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.
Hydrogels that can be rapidly cross-linked under physiological conditions are beneficial for the engineering of vascularized 3-dimensional (3D) tissues and organs, in particular when cells are embedded at a high cell density or tissues are fabricated using bottom-up processes, including bioprinting and micromolding. Here, we prepared a gelatin-carboxymethylcellulose (CMC) hydrogel that cross-linked rapidly (<30 s) by mixing hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified CMC (CMC−CHO). Vascular endothelial cells encapsulated in the gelatin-CMC hydrogels were viable and sprouted readily, indicating that the hydrogels and their crosslinking reactions were cytocompatible and provided a suitable microenvironment for angiogenesis. Sprouting length of the vascular endothelial cells was modulated by altering the stiffness of the hydrogels and varying the concentrations of the two hydrogel components. Furthermore, we used an electrochemical reaction to detach cells from a gold electrode surface. In this approach, cells that were seeded on a gold surface via the oligopeptide layer, detached rapidly along with the electrochemical desorption of the layer and transferred to the hydrogel. Owing to the rapid gelation of the hydrogels and rapid electrochemical detachment of cells, cell transfer was completed within 10 min (including 30 s of gelation and 5 min of potential application). Rapid cell transfer was observed not only on a flat surface but also on different shapes, such as cylindrical needles. Vascular endothelial cells were transferred from needles onto the hydrogel to fabricate endothelial cell-enveloped microchannels. In subsequent perfusion culture, the transferred endothelial cells migrated and formed luminal structures in the hydrogel. This in situ cross-linkable hydrogel may be useful for the rapid fabrication of perfusable vascular networks to engineer vascularized and cell-dense 3D tissues and organs.
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.
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.
Super-dense transfected cell microarrays (TCMs) were created by a piezoelectric inkjet printer on a glass substrate that had been grafted with poly(ethylene glycol) (PEG). The micro-spots that contained plasmid and extra-cellular matrix (ECM) protein were separated from one another by a hydrophilic barrier generated by PEG. We successfully constructed the densest TCMs with spots of 50 μm in diameter and 150 μm in pitch.
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