Kenta Shimba

Microcasting with agarose gel via degassed polydimethylsiloxane molds for repellency-guided cell patterning

Hydrogel patterning methods are widely used for cell patterning because they offer better long-term stability than protein patterning methods such as micro-contact printing, but conventional hydrogel patterning methods require special apparatuses such as a laser or an electron beam lithography system or they have complicated chemical operations which prevent their practical use in biological laboratories. A simple method was developed to cast a hydrogel solution without external power sources using a polydimethylsiloxane (PDMS) mold with micro-channels. This study employed “the accumulation of vacuum pressure” in a degassed lump of PDMS as a driving force for introducing agarose solution into the micro-channels. Sufficient vacuum pressure could be accumulated within 1 h in the PDMS elastomer that was acting as a vacuum tank, and 2 w/v% agarose solution could be aspirated into the micro-channels with widths from 100 to 2000 μm and a height of 19 μm, fully filling them. After the gelation and dehydration of agarose solution in the micro-channels, the patterns of agarose gel on the channels were successfully cast with a 90%-width accuracy. By using the repellency of agarose gel toward cell adhesion, patterned cultures of myoblasts and cortical neurons were successfully prepared. This technique is expected to be useful in repellency-guided cell patterning for various types of cells, with applications to cell–cell interactions and axon guidance.

Microfabricated device for co-culture of sympathetic neuron and iPS-derived cardiomyocytes

Induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CMs) has been expected as a cell source for therapy of serious heart failure. However, it is unclear whether the function of iPS-CMs is modulated by the host sympathetic nervous system. Here we developed a device for co-culture of sympathetic neurons and iPS-CMs using microfabrication technique. The device consisted of a culture chamber and a microelectrode-array (MEA) substrate. The superior cervical ganglion (SCG) neurons were co-cultured with iPS-CMs in a microfabricated device, which had multiple compartments. Several days after seeding, synapses were formed between SCG neurons and iPS-CMs, as confirmed by immunostaining. Spontaneous electrical activities of the SCG neurons and the iPS-CMs were observed from the electrode of the MEA substrate. The beat rate of iPS-CMs increased after electrical stimulation of the co-cultured SCG neurons. Such changes in the beat rate were prevented in the presence of propranolol, a β-adrenoreceptor antagonist. These results suggest that the microfabricated device will be utilized for studying the functional modulation of iPS-CMs by connected sympathetic neurons.

Autonomic nervous system driven cardiomyocytes in vitro

Rat superior cervical ganglia (SCG), which are sympathetic ganglia, neurons and ventricular myocytes (VMs) were co-cultured separately in a minichamber placed on a microelectrode-array (MEA) substrate. The minichamber was fabricated photolithographically and had 2 compartments, 16 microcompartments and 8 microconduits. The SCG neurons were seeded into one of the compartments and all of the microcompartments using a glass pipette controlled by a micromanipulator and a microinjector. The VMs were seeded into the other compartment. Three days after seeding of the VMs, the neurites of the SCG neurons had connected with the VMs via the microconduits. Electrical stimulations, trains of biphasic square pulses, were applied to the SCG neurons in the microcompartments using 16 electrodes. Evoked responses were observed in several electrodes while electrical stimulation was applied to the SCG neurons. According to the two-way analysis of variance (ANOVA), the beat rate after electrical stimulation was affected by the frequency and the number of the stimulation pulses. These results suggest that pulse number and the frequency of the electrical stimulation contribute to modulation of the beat rate of the cardiomyocytes.

Signal transfer within a cultured asymmetric cortical neuron circuit

OBJECTIVE Simplified neuronal circuits are required for investigating information representation in nervous systems and for validating theoretical neural network models. Here, we developed patterned neuronal circuits using micro fabricated devices, comprising a micro-well array bonded to a microelectrode-array substrate. APPROACH The micro-well array consisted of micrometre-scale wells connected by tunnels, all contained within a silicone slab called a micro-chamber. The design of the micro-chamber confined somata to the wells and allowed axons to grow through the tunnels bidirectionally but with a designed, unidirectional bias. We guided axons into the point of the arrow structure where one of the two tunnel entrances is located, making that the preferred direction. MAIN RESULTS When rat cortical neurons were cultured in the wells, their axons grew through the tunnels and connected to neurons in adjoining wells. Unidirectional burst transfers and other asymmetric signal-propagation phenomena were observed via the substrate-embedded electrodes. Seventy-nine percent of burst transfers were in the forward direction. We also observed rapid propagation of activity from sites of local electrical stimulation, and significant effects of inhibitory synapse blockade on bursting activity. SIGNIFICANCE These results suggest that this simple, substrate-controlled neuronal circuit can be applied to develop in vitro models of the function of cortical microcircuits or deep neural networks, better to elucidate the laws governing the dynamics of neuronal networks.

Functional innervation of human induced pluripotent stem cell-derived cardiomyocytes by co-culture with sympathetic neurons developed using a microtunnel technique

Microelectrode array (MEA) based-drug screening with human induced pluripotent stem cell-derived cardiomyocytes (hiPSCM) is a potent pre-clinical assay for efficiently assessing proarrhythmic risks in new candidates. Furthermore, predicting sympathetic modulation of the proarrhythmic side-effects is an important issue. Although we have previously developed an MEA-based co-culture system of rat primary cardiomyocyte and sympathetic neurons (rSNs), it is unclear if this co-culture approach is applicable to develop and investigate sympathetic innervation of hiPSCMs. In this study, we developed a co-culture of rSNs and hiPSCMs on MEA substrate, and assessed functional connections. The inter-beat interval of hiPSCM was significantly shortened by stimulation in SNs depending on frequency and pulse number, indicating functional connections between rSNs and hiPSCM and the dependency of chronotropic effects on rSN activity pattern. These results suggest that our co-culture approach can evaluate sympathetic effects on hiPSCMs and would be a useful tool for assessing sympathetic modulated-cardiotoxicity in human cardiac tissue.

Long term observation of propagating action potentials along the axon in a microtunnel device

The axon has a crucial role in the signal processing in the central nervous system. However, little is known about relationship between the structure of axons and conduction property of the axon. In this study, we developed a culture device to evaluate conduction velocity of relatively proximal (near the soma) and distal (far from the soma) parts of axons. The device has electrode units aligned at the bottom of the microtunnel structure. The electrode units enable the recording of action potentials of a single axon from three electrodes. Mouse cortical neurons were cultured in the device. The activity was recorded every five days. By employing an effective spike sorting method and a histogram method, propagating action potentials along the individual axon were successively detected from the spike trains which included the bursting activity. Finally, the change of the conduction delay at the distal part of axons was evaluated. The results show that our experimental system is feasible to study the conduction property of the axon.

Soft magnetic material based localized magnetic stimulation to cultured neuronal cells and modulation of network activities

Magnetic stimulation is able to modulate the neuronal network activity using the non-invasive magnetically induced current. However, it is unknown how stimulation modulates the neuronal network activity. Therefore, we considered that precise stimulation and evaluation of the modulation of network activities in the vicinity of stimulated sites is required. Here, to establish precisely magnetic stimulation, we developed a Mu-metal that has high magnetic permeability soft magnetic material based localized magnetic stimulation (LMS) system with micro-fabricated dual cell-culture chambers. And, combining this device with a microelectrode array (MEA) permitted the evaluation of the stimulus effects at the stimulated and non-stimulated sites. Here, the dual cell-culture chambers were arranged in a concentric circle manner. Between the inner and outer chambers, 4, 8 and 12 connecting microfluid channels were fabricated using polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in outer and inner chambers. Through the micro-conduits, functional synaptic connections were formed. Mu-metal was aligned along the outer circle, which allowed us of focal magnetic stimulation to the cells in the outer chamber. Applying low frequency magnetic field to the Mu-metal, induced currents were generated and the electrical activity of the cells in the outer chamber was modified depending on the stimulation intensity. Following the modified activity in the outer circles, the cells in the inner chamber also showed slightly depressed activity patterns. These results suggested that our system would be promising for highly regulated neural stimulation.

Neurogenesis Enhances Response Specificity to Spatial Pattern Stimulation in Hippocampal Cultures

Objective: Adult neurogenesis in the hippocampus facilitates cognitive functions such as pattern separation in mammals. However, it remains unclear how newborn neurons mediate changes in neural networks to enhance the pattern separation ability. Here, we developed an in vitro model of adult neurogenesis using rat hippocampal cultures in order to investigate whether newborn neurons can be directly incorporated into neural networks related to pattern separation to produce functional improvements. Method: We optimized at schedule of basic fibroblast growth factor (bFGF) administration to enhance neurogenesis, and then used a microelectrode array system to evaluate the responses of neural cultures to two different spatial pattern stimuli (L and inverted L shapes) before and after training. Results: We found that early synaptic response times to a given pattern were shortened after training, and that this effect was more pronounced in cultures treated with bFGF. Furthermore, bFGF-treated cultures showed improved response specificity after training as indicated by calculated Kullback–Leibler divergence values, suggesting that pattern separation was better achieved in cultures with enhanced neurogenesis. Conclusion: Neural networks containing greater numbers of immature neurons exhibited higher response specificity to spatial pattern stimulation, suggesting the improvement of the pattern separation by neurogenesis enhancement. Significance: These results are the first in vitro demonstration that neurogenesis improves pattern separation. Our novel in vitro system will be a useful tool for investigating the contribution of adult neurogenesis to cognitive functions.OBJECTIVE Adult neurogenesis in the hippocampus facilitates cognitive functions such as pattern separation in mammals. However, it remains unclear how newborn neurons mediate changes in neural networks to enhance pattern separation ability. Here, we developed an in vitro model of adult neurogenesis using rat hippocampal cultures in order to investigate whether newborn neurons can be directly incorporated into neural networks related to pattern separation to produce functional improvements. METHOD We optimized at schedule of basic fibroblast growth factor (bFGF) administration to enhance neurogenesis, and then used a microelectrode array system to evaluate the responses of neural cultures to two different spatial pattern stimuli (L and inverted L shapes) before and after training. RESULTS We found that early synaptic response times to a given pattern were shortened after training, and that this effect was more pronounced in cultures treated with bFGF. Furthermore, bFGF-treated cultures showed improved response specificity after training as indicated by calculated Kullback-Leibler divergence values, suggesting that pattern separation was better achieved in cultures with enhanced neurogenesis. CONCLUSION Neural networks containing greater numbers of immature neurons exhibited higher response specificity to spatial pattern stimulation, suggesting the improvement of the pattern separation by neurogenesis enhancement. SIGNIFICANCE These results are the first in vitro demonstration that neurogenesis improves pattern separation. Our novel in vitro system will be a useful tool for investigating the contribution of adult neurogenesis to cognitive functions.

Cell-cycle-dependent Ca2+ transients in human induced pluripotent stem cells revealed by a simultaneous imaging of cell nuclei and intracellular Ca2+ level

Cell cycle phase and [Ca2+]i are key determinants of self-renewal and differentiation in pluripotent stem cells. However, little is known about their relationship in human pluripotent stem cells owing to the lack of an effective method. Here, we applied an imaging-based approach for evaluating the relationship between the cell cycle and Ca2+ transients in human induced pluripotent stem (iPS) cells. Ca imaging and DNA staining was simultaneously performed at the same site. Then, individual cells were recognized and the cell cycle phase was estimated from the image of nuclei. We found that 18 ± 4% of human iPS cells exhibited spontaneous Ca2+ transients and their inter-transient interval was 119 ± 19 s. Ca wave events were observed in 64% of the sample and the [Ca2+]i elevation propagated among 47 ± 30 cells with a duration of 57 ± 22 s. With the imaging-based approach, we demonstrated that the ratio of cells exhibiting Ca2+ transients significantly decreased during cell cycle progression, suggesting that the relationship previously described in mouse cells holds true in the human context as well. These results suggest that our method is suitable for evaluating Ca2+ transients, the cell cycle phase, and their relationship with densely cultured cells.

Microfabricated multi-electrode device for detecting oligodendrocyte-regulated changes in axonal conduction velocity

Myelin disorders cause cognitive dysfunction, but little is known about how abnormal myelin sheath affects neural activities at the network level. One reason for the lack is a technical difficulty in simultaneous monitoring of changes in both the axonal conduction and network activity. Then, we aimed to develop a culture device to detect myelination dependent changes in axonal conduction velocity in a neuronal network. The photolithographically fabricated device has microtunnels for guiding axons. Two microelectrodes and an oligodendrocyte (OL) culture compartment are set at each microtunnel. This configuration allows us to monitor changes in conduction velocity of axons wrapped by OLs. Neurons and OLs dissected from rat cortical tissues were cultured in the culture device. An immunocytochemical study indicated axonal growth and maturation of OL at 42 days in vitro (DIV), suggesting that neuron-OL co-culture was maintained in microtunnels. Propagating action potentials of individual axons were detected from spontaneous neural activities with a spike sorting method and their conduction velocities were examined. Conduction velocity without seeding OLs was 0.31 m/s, which was consistent with that of previous reports with unmyelinated axons. Although no apparent myelin sheath was observed in OL culture compartments, conduction delay with seeding OLs was approximately half as long as that without seeding OLs at 45 DIV. These results suggest that the culture device enables us to detect the OL-regulated changes in axonal conduction in the neuronal network.