective generates a weakly focused laser beam, which traps a particle in the beam axis and guides it to move along the direction of beam propagation.Based on these techniques, we have developed a laser cell-micropatterning system in which the laser beam is focused in a transition state between generating an optical trap and optical guidance [5]. Using this system, a single biological cell can be trapped in a typical (e.g., 30mm) cell culture dish and patterned onto a designated cell culture niche with very high spatial and temporal resolution [6][7][8].However, this and the other currently available laser-based single-cell-manipulation techniques can be used to pattern only spherical cells, not irregularly shaped cells such as rod-shape cardiomyocytes. The work stems from the use of a spatial light modulator (SLM) loaded with a computer generated phase map to shape a single laser beam into multiple microbeams analogous to techniques used in holographic optical tweezers (HOT) [9]. In contrast to the optical configuration in HOT, our system uses a low NA objective to produce multiple weakly focused laser microbeams in our multiple beam laser guidance system. Based on the cell image acquired during laser cell patterning, the multiple beams can be distributed around the outer contour of an irregularly shaped cell to achieve accurate cell patterning. In addition to describing the principle and practice of the system design, here we present what is to our knowledge the first achievement of patterning large, irregularly (rod) shaped adult rat cardiomyocytes (ACMs) in an end-to-end connected alignment to replicate the in vivo heart-muscle structure without use of substrate surface modifications that may interfere with in vivo-like cell-cell and cell-extracellular matrix interactions.The basic structure of the multiple-beam laser cell-patterning system is schematically shown in Figure 1. The cell suspension was loaded into the microfluidics-based cell feeding system built in the celldeposition chamber, with a microfluidic channel width of 200 µm. The guidance region (also the imaging region of the objective) was initially focused onto the outlet of the cell-feeding microfluidic channel by controlling the movement of the ce...
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