Polyanion-induced, microfluidic engineering of fragmented collagen microfibers for reconstituting extracellular environments of 3D hepatocyte culture.

Artificial biological scaffolds made of extracellular matrix (ECM) components, such as type I collagen, provide ideal physicochemical cues to various cell culture platforms. However, it remains a challenge to fabricate micrometer-sized ECM materials with precisely controlled morphologies that could reconstitute the 3-dimensional (3D) microenvironments surrounding cells. In the present study, we proposed a unique process to fabricate fragmented collagen microfibers using a microfluidic laminar-flow system. The continuous flow of an acidic collagen solution was neutralized to generate solid fibers, which were subsequently fragmented by applying a gentle shear stress in a polyanion-containing phosphate buffer. The morphology of the fiber fragment was controllable in a wide range by changing the type and/or concentration of the polyanion and by tuning the applied shear stress. The biological benefits of the fragmented fibers were investigated through the formation of multicellular spheroids composed of primary rat hepatocytes and microfibers on non-cell-adhesive micro-vessels. The microfibers enhanced the survival and functions of the hepatocytes and reproduced proper cell polarity, because the fibers facilitated the formation of cell-cell and cell-matrix interactions while modulating the close packing of cells. These results clearly indicated that the microengineered fragmented collagen fibers have great potential to reconstitute extracellular microenvironments for hepatocytes in 3D culture, which will be of significant benefit for cell-based drug testing and bottom-up tissue engineering.

One-Step Formation of Microporous Hydrogel Sponges Encapsulating Living Cells by Utilizing Bicontinuous Dispersion of Aqueous Polymer Solutions.

With the recent progress in three-dimensional (3D) cell culture techniques for regenerative medicine and drug development, hydrogel-based tissue engineering approaches that can precisely organize cells into functional formats have attracted increasing attention. However, challenges remain in creating continuous microconduits within hydrogels to effectively deliver oxygen and nutrients to the embedded cells. Here we propose a one-step, fully liquid state, and all-aqueous process to create porous hydrogels that can encapsulate living cells without the need for extensive processing protocols, including the incorporation and removal of sacrificial materials. An unusual bicontinuous state of aqueous two-phase dispersion was utilized, and one of the two phases, encapsulating living cells, was rapidly photo-cross-linked to form hydrogel sponges. We optimized the volumetric mixing ratio of gelatin methacrylate (GelMA)-rich and polyethylene glycol (PEG)-rich solutions and investigated the effects of the formed continuous microconduits on the cell functions by creating liver-tissue mimetic 3D constructs. The presented technology provides a facile and versatile strategy for fabricating microstructured hydrogels for cell culture and would bring new insights for the development of porous materials by fully aqueous bicontinuous dispersions.

Formation of pressurizable hydrogel-based vascular tissue models by selective gelation in composite PDMS channels.

Vascular tissue models created in vitro are of great utility in the biomedical research field, but versatile, facile strategies are still under development. In this study, we proposed a new approach to prepare vascular tissue models in PDMS-based composite channel structures embedded with barium salt powders. When a cell-containing hydrogel precursor solution was continuously pumped in the channel, the precursor solution in the vicinity of the channel wall was selectively gelled because of the barium ions as the gelation agent supplied to the flow. Based on this concept, we were able to prepare vascular tissue models, with diameters of 1-2 mm and with tunable morphologies, composed of smooth muscle cells in the hydrogel matrix and endothelial cells on the lumen. Perfusion culture was successfully performed under a pressurized condition of ∼120 mmHg. The presented platform is potentially useful for creating vascular tissue models that reproduce the physical and morphological characteristics similar to those of vascular tissues in vivo.

Micropassage-embedding composite hydrogel fibers enable quantitative evaluation of cancer cell invasion under 3D coculture conditions.

Cell migration and invasion are of significant importance in physiological phenomena, including wound healing and cancer metastasis. Here we propose a new system for quantitatively evaluating cancer cell invasion in a three-dimensional (3D), in vivo tissue-like environment. This system uses composite hydrogel microfibers whose cross section has a relatively soft micropassage region and that were prepared using a multilayered microfluidic device; cancer cells are encapsulated in the core and fibroblasts are seeded in the shell regions surrounding the core. Cancer cell proliferation is guided through the micropassage because of the physical restriction imposed by the surrounding solid shell regions. Quantitative analysis of cancer cell invasion is possible simply by counting the cancer cell colonies that form outside the fiber. This platform enables the evaluation of anticancer drug efficacy (cisplatin, paclitaxel, and 5-fluorouracil) based on the degree of invasion and the gene expression of cancer cells (A549 cells) with or without the presence of fibroblasts (NIH-3T3 cells). The presented hydrogel fiber-based migration assays could be useful for studying cell behaviors under 3D coculture conditions and for drug screening and evaluation.

Development of a perfusable 3D liver cell cultivation system via bundling-up assembly of cell-laden microfibers.

Although the reconstruction of functional 3D liver tissue models in vitro presents numerous challenges, it is in great demand for drug development, regenerative medicine, and physiological studies. Here we propose a new approach to perform perfusion cultivation of liver cells by assembling cell-laden hydrogel microfibers. HepG2 cells were densely packed into the core of sandwich-type anisotropic microfibers, which were produced using microfluidic devices. The obtained microfibers were bundled up and packed into a perfusion chamber, and perfusion cultivation was performed. We evaluated cell viability and functions, and also monitored the oxygen consumption. Furthermore, fibers covered with vascular endothelial cells were united during the perfusion culture, to form vascular network-like conduits between fibers. The presented technique can structurally mimic the hepatic lobule in vivo and could prove to be a useful model for various biomedical research applications.

Collagen Microparticle-Mediated 3D Cell Organization: A Facile Route to Bottom-up Engineering of Thick and Porous Tissues.

In closely packed artificial 3D cellular constructs, cells located near the center of the constructs are not functional because of the limited supply of oxygen and nutrition. Here we describe a simple, unique, and highly versatile approach to organizing cells into thick but porous 3D tissues, using cell-sized collagen microparticles as particulate scaffolds. When cells and particles are mixed and seeded in a noncell-adhesive planar chamber, they gather to form sheet-shaped structures with a thickness of 100-150 µm. In the construct, uniformly distributed particles work as a binder between cells and modulate the strong intercellular contraction. We confirmed that several factors, including the particle/cell ratio and particle size, critically affect the stability and shrink behaviors of porous tissues prepared using mouse embryonic fibroblasts (NIH-3T3 cells). Cross-sectional observation, together with cell proliferation and viability assays, revealed that the cells composing the tissues are functional primarily because interior pores between cells/particles worked as a path for efficient molecular transport. Furthermore, we prepared thick cell tissues of a liver model using human hepatocarcinoma cells (HepG2 cells), and confirmed that liver-specific functions were upregulated when composite tissues were formed using collagen microparticles prepared with several different stabilization protocols by glutaraldehyde, genipin, and methyl acetate). The process presented would be highly useful in enabling one-step production of thick cellular constructs in which porosity and morphology are tunable.

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.

Cell-sized condensed collagen microparticles for preparing microengineered composite spheroids of primary hepatocytes.

The reconstitution of extracellular matrix (ECM) components in three-dimensional (3D) cell culture environments with microscale precision is a challenging issue. ECM microparticles would potentially be useful as solid particulate scaffolds that can be incorporated into 3D cellular constructs, but technologies for transforming ECM proteins into cell-sized stable particles are currently lacking. Here, we describe new processes to produce highly condensed collagen microparticles by means of droplet microfluidics or membrane emulsification. Droplets of an aqueous solution of type I collagen were formed in a continuous phase of polar organic solvent followed by rapid dissolution of water molecules into the continuous phase because the droplets were in a non-equilibrium state. We obtained highly unique, disc-shaped condensed collagen microparticles with a final collagen concentration above 10% and examined factors affecting particle size and morphology. After testing the cell-adhesion properties on the collagen microparticles, composite multicellular spheroids comprising the particles and primary rat hepatocytes were formed using microfabricated hydrogel chambers. We found that the ratio of the cells and particles is critical in terms of improvement of hepatic functions in the composite spheroids. The presented methodology for incorporating particulate-form ECM components in multicellular spheroids would be advantageous because of the biochemical similarity with the microenvironments in vivo.

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.

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.

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.