Sari Sugaya

Microfluidic synthesis of chemically and physically anisotropic hydrogel microfibers for guided cell growth and networking

Hydrogel materials with microscale heterogeneity are of great interest in the effort to spatially control cellular microenvironments in tissue engineering applications. Here we present a microfluidic system to continuously synthesize chemically and physically anisotropic Ca–alginate hydrogel microfibers enabling the guidance of cell proliferation to form linear cell colonies and intracellular networks. The microfluidic gelation process involves 2 critical steps to obtain alginate microfibers using axisymmetric microchannels with uniform depth: introduction of a buffer solution between the sodium alginate (NaA) and CaCl2 solutions to modulate the gelation speed, and use of a thickener to balance the viscosities of the solutions. We synthesized hydrogel fibers with diameters of ∼7 to 200 μm, maintaining the anisotropy in the cross-section, and examined factors affecting the fiber diameter and uniformity. Moreover, parallel alginate flows with and without propylene glycol alginate (PGA) enabled the formation of sandwich-type solid-soft-solid hydrogel fibers, which were used to guide the direction of growth of cells inoculated in the soft-core, with the help of outer polycation membranes made of poly-L-lysine. We demonstrated the formation of linear colonies of 3T3 and HeLa cells inside the anisotropic fiber and observed elongated nuclei along the fiber direction. In addition, the heterogeneous morphology of the fiber was utilized to guide neurite elongation and generate cellular networks by using neuron-like PC12 cells. The hydrogel fibers reported here can be used as an innovative tool for investigating cell and tissue morphogenesis in heterogeneous microenvironments, and for creating tissue models with precise control of cellular alignment and elongation.

Observation of nonspherical particle behaviors for continuous shape-based separation using hydrodynamic filtration

Selection of particles or cells of specific shapes from a complex mixture is an essential procedure for various biological and industrial applications, including synchronization of the cell cycle, classification of environmental bacteria, and elimination of aggregates from synthesized particles. Here, we investigate the separation behaviors of nonspherical and spherical particles∕cells in the hydrodynamic filtration (HDF) scheme, which was previously developed for continuous size-dependent particle∕cell separation. Nonspherical particle models were prepared by coating the hemisphere of spherical polymer particles with a thin Au layer and by bonding the Janus particles to form twins and triplets resembling dividing and aggregating cells, respectively. High-speed imaging revealed a difference in the separation behaviors of spherical and nonspherical particles at a branch point; nonspherical particles showed rotation behavior and did not enter the branch channel even when their minor axis was smaller than the virtual width of the flow region entering the branch channel, w(1). The confocal-laser high-speed particle intensity velocimetry system visualized the flow profile inside the HDF microchannel, demonstrating that the steep flow-velocity distribution at the branch point is the main factor causing the rotation behavior of nonspherical particles. As applications, we successfully separated spherical and nonspherical particles with various major∕minor lengths and also demonstrated the selection of budding∕single cells from a yeast cell mixture. We therefore conclude that the HDF scheme can be used for continuous shape-based particle∕cell separation.

Microfluidic production of single micrometer-sized hydrogel beads utilizing droplet dissolution in a polar solvent

In this study, a microfluidic process is proposed for preparing monodisperse micrometer-sized hydrogel beads. This process utilizes non-equilibrium aqueous droplets formed in a polar organic solvent. The water-in-oil droplets of the hydrogel precursor rapidly shrunk owing to the dissolution of water molecules into the continuous phase. The shrunken and condensed droplets were then gelled, resulting in the formation of hydrogel microbeads with sizes significantly smaller than the initial droplet size. This study employed methyl acetate as the polar organic solvent, which can dissolve water at 8%. Two types of monodisperse hydrogel beads-Ca-alginate and chitosan-with sizes of 6-10 μm (coefficient of variation < 6%) were successfully produced. In addition, we obtained hydrogel beads with non-spherical morphologies by controlling the degree of droplet shrinkage at the time of gelation and by adjusting the concentration of the gelation agent. Furthermore, the encapsulation and concentration of DNA molecules within the hydrogel beads were demonstrated. The process presented in this study has great potential to produce small and highly concentrated hydrogel beads that are difficult to obtain by using conventional microfluidic processes.

Micropatterning of hydrogels on locally hydrophilized regions on PDMS by stepwise solution dipping and in situ gelation.

This study presents a simple but highly versatile method of fabricating picoliter-volume hydrogel patterns on poly(dimethylsiloxane) (PDMS) substrates. Hydrophilic regions were prepared on hydrophobic PDMS plates by trapping and melting functional polymer particles and performing subsequent reactions with partially oxidized dextran. Small aliquots of a gelation solution were selectively trapped on the hydrophilic areas by a simple dipping process that was utilized to make thin hydrogel patterns by the in situ gelation of a sol solution. Using this process, we successfully formed calcium alginate, collagen I, and chitosan hydrogels with a thickness of several micrometers and shapes that followed the hydrophilized regions. In addition, alginate and collagen gel patterns were used to capture cells with different adhesion properties selectively on or off the hydrogel structures. The presented strategy could be applicable to the preparation of a variety of hydrogels for the development of functional biosensors, bioreactors, and cell cultivation platforms.

A droplet-based microfluidic process to produce yarn-ball-shaped hydrogel microbeads

A simple microfluidic process for producing yarn-ball-shaped hydrogel microbeads was devised. The mechanism employed parallel flows of aqueous solutions of a hydrogel precursor, a gelation agent, and a buffer to generate incompletely gelled alginate hydrogel microfibers. Water-in-oil droplets were generated simultaneously to fragment the fibers and fold the fragments into a yarn-ball-like shape. Alginate beads with an average outer diameter of ∼200 μm and fiber width of 10–30 μm were fabricated, which had a relatively large void volume and a high surface-to-volume ratio, enabling the efficient transport of molecules in and out of the hydrogel matrix. It was found that the concentration of the gelation agent was critical for obtaining the yarn-ball-shaped beads. In addition, to test the feasibility of the beads for use in bioencapsulation, mammalian cells were encapsulated within the hydrogel matrices of the beads at high densities, and the proliferation ability of the cells was investigated by changing the precursor concentration. The fabricated hydrogel beads should be useful as a new material for bioimmobilization and bioencapsulation and could find applications in cell transplantation therapies.

Manipulation of cells and cell spheroids using collagen hydrogel microbeads prepared by microfluidic devices

We present a manipulation technique of cells and cell spheroids by utilizing cell-size collagen hydrogel microbeads. Collagen hydrogel beads were prepared by using non-equilibrium microfluidic W/O droplets containing hydrogel molecules, formed in a continuous phase of a water-soluble organic solvent. Monodisperse collagen hydrogel beads with sizes smaller than 20 µm were produced, and their morphologies were controlled by changing the flow rates and the types of collagen molecules, and by adding cross-linking reagents. As applications to cell and cell spheroid manipulation, hydrogel beads containing magnetic nanoparticles were produced and then utilized to form cell-bead complexes and heterogeneous cell spheroids. The presented micrometer-size collagen hydrogel beads would be highly useful as new types of cell handling tools, cell cultivation matrices, and building blocks for tissue engineering.

Fabrication of functional hydrogel microbeads utilizing non-equilibrium microfluidics for biological applications

In this report, we present two processes to fabricate unique hydrogel beads, (1) yarn-ball-shape beads and (2) extremely-small beads, utilizing non-equilibrium microfluidics. First, to prepare the yarn-ball-shape microbeads, an incompletely-gelled Ca-alginate hydrogel fiber in a microchannel was cut into pieces and incorporated into water-in-oil droplets. Then, non-equilibrium W/O droplets, composed of water containing hydrogel molecules as the disperse phase and a water-soluble organic solvent as the continuous phase, were used to prepare extremely small hydrogel Ca-alginate hydrogel beads (Φ = ∼10 µm). As an application, high-density cell cultivation (∼1×108 cells/mL) in the yarn-ball-shape hydrogel beads was demonstrated. These hydrogel beads would be highly useful as unique carriers or matrices for biological immobilization, cultivation, and transplantation.

Morphology control of protein microparticles produced using microfluidic droplets in a non-equilibrium state

Here we present microfluidic systems to produce micrometer-sized particles made of proteins, using monodisperse droplets in a non-equilibrium state. Droplets of an aqueous solution of protein were formed in the continuous phase of a water-dissolving polar organic solvent, and then the droplets were shrunk because of the dissolution of water molecules. Protein molecules were precipitated, and then stable protein microparticles were obtained after the crosslinking reaction. We obtained spherical/non-spherical protein microparticles using several types of proteins, and examined factors affecting the particle morphology. In particular, highly unique tear-drop-shaped particles and hemispherical particles were obtained when incompletely dehydrated droplets were crosslinked. In addition, we cultured mammalian cells on the particles made of collagen, demonstrating the applicability of the particles as cell-culture scaffolds. The presented protein microparticles would be useful as carriers or scaffolds for various biological/medical applications.

Fluidic preparation of patterned hydrogel fibers using micronozzle-array devices for neural cell guidance

Micronozzle array-combined microfluidic devices have been developed to form anisotropically-patterned hydrogel fibers composed of soft and solid regions, which are highly useful to form linear cell colonies and neurite networks. The patterns of the vertical micronozzles determine the cross-sectional compositions of the Ca-alginate microfibers, with the widths of the fiber and the soft regions of 60~100 and 10~20 μm, respectively. Neuron-like cells (PC12 cells) were inoculated in the soft region of the fiber, which showed unprecedented behaviors of linear colony formation and guided neurite outgrowth, and cellular networks were formed. The presented anisotropic microfibers are highly useful as a useful material for nerve regeneration therapies.