Device for co-culture of sympathetic neurons and cardiomyocytes using microfabrication

Takeuchi, Akiteru; Nakafutami, Shingo; Tani, Hiromasa; Mori, Masahide; Takayama, Yoichiro; Moriguchi, Hiroyuki; Kotani, Kiyoshi; Miwa, Keiko; Lee, Jong-Kook; Noshiro, Makoto; Jimbo, Yasuhiko

doi:10.1039/c0lc00327a

Cited by 61 publications

(69 citation statements)

References 32 publications

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“…We found changes in pathways regulating gap junction protein expression along numerous changes in pathways associated with metabolism and development (Supplement Figure S1). The results of our proteomics screen is consistent with other studies which link innervation to developmental processes [21,[25][26][27][28]. Shortly after birth, cardiomyocyte hyperplasia decreases and CV increases [25], which correlate to increased βadrenoreceptor density on cardiomyocytes and higher levels of catecholamines in the circulation [29].…”

Section: Discussionsupporting

confidence: 90%

Optical interrogation of sympathetic neuronal effects on macroscopic cardiac monolayer dynamics

Burton

Tomek

Ambrosi

et al. 2019

Preprint

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Alterations in autonomic function are known to occur in cardiac conditions including sudden cardiac death. Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. Dissecting direct neural-cardiac interactions at the cellular level is technically challenging and understudied due to the lack of experimental model systems and methodologies. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiac monolayer dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac wave behaviour and the applicability of optogenetics in mechanistic in vitro studies. We combine photo-uncaging or optogenetic neural stimulation with imaging of cardiac monolayers to measure electrical activity in an automated fashion, illustrating the power and high throughput capability of such interrogations. The methods described highlight the challenges and benefits of co-cultures as experimental model systems. RESULTS:1] Stellate Sympathetic Neurons make contacts with cardiomyocytes: Scanning electron microscopy of sympathetic neurons growing in co-culture with cardiomyocytes in vitro shows connections between neurite extension and cardiac syncytium (Figure 1 A). The neuron bodies and extensions clearly make physical contact with myocytes (Figure 1 B, C, D, E, F, G, H, I, J). Immuno staining of co-cultures shows co-localisation of Synpasin and Beta2 receptors (Figure 1 K), sympathetic neurons show positive staining for Th (Figure 1 L), and fibronectin contamination in the co-cultures was assessed by staining with Vimentin (Figure 1 M), which showed low abundance. 2] Pattern formation is affected by the presence of neurons: Using dye-free imaging (Figure 2 A, B, C), we investigated how neuronal activation modulates cardiac patterns of activation in monolayer culture. We chose experimental conditions that spontaneously yield a wide variety of wavefront topologies within the imaging system's 16 x 16 mm field of view. Introduction of an additional cell type can potentially introduce heterogeneities that would impact wave front stability. Surprisingly, co-cultures displayed fewer wave breaks than their monoculture counterparts at similar plating densities (Figure 2 D). We broadly classified wave dynamics as simple (periodic target waves and single spiral wave reentry, Figure 2 D top row), or complex (single dominant spirals with additional irregular waves and multiple equally sized wavelets, Figure 2 D bottom row). While monocultures frequently displayed complex dynamics, co-cultures rarely displayed this behavior (Figure 2 E, P<0.05, chi-square test). Indeed, we observed wavelet reentry in only one of the co-culture preparations (6 isolations, 20 preparations). 3] Conduction velocity in spontaneously active co-cultures is faster than myocyte monocultures:We measured conduction velocity in unstimulated co-cultures (n=19) and myocyte monocultures (n=9). Figure 2 F s...

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Section: Discussionsupporting

confidence: 90%

Optical interrogation of sympathetic neuronal effects on macroscopic cardiac monolayer dynamics

Burton

Tomek

Ambrosi

et al. 2019

Preprint

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“…The first microfluidic device allowing for neuromuscular coculture was developed by Takeuchi et al (Takeuchi et al 2011), adapted from (I. Suzuki, Y. Sugio, Y. Jimbo and K. Yasuda, Lab Chip, 2005, 5, 241-247.).…”

Section: A Neuronal Coculturementioning

confidence: 99%

“…a) Microfluidic device allowing precise application of neural agrin to arrays of myotubes (reproduced from(Tourovskaia, Li, and Folch 2008) with permission). b) Coculture of spatially segregated sympathetic neurons and cardiomyocytes (reproduced from(Takeuchi et al 2011) with permission). c) Microfluidic devices designed for 3D muscle strip innervation by motor neurons (reproduced from(Morimoto et al 2013) with permission).…”

mentioning

confidence: 99%

Microfabrication and microfluidics for muscle tissue models

Uzel

Pavesi

Kamm

2014

Progress in Biophysics and Molecular Biology

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The relatively recent development of microfluidic systems with wide-ranging capabilities for generating realistic 2D or 3D systems with single or multiple cell types has given rise to an extensive collection of platform technologies useful in muscle tissue engineering. These new systems are aimed at (i) gaining fundamental understanding of muscle function, (ii) creating functional muscle constructs in vitro, and (iii) utilizing these constructs a variety of applications. Use of microfluidics to control the various stimuli that promote differentiation of multipotent cells into cardiac or skeletal muscle is first discussed. Next, systems that incorporate muscle cells to produce either 2D sheets or 3D tissues of contractile muscle are described with an emphasis on the more recent 3D platforms. These systems are useful for fundamental studies of muscle biology and can also be incorporated into drug screening assays. Applications are discussed for muscle actuators in the context of microrobotics and in miniaturized biological pumps. Finally, an important area of recent study involves coculture with cell types that either activate muscle or facilitate its function. Limitations of current designs and the potential for improving functionality for a wider range of applications is also discussed, with a look toward using current understanding and capabilities to design systems of greater realism, complexity and functionality.

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“…We are currently exploring a variety of different techniques pioneered by other research groups, ranging from maintaining neurons and myocytes in separate but connected compartments (Takeuchi et al . ), to virally transfecting neurons with calcium‐sensitive markers (Looger & Griesbeck, ) and imaging cardiac motion with a second camera using a macroscopic phase contrast approach (Hwang et al . ).…”

Section: Introductionmentioning

confidence: 99%

Macro‐micro imaging of cardiac–neural circuits in co‐cultures from normal and diseased hearts

Bub

Burton

2014

The Journal of Physiology

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The autonomic nervous system plays an important role in the modulation of normal cardiac rhythm, but is also implicated in modulating the heart's susceptibility to re-entrant ventricular and atrial arrhythmias. The mechanisms by which the autonomic nervous system is pro-arrhythmic or anti-arrhythmic is multifaceted and varies for different types of arrhythmia and their cardiac substrates. Despite decades of research in this area, fundamental questions related to how neuron density and spatial organization modulate cardiac wave dynamics remain unanswered. These questions may be ill-posed in intact tissues where the activity of individual cells is often experimentally inaccessible. Development of simplified biological models that would allow us to better understand the influence of neural activation on cardiac activity can be beneficial. This Symposium Review summarizes the development of in vitro cardiomyocyte cell culture models of re-entrant activity, as well as challenges associated with extending these models to include the effects of neural activation.

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Device for co-culture of sympathetic neurons and cardiomyocytes using microfabrication

Cited by 61 publications

References 32 publications

Optical interrogation of sympathetic neuronal effects on macroscopic cardiac monolayer dynamics

Optical interrogation of sympathetic neuronal effects on macroscopic cardiac monolayer dynamics

Microfabrication and microfluidics for muscle tissue models

Macro‐micro imaging of cardiac–neural circuits in co‐cultures from normal and diseased hearts

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