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Construction of three-dimensional 3D microenvironment become an important issue in recent biological studies due to their biological relevance compared to conventional two-dimensional 2D microenvironment. Various fabrication techniques have been employed to construct a 3D microenvironment, however, it is difficult to fully satisfy the biological and mechanical properties required for the 3D cell culture system, such as heterogeneous tissue structures generated from the functional differences or diseases.

We propose here an assembly method for facile construction of 3D microenvironment in a poly dimethylsiloxane PDMS channel using hydrogel units. The high-aspect-ratio of hydrogel units was achieved by fabricating these units using a 2D mold.

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With this approach, 3D heterogeneous hydrogel units were produced and assembled in a PDMS channel by structural hookup. In vivo-like 3D heterogeneous microenvironment in a precisely controllable fluidic system was also demonstrated using a controlled assembly of different types of hydrogel units, which was difficult to obtain from methods. By regulating the flow condition, the mechanical stability of the assembled hydrogel units was verified by the flow-induced deformation of hydrogel units.

In addition, in vivo-like cell culture environment was demonstrated using an assembly of cell-coated hydrogel units in the fluidic channel. Based on these features, our method expects to provide a beneficial tool for the 3D cell culture module and biomimetic engineering.


Three-dimensional 3D environment in a microfluidic channel has received much attention in both biology and bioengineering field since the demand for mimicking an in vivo like nature condition on a miniaturized system increases [ 1 ]. In contrast with standard flat cell culture, microfluidics-based cell culture systems provide advantages such as reduced sample and reagent consumption, precise control of culture environment and automated operation.

Furthermore, an artificial 3D environment can be used to improve the physiological relevance of cell or tissue and to reduce the gap between the in vitro system and the in vivo state, which emphasizes the importance of 3D environment [ 23 ]. These features overcome the limitations of conventional in vitro cell culture system and facilitate lots of applications such as in vitro biological study [ 4 — 6 ], drug development [ 7 — 9 ] and cancer research [ 10 — 12 ].

Assembly of hydrogel units for 3d microenvironment in a poly(dimethylsiloxane) channel

To date, various methods of microfabrication techniques have been applied to construct a microfluidic 3D environment based on the purpose, de and function of its device. Each of these techniques is available to construct a simple microfluidic 3D environment, however, it is difficult to obtain multiple criteria such as complex geometry, mechanical property, biocompatibility, usability and durability, which are crucial to the cell culture environment in a microfluidic system.

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Although the trapped cells can be easily applied for the facile supply and retrieval of media, reagents, and other chemicals, the lack of extracellular matrix that is indispensable for binding, interaction, and proliferation of cells causes a different behavior of the cultured cell compared with in vivo state.

In addition, structures and functions of hydrogel scaffolds are limited due to intrinsic characteristics of hydrogel materials.

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To obtain better mechanical properties, there is a certain loss of biocompatibility, and vice versa. Thus, a new method is needed to develop a new model for the construction of a 3D environment to overcome these limitations. Here, we present a novel method for the construction of a 3D environment by assembling 3D hydrogel units in a poly dimethylsiloxane PDMS channel.

In general, a high-aspect-ratio structure has been formed using a complex fabrication process based on the z axis 3D structure. In contrast, we deed our high-aspect-ratio structures on a two-dimensional 2D plane. After fabricating the planar structures, we formed 3D structures, whereby the planar structures stand vertically by geometrical fitting in a fluidic channel. In this study, we have mimicked villi structure of intestine as an example of high-aspect-ratio structures. We also investigated the mechanical durability of hydrogel units under the flow-induced mechanical stimulus in the hydrogel unit-assembled PDMS channel.

Finally, we demonstrated in vivo-like 3D environment by assembling cell-coated hydrogel units in the fluidic channel. The purpose of our platform is to construct an on-chip controllable 3D cell culture environment using a cell-culturable hydrogel and a supporting polymer channel.

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For the 3D cell culture environment under flow conditions, we developed an assembly method in which 3D hydrogel units can be geometrically intermeshed with a polymer channel device. In this study, we have mimicked a human intestinal microenvironment that has complex 3D structures under controlled flow conditions. As shown in Fig. De of hydrogel unit, fluidic channel and assembly process.

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Hydrogel units are assembled in a fluidic channel by intermeshing latch blocks and latch structures. After assembly, the fluidic channel is covered with a glass slide to seal the microfluidic environment.

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The hydrogel unit consists of three parts: basal part, villi and latch blocks Fig. The basal part is a long rectangular block, which plays a role as a structural backbone of hydrogel unit. Latch blocks at both ends of the basal part are protruded perpendicular to the basal part and villi structures. Villi are protruded from poly speed dating Midland basal part and become narrow toward the rounded-end of the villi. The fluidic channel is deed to be integrated with a hydrogel unit and used to build up a controllable fluidic condition Fig.

The fluidic channel consists of two parts: fluidic chamber and latch structure. Latch structures are located along the fluidic chamber at both sides as the shape of the long groove and the narrow slots. To fabricate protruded latch blocks on the basal part, we performed two-step photolithography. Compared to the molds used in other 3D structure studies, we exploited a conventional fabrication process to make high-aspect-ratio structures by patterning on a 2D plane.

This method makes it to produce various high-aspect-ratio structures by a simple photolithography process. The basal part and villi structures were patterned on a 2D plane y — z plane shown in Fig. The fabrication of a fluidic channel was performed in a similar way to the fabrication of hydrogel mold unit, however, several more steps were needed to make a deep fluidic chamber. After that, the ceiling of the fluidic chamber was cut off to make an open chamber with 1.

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Then, a flat PDMS plate was bonded on the opposite side of the latch structures to make a ceiling of the fluidic chamber. Rigid mechanical properties of alginate are suitable for the structural backbone of hydrogel units and collagen ensures enhanced cell adhesion and proliferation. To satisfy these properties, we set the final concentration of alginate as 1. The hydrogel mixture was loaded to a surface-treated PDMS mold using a pipette or a syringe.

Then, mM CaCl 2 solution dissolved in distilled water was nebulized on the mold for 2 min to crosslink alginate. Fully crosslinked hydrogel units were peeled off from the PDMS mold by mild pipetting. For the hydrogel visualization, 50 mM methylene blue solution was used. The culture media was replaced after 1 day of seeding and every 2 days in a culture period. To prepare the cell coating on a hydrogel unit, the cells were detached and concentrated to enhance the efficiency of cell coating.

To detach the cells from the dish, remove the media from poly speed dating Midland dish and wash with 10 mL of PBS. Then, 1 mL of 0. The detached cells were collected with 10 mL of media, centrifuged to collect cells in the bottom of poly speed dating Midland tube and to remove the supernatant media and trypsin—EDTA. This concentrated suspension of Caco-2 cells was incubated with prepared hydrogel units in a microtube overnight inside a cell culture incubator.

The assembly process of hydrogel units in a fluidic channel was carried out by intermeshing of the latch structure and the latch block. A schematic of the assembly process is shown in Fig. First, the hydrogel unit is aligned perpendicular to the direction of the fluidic channel. Then the latch blocks at both ends are fitted to groove and slot structures. The assembled latch blocks in the groove are structurally fixed by slot structures.

This geometrical locking between the fluidic channel and the hydrogel unit makes a structural support inside a channel and maintains its condition against the flow. The plasma-treated channel was filled with PBS, and then fabricated hydrogel units were assembled by being adjusted to enter into grooves and bridge structures. The spacing between the hydrogel units were controlled by the gap in the bridge structures. After the assembly process, the channel was covered with a glass slide and tightly sealed by weight blocks on an assembly jig.

To fabricate hydrogel villi structures using a hydrogel mixture, we used a replica molding technique without complicated or expensive fabrication processes. Several PDMS molds with laterally patterned villi structure were used to fabricate hydrogel villi structures having a long villi length with various aspect ratios. It is difficult to observe hydrogel villi structures under the bright field imaging using a conventional microscope due to its bright color and transparency.

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To demonstrate an easy fabrication for size-controllable hydrogel villi, we fabricated several types of hydrogel villus of different dimensions. This suggests that a conventional fabrication process for high-aspect-ratio structures would be circumvented by the simple fabrication process. Various size of the hydrogel villi structure on the PDMS mold. We verified that hydrogel villi were successfully fabricated irrespective of their long dimension and aspect ratio, which was limited by conventional methods.

The shrinking phenomenon of hydrogel scaffold was observed just after the nebulization of CaCl 2 for alginate crosslinking. After the mold was immersed in the media, however, the reduced hydrogel villi returned to their original size. A few minutes after the media supply, the hydrogel villi were tightly fitted to the mold. It seems that these phenomena resulted from the movement of water across hydrogel villi structure.

In this work, hydrogel villi structures were fabricated by an alginate—collagen mixture and the gelation of alginate was based on electrostatic crosslinking by calcium ion. Accordingly, the difference in the ion concentration between the hydrogel and the surrounding media causes an ion concentration gradient which induces the movement of water.

It seems poly speed dating Midland this movement of water causes swelling and shrinkage of the hydrogel villi structure. Each hydrogel unit was assembled in parallel to form a hydrogel villi array structure inside a fluidic chamber Fig. Methylene blue solution was used to aid the visualization of hydrogel units.

Each hydrogel unit was upright in the chamber and its shape remained stable under the flow in the chamber.

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