Tatsuya Osaki

Acceleration of Vascular Sprouting from Fabricated Perfusable Vascular-Like Structures

Fabrication of vascular networks is essential for engineering three-dimensional thick tissues and organs in the emerging fields of tissue engineering and regenerative medicine. In this study, we describe the fabrication of perfusable vascular-like structures by transferring endothelial cells using an electrochemical reaction as well as acceleration of subsequent endothelial sprouting by two stimuli: phorbol 12-myristate 13-acetate (PMA) and fluidic shear stress. The electrochemical transfer of cells was achieved using an oligopeptide that formed a dense molecular layer on a gold surface and was then electrochemically desorbed from the surface. Human umbilical vein endothelial cells (HUVECs), adhered to gold-coated needles (ϕ600 μm) via the oligopeptide, were transferred to collagen gel along with electrochemical desorption of the molecular layer, resulting in the formation of endothelial cell-lined vascular-like structures. In the following culture, the endothelial cells migrated into the collagen gel and formed branched luminal structures. However, this branching process was strikingly slow (>14 d) and the cell layers on the internal surfaces became disrupted in some regions. To address these issues, we examined the effects of the protein kinase C (PKC) activator, PMA, and shear stress generated by medium flow. Addition of PMA at an optimum concentration significantly accelerated migration, vascular network formation, and its stabilization. Exposure to shear stress reoriented the cells in the direction of the medium flow and further accelerated vascular network formation. Because of the synergistic effects, HUVECs began to sprout as early as 3 d of perfusion culture and neighboring vascular-like structures were bridged within 5 d. Although further investigations of vascular functions need to be performed, this approach may be an effective strategy for rapid fabrication of perfusable microvascular networks when engineering three-dimensional fully vascularized tissues and organs.

Rapid engineering of endothelial cell-lined vascular-like structures in in situ crosslinkable hydrogels

Fabrication of perfusable vascular networks in vitro is one of the most critical challenges in the advancement of tissue engineering. Because cells consume oxygen and nutrients during the fabrication process, a rapid fabrication approach is necessary to construct cell-dense vital tissues and organs, such as the liver. In this study, we propose a rapid molding process using an in situ crosslinkable hydrogel and electrochemical cell transfer for the fabrication of perfusable vascular structures. The in situ crosslinkable hydrogel was composed of hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified hyaluronic acid (HA-CHO). By simply mixing these two solutions, the gelation occurred in less than 20 s through the formation of a stable hydrazone bond. To rapidly transfer cells from a culture surface to the hydrogel, we utilized a zwitterionic oligopeptide, which forms a self-assembled molecular layer on a gold surface. Human umbilical vein endothelial cells adhering on a gold surface via the oligopeptide layer were transferred to the hydrogel within 5 min, along with electrochemical desorption of the oligopeptides. This approach was applicable to cylindrical needles 200-700 µm in diameter, resulting in the formation of perfusable microchannels where the internal surface was fully enveloped with the transferred endothelial cells. The entire fabrication process was completed within 10 min, including 20 s for the hydrogel crosslinking and 5 min for the electrochemical cell transfer. This rapid fabrication approach may provide a promising strategy to construct perfusable vasculatures in cell-dense tissue constructs and subsequently allow cells to organize complicated and fully vascularized tissues while preventing hypoxic cell injury.

Tissue engineering based on electrochemical desorption of an RGD-containing oligopeptide.

This paper describes a non‐invasive approach for efficient detachment of cells adhered to a gold substrate via a specific oligopeptide. Detachment is effected by an electrical stimulus. The oligopeptide contains cysteine, which spontaneously forms a gold–thiolate bond on a gold surface. This chemical adsorption reaches > 95% equilibrium within 10 min after immersion of a gold‐coated substrate in a solution containing the peptide. The peptide is reversibly desorbed from the surface within 5 min of application of a negative electrical potential. By taking advantage of this simple adsorption and desorption mechanism, cells can be grown on an oligopeptide‐functionalized gold surface and can be efficiently detached as single cells or cell sheets by application of a negative electrical potential. This approach was also applied to the surface of gold‐coated microrods. Capillary‐like microchannels were formed in collagen gel by transferring endothelial cells to the internal surfaces of the microchannels. During subsequent perfusion culture, the enveloped endothelial cells migrated into the collagen gel and formed luminal structures, which sprouted from the microchannels. This technique has the potential to provide a fundamental tool for the engineering of thick cell sheets as well as vascularized tissues and organs. Copyright

Engineering thick cell sheets by electrochemical desorption of oligopeptides on membrane substrates

We developed a gold-coated membrane substrate modified with an oligopeptide layer that can be used to grow and subsequently detach a thick cell sheet through an electrochemical reaction. The oligopeptide CCRRGDWLC was designed to contain a cell adhesive domain (RGD) in the center and cysteine residues at both terminals. Cysteine contains a thiol group that forms a gold–thiolate bond on a gold surface. Cells attached to gold-coated membrane substrates via the oligopeptide layer were readily and noninvasively detached by applying a negative electrical potential to cleave the gold–thiolate bond. Because of the effective oxygen supply, fibroblasts vigorously grew on the membrane substrate and the thickness of the cell sheets was ∼60 μm at 14 days of culture, which was 2.9-fold greater than that of cells grown on a conventional culture dish. The cell sheets were detached after 7 min of electrical potential application. Using this approach, five layers of cell sheets were stacked sequentially with thicknesses reaching >200 μm. This approach was also beneficial for rapidly and readily transplanting cell sheets. Grafted cell sheets secreted collagen and remained at the transplanted site for at least 2 months after transplantation. This simple electrochemical cell sheet engineering technology is a promising tool for tissue engineering and regenerative medicine applications.

In Vitro Microfluidic Models for Neurodegenerative Disorders

Microfluidic devices enable novel means of emulating neurodegenerative disease pathophysiology in vitro. These organ-on-a-chip systems can potentially reduce animal testing and substitute (or augment) simple 2D culture systems. Reconstituting critical features of neurodegenerative diseases in a biomimetic system using microfluidics can thereby accelerate drug discovery and improve our understanding of the mechanisms of several currently incurable diseases. This review describes latest advances in modeling neurodegenerative diseases in the central nervous system and the peripheral nervous system. First, this study summarizes fundamental advantages of microfluidic devices in the creation of compartmentalized cell culture microenvironments for the co-culture of neurons, glial cells, endothelial cells, and skeletal muscle cells and in their recapitulation of spatiotemporal chemical gradients and mechanical microenvironments. Then, this reviews neurodegenerative-disease-on-a-chip models focusing on Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis. Finally, this study discusses about current drawbacks of these models and strategies that may overcome them. These organ-on-chip technologies can be useful to be the first line of testing line in drug development and toxicology studies, which can contribute significantly to minimize the phase of animal testing steps.

Catch-and-Release of Target Cells Using Aptamer-Conjugated Electroactive Zwitterionic Oligopeptide SAM

Nucleic acid aptamers possess attractive features such as specific molecular recognition, high-affinity binding, and rapid acquisition and replication, which could be feasible components for separating specific cells from other cell types. This study demonstrates that aptamers conjugated to an oligopeptide self-assembled monolayer (SAM) can be used to selectively trap human hepatic cancer cells from cell mixtures containing normal human hepatocytes or human fibroblasts. Molecular dynamics calculations have been performed to understand how the configurations of the aptamers are related to the experimental results of selective cell capture. We further demonstrate that the captured hepatic cancer cells can be detached and collected along with electrochemical desorption of the oligopeptide SAM, and by repeating these catch-and-release processes, target cells can be enriched. This combination of capture with aptamers and detachment with electrochemical reactions is a promising tool in various research fields ranging from basic cancer research to tissue engineering applications.

Engineered 3D vascular and neuronal networks in a microfluidic platform

Neurovascular coupling plays a key role in the pathogenesis of neurodegenerative disorders including motor neuron disease (MND). In vitro models provide an opportunity to understand the pathogenesis of MND, and offer the potential for drug screening. Here, we describe a new 3D microvascular and neuronal network model in a microfluidic platform to investigate interactions between these two systems. Both 3D networks were established by co-culturing human embryonic stem (ES)-derived MN spheroids and endothelial cells (ECs) in microfluidic devices. Co-culture with ECs improves neurite elongation and neuronal connectivity as measured by Ca2+ oscillation. This improvement was regulated not only by paracrine signals such as brain-derived neurotrophic factor secreted by ECs but also through direct cell-cell interactions via the delta-notch pathway, promoting neuron differentiation and neuroprotection. Bi-directional signaling was observed in that the neural networks also affected vascular network formation under perfusion culture. This in vitro model could enable investigations of neuro-vascular coupling, essential to understanding the pathogenesis of neurodegenerative diseases including MNDs such as amyotrophic lateral sclerosis.

Processing of nanolitre liquid plugs for microfluidic cell-based assays

Abstract Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.

Flatbed epi relief-contrast cellular monitoring system for stable cell culture

Consistent cell preparation is a fundamental preliminary step for understanding complex cellular mechanisms in various cell-based research fields, including basic cell biology, cancer research, and tissue engineering. However, certain elusive factors, such as cellular de-differentiation and contamination with mycoplasma or other types of cells, have compromised the reproducibility and reliability of cell-based approaches. Here, we propose an epi relief-contrast cellular monitoring system (eRC-CMS) that allows images of cells in a typical culture plate to be acquired, stored, and analysed for daily cell quality control. Due to its full flatbed nature and automated system, cells placed at any location on the stage can be analysed without special attention. Using this system, changes in the size, circularity, and proliferation of endothelial cells in subculture were recorded. Analyses of images of ~9,930,000 individual cells revealed that the growth activity and cell circularity in subcultures were closely correlated with their angiogenic activity in a subsequent hydrogel assay, demonstrating that eRC-CMS is useful for assessing cell quality in advance. We further demonstrated that eRC-CMS was feasible for the imaging of neurite elongation and spheroid formation. This system may provide a robust and versatile approach for daily cell preparation to facilitate reliable and reproducible cell-based studies.

Electrical detachment of cells for engineering capillary-like structures in a photocrosslinkable hydrogel

A major challenge in tissue engineering is the fabrication of vascular networks capable of delivering oxygen and nutrients throughout tissue constructs. Because cells located more than a few hundred micrometers away from the nearest capillaries are susceptible to oxygen shortages, it is crucial to develop microscale technologies for engineering a vascular structure in three-dimensionally thick tissues. This study describes an electrochemical approach for fabricating capillary-like structures precisely aligned within micrometer distances, the internal surfaces of which are covered with vascular endothelial cells in a photocrosslinkable hydrogel.