Yuya Yajima

Preparation of stripe-patterned heterogeneous hydrogel sheets using microfluidic devices for high-density coculture of hepatocytes and fibroblasts

Here we demonstrate the production of stripe-patterned heterogeneous hydrogel sheets for the high-density 3D coculture of multiple cell types, by using microchannel-combined micronozzle devices. The prepared hydrogel sheet, composed of multiple regions with varying physical stiffness, regulates the direction of proliferation of encapsulated cells and enables the formation of arrays of rod-like heterotypic organoids inside the hydrogel matrix. We successfully prepared stripe-patterned hydrogel sheets with a uniform thickness of ~100 μm and a width of several millimeters. Hepatoma cells (HepG2) and fibroblasts (Swiss 3T3) were embedded inside the hydrogel matrix and cocultured, to form heterotypic micro-organoids mimicking in vivo hepatic cord structures. The upregulation of hepatic functions by the 3D coculture was confirmed by analyzing liver-specific functions. The presented heterogeneous hydrogel sheet could be useful, as it provides relatively large, but precisely-controlled, 3-dimensional microenvironments for the high-density coculture of multiple types of cells.

Patterned hydrogel microfibers prepared using multilayered microfluidic devices for guiding network formation of neural cells

Multilayered microfluidic devices with a micronozzle array structure have been developed to prepare unique hydrogel microfibers with highly complex cross-sectional morphologies. Hydrogel precursor solutions with different compositions are introduced through vertical micronozzles, united and focused, and continuously gelled to form hydrogel fibers with multiple regions of different physicochemical composition. We prepared alginate hydrogel microfibers with diameters of 60 ~ 130 μm and 4/8 parallel regions in the periphery. Neuron-like PC12 cells encapsulated in the parallel region, which was made of a soft hydrogel matrix, proliferated and formed linear intercellular networks along the fiber length because of the physical restrictions imposed by the relatively rigid regions. After cultivation for 14 days, one-millimeter-long intercellular networks that structurally mimic complex nerve bundles found in vivo were formed. The proposed fibers should be useful for producing various in vivo linear tissues and should be applicable to regenerative medicine and physiological studies of cells.

Facile fabrication processes for hydrogel-based microfluidic devices made of natural biopolymers.

We present facile strategies for the fabrication of two types of microfluidic devices made of hydrogels using the natural biopolymers, alginate, and gelatin as substrates. The processes presented include the molding-based preparation of hydrogel plates and their chemical bonding. To prepare calcium-alginate hydrogel microdevices, we suppressed the volume shrinkage of the alginate solution during gelation using propylene glycol alginate in the precursor solution along with sodium alginate. In addition, a chemical bonding method was developed using a polyelectrolyte membrane of poly-L-lysine as the electrostatic glue. To prepare gelatin-based microdevices, we used microbial transglutaminase to bond hydrogel plates chemically and to cross-link and stabilize the hydrogel matrix. As an application, mammalian cells (fibroblasts and vascular endothelial cells) were cultivated on the microchannel surface to form three-dimensional capillary-embedding tissue models for biological research and tissue engineering.

Fabrication of multilayered vascular tissues using microfluidic agarose hydrogel platforms

Vascular tissues fabricated in vitro are useful tools for studying blood vessel‐related cellular physiologies and for constructing relatively large 3D tissues. An efficient strategy for fabricating vascular tissue models with multilayered, branched, and thick structures through the in situ hydrogel formation in fluidic channels is proposed. First, an aqueous solution of RGD‐alginate containing smooth muscle cells (SMCs) is introduced into channel structures made of agarose hydrogel, forming a cell‐embedding Ca‐alginate hydrogel layer with a thickness of several hundred micrometers on the channel surface because of the Ca2+ ions diffused from the agarose hydrogel matrix. Next, endothelial cells (ECs) are introduced and cultured for up to seven days to form hierarchically organized, multilayered vascular tissues. The factors affecting the thickness of the Ca‐alginate hydrogel layer, and prepared several types of microchannels with different morphologies are examined. The fabricated vascular tissue models are easily recovered from the channel by simply detaching the agarose hydrogel plates. In addition, the effect of O2 tension (20 or 80%) on the viability and elastin production of SMCs during the perfusion culture is evaluated. This technique would pave a new way for vascular tissue engineering because it enables the facile production of morphologically in vivo vascular tissue‐like structures that can be employed for various biomedical applications.

Shape control of cell-embedding hydrogel microstructures utilizing non-equilibrium aqueous two-phase systems

Here we demonstrate the fabrication of non-spherical hydrogel microstructures that are useful as building blocks for tissue engineering, by utilizing non-equilibrium aqueous two-phase systems (ATPS) in microchambers. A dextran (Dex) solution containing cells and hydrogel precursor was introduced into microfabricated chambers, which was gradually shrunk and deformed after pouring a polyethylene glycol (PEG) solution. By further adding a gelation agent, we successfully obtained non-spherical (toroidal or cup-shaped) cell-laden hydrogel microstructures. We were able to control the hydrogel morphology by using microchambers with different geometries, changing the compositions of the solutions, and modifying the surface property of the microchamber.

Microfluidics-based wet spinning of protein microfibers as solid scaffolds for 3D cell cultivation

Here we propose a facile and versatile process to fabricate cell-sized protein microfibers using microfluidic spinning system and sacrificial layers of alginate. A precursor solution containing protein molecules and sodium alginate (Na-Alg), a buffer solution, and a gelation solution were introduced into the microfluidic devices, forming composite microfibers comprising Ca-Alg and the protein. After chemically cross-linking the protein molecules and removing the alginate polymer, microfibers made of proteins were obtained. We demonstrated two processes, uniform fiber production using a simple microchannel and parallel production of narrow fibers using multilayered microfluidic devices. As an application, we cultured mammalian cells within hydrogel matrices incorporating gelatin microfibers, and evaluated the effects of the fibers on cell proliferation and network formation. The presented microfluidic process would be useful for fabricating cell-sized microfibers made of proteins which are applicable to 3D cell cultivation and tissue engineering.

One-step microfluidic spinning of collagen microfibers and their application to cell cultivation

Here we propose a simple but efficient method to produce collagen hydrogel microfibers using a microfluidic spinning system and a formation technique of collagen gels in a phosphate buffer. An aqueous solution of type I collagen was introduced into the microfluidic devices together with a phosphate buffer and distilled water. Parallel laminar flow was stably formed in the microchannel, and the collagen solution was transformed into microfibers with a uniform diameter. We examined factors affecting the fiber diameter and successfully obtained microfibers with a diameter from ~8 to ~30 μm. In addition, we confirmed that cells adhered on the surface of the microfibers and proliferated after culturing for several days. The presented method is advantageous because it enables the production of collagen microfibers under a wet condition without using complicated operations, organic solvents, or crosslinking reagents.