HYDROGEL-BASED MICROFLUIDIC CHIP FOR CO-CULTURING CELLS

Abstract

Provided are a hydrogel-based microfluidic chip for cell co-culture and a use thereof, wherein the microfluidic chip allows the co-culture of cancer cells and vascular endothelial cells; can be widely applied in various studies associated with cancer; is suitable in studies on the photothermal therapy effect on, especially, cancer cells; and has excellent biocompatibility, mechanical properties, and economical feasibility.

Claims

1. A microfluidic chip for co-culture of cancer cells comprising: (a) one or more microchambers as cell culture sections, including sample inlets; (b) bridge channels connected to the microchambers; and (c) a microfluidic channel connected to the bridge channels and including a hydrogel inlet, wherein the microfluidic chip comprises a barrier formed by hydrogels and vascular endothelial cells, wherein the hydrogels comprise gelatin-acryl polymer prepared by mixing gelatin and an acryl polymer, wherein the hydrogels and the vascular endothelial cells are injected through the hydrogel inlet.

2. The microfluidic chip of claim 1, wherein the acryl polymer is selected from the group consisting of an acrylate and methacrylate copolymer, a methacrylate copolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylate copolymer, a cyanoethyl methacrylate copolymer, an aminoalkyl methacrylate copolymer, a poly(acrylate) copolymer, a polyacrylamide copolymer, a glycidyl methacrylate copolymer and a mixture thereof.

3. The microfluidic chip of claim 1, wherein the hydrogels comprise a 5-15 wt % concentration of gelatin-acryl polymer.

4. The microfluidic chip of claim 1, wherein the gelatin and the acryl polymer are photo-crosslinked in the hydrogels.

5. The microfluidic chip of claim 1, wherein the microfluidic chip is fabricated by using a polymer material selected from the group consisting of poly(dimethylsiloxane) (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins, polyimides and polyurethanes.

6. The microfluidic chip of claim 1, wherein the microfluidic chip is joined to an upper portion of a plate facilitating optical measurement, which is selected from the group consisting of slide glass, crystal and glass.

7. The microfluidic chip of claim 1, wherein the microchambers are arranged in one or more columns and one or more rows.

8. A method for cell co-culture comprising: (a) preparing a microfluidic chip for cell co-culture, comprising: (i) one or more microchambers as cell culture sections, including sample inlets; (ii) bridge channels connected to the microchambers; and (iii) a microfluidic channel connected to the bridge channels and including a hydrogel inlet; (b) preparing hydrogels that comprise gelatin-acryl polymer prepared by mixing gelatin and an acryl polymer; (c) injecting hydrogels and vascular endothelial cells into the hydrogel inlet, (d) inducing photo-crosslinking to construct a barrier; and (e) injecting cancer cells into the sample inlets, followed by culturing.

9. A method for analyzing a photothermal therapy effect on cancer cells comprising: (a) preparing a microfluidic chip for cell co-culture comprising: (i) one or more microchambers as cell culture sections including sample inlets; (ii) bridge channels connected to the microchambers; and (iii) a microfluidic channel connected to the bridge channels and including a hydrogel inlet; (b) preparing hydrogels that comprise gelatin-acryl polymer prepared by mixing gelatin and an acryl polymer; (c) injecting the hydrogels and vascular endothelial cells into the hydrogel inlet, (d) inducing photo-crosslinking to construct a barrier; (e) injecting cancer cells through the sample inlets, followed by culturing; (f) injecting nanoparticles exhibiting a photothermal effect through the sample inlets, followed by culturing; and (g) irradiating a laser to the microchambers to analyze the extent of survival or death of the cancer cells.

10. The method of claim 9, wherein the nanoparticles are gold nanorods.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 shows a gelatin methacrylate hydrogel-based microfluidic chip for co-culture: (A) Schematic diagram of gelatin methacrylate hydrogel-based microfluidic chip for co-culture, including a microfluidic channel and microchambers; and (B) Image of gelatin methacrylate hydrogel-based microfluidic chip for co-culture;

[0052] FIGS. 2A to 2C show SEM images of 5 w/v %, 15 w/v %, and 25 w/v % photo-crosslinkable GelMA hydrogels. Scale bars indicate 20 m;

[0053] FIGS. 3A and 3B show effects of GelMA hydrogel concentrations (5-25 w/v %). FIGS. 3A and 3B show pore size and aspect ratio, respectively. The aspect ratio means the value of the length of pores divided by width of pores (*p<0.05, **p<0.01);

[0054] FIG. 4 shows analysis results of 10 w/v % gelatin methacrylate hydrogels for barrier and cell encapsulation: (A) SEM image of 10 w/v % gelatin methacrylate hydrogels; (B) fluorescent images of molecular diffusion of four square-shaped microchambers (Left-up (LU), Right-up (RU), Left-down (LD), and Right-Down (RD)). Rhodamine B-dextran was only injected into RU microchamber, and was diffused to LD microchamber. (C) Analysis graph of the molecular diffusion through 10 w/v % gelatin methacrylate hydrogels for 1 day and 5 days;

[0055] FIG. 5 shows synthesis results of gold nanorods. (A) TEM image of synthesized gold nanorods; (B) UV-visible spectrum results of gold nanorods stabilized with CTAB; and (C) Schematic diagram of injection of synthesized gold nanoparticles into square-shaped microchambers;

[0056] FIG. 6 shows analysis results of photothermal therapy effect of gold nanorods. (A) Analysis of temperature increase depending on gold nanorod concentration after NIR laser irradiation (808 nm, 7 W); (B) CCK-8 live/dead assay graphs of photothermal therapy effects on glioblastoma cells and breast cancer cells in 96-well plate; and (C) live/dead assay fluorescent images of glioblastoma cells and breast cancer cells in co-culture microfluidic chip; and

[0057] FIG. 7 shows confocal microscopic images with respect to metastasis of cancer cells. (A) Schematic diagram of hydrogel-based co-culture microfluidic chip for study of cancer cell metastasis; (B) Confocal microscopic image of MCF7 cells; (C) Confocal microscopic image of U87MG cells on glass substrate; (D) Confocal microscopic image of U87MG cells metastasized to GelMA barrier from chamber in device; (E) Confocal microscopic image of GelMA barrier chamber containing metastatic U87MG cells; (F) Confocal microscopic image of MCF7 cells cultured in chamber; and (G) High-magnification confocal microscopic image of bridge channel containing U87MG cells metastasized to GelMA barrier from chamber in device.

MODE FOR CARRYING OUT THE INVENTION

[0058] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Materials and Methods

Fabrication of 3D Microfluidic Co-Culture Device

[0059] The microchambers and bridge channels were manufactured by two-step photolithography methods known in the art. To fabricate 3D microfluidic co-culture device, microchambers and bridge channels were designed by AutoCAD program. To manufacture bridge channels, SU-8 25 photoresist was spin-coated on a silicon wafer (1,000 rpm, 60 s, and 40 m in thickness). To manufacture microchambers, SU-8 100 was spin-coated on SU-8 25 photoresist-patterned substrates (3,000 rpm, 60 s, and 250 m in thickness). The poly(dimethylsiloxane) (PDMS) precursor solution was molded from the photoresist-patterned silicon wafer, and PDMS-based 3D microfluidic culture device was bonded into glass slides using oxygen plasma treatment (Femto Science, Korea).

[0060] The microfluidic chip including four square-shaped microchambers (Left-up (LU), Right-up (RU), Left-down (LD), and Right-Down (RD)) and a cruciform microfluidic channel connected to bridge microfluidic channels. The four square-shaped microchambers (250 m in thickness) are connected by the bridge microchannels (40 m in thickness), and the bridge microchannels are connected with the cruciform microfluidic channel (250 m in thickness). The cruciform microfluidic channel was manufactured in order to prevent the encapsulation of vascular endothelial cells in gelatin methacrylate hydrogels and the molecular diffusion between square-shaped microchambers, and the bridge microchannels were designed to increase the resistance of fluid. Resultantly, the gelatin methacrylate hydrogels were crosslinked by UV light in only the cruciform microfluidic channel, and breast cancer cells and glioblastoma cells were injected across each other in the square microchambers. Then, the molecular diffusion effect of 10 w/v % gelatin methacrylate hydrogels was investigated. By injecting rhodamine B-dextran into RU microchamber, the molecular diffusion of rhodamine B-dextran to LD microchamber was verified, and the molecular diffusion of gelatin methacrylate hydrogels was verified for 1 day and 5 days. Therefore, the gelatin methacrylate hydrogels were used for cell encapsulation and a barrier in the cruciform microfluidic channel.

Gelatin Methacrylate (GelMA) Hydrogels Synthesis

[0061] For the photo-crosslinkable GelMA hydrogels, type A porcine skin gelatin was stirred at 50 C. and phosphate buffered saline (PBS, GIBCO, USA) was mixed until fully dissolved. Methacrylic anhydride was added at a rate of 0.5 mL/min under stirring conditions for 2 h. The mixture was dialyzed against distilled water using 12-14 kDa-cutoff dialysis tubing for 3-4 days at 40 C. to remove salts and methacrylic acids. The solution was lyophilized for 1 week and was subsequently stored at 80 C.

Gold Nanorod Synthesis

[0062] Gold nanorods were synthesized by the seed-growth method. First, the seed solution was prepared by adding 0.25 mL of 0.01 M aqueous HAuCl.sub.4 solution and 0.6 mL of 0.01 M NaBH4 solution to 7.5 mL of 0.1 M CTAB solution. Here, the seed solution was stabilized at room temperature for 2 hours or longer before use. The growth solution was prepared by adding 0.2 ml of 0.01 M HAuCl.sub.4, 0.03 mL of 0.01 M AgNO3, and 0.032 mL of 1 M ascorbic acid to 4.75 mL of 0.1 M CTAB. 0.01 mL of the prepared seed solution was added to the growth solution, and then the mixture was stabilized at room temperature for 3 hours or longer, thereby synthesizing gold nanorods.

Scanning Electron Microscope

[0063] The structure of GelMA hydrogels was analyzed by using a scanning electron microscope (SEM). The swollen hydrogels were frozen and were subsequently lyophilized. The lyophilized samples were cut and their cross-sections were coated with platinum using a turbo sputter coater (EMITECH, K575X). SEM images were obtained at a high voltage of 30 kV.

Culture of Cancer Cells

[0064] Endothelial cells were cultured together with an endothelial cell culture medium (EGM2+Single Quot Kit Components, Lonza, Switzerland) in flask coated with 2% gelatin, and breast cancer cells (MCF7) and glioblastoma cells (U87MG) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Loading of GelMA Hydrogels and Cell-Encapsulated Collagen Gels

[0065] To culture vascular endothelial cells in a 3D manner, 210.sup.6 cells/mL were suspended and encapsulated within 100 L of the GelMA hydrogel solution. Of these, 20 L of endothelial cell-encapsulated GelMA hydrogel solution was injected into the cruciform channel. Through UV irradiation for 20 seconds, the GelMA hydrogels form a barrier in the microfluidic chip by photo-crosslinkage. Then, 210.sup.6 cells/mL of MCF7 cells and U87MG cells were injected into square-shaped LU, RU, RD, and LD chambers, crossing each other, together with 10 L of culture medium.

Analysis of Photothermal Therapy Effect

[0066] The cells were injected into the chambers, and cultured for 1 day to make cells adhere to the chambers, and 20, 30, and 40 l of gold nanorods were mixed with 200 l of the cell culture medium, and the mixture was injected through the chamber inlet, followed by NIR laser irradiation, and then the temperature increase was investigated. In addition, glioblastoma cells and breast cancer cells were cultured in the chip for one day, and in a similar manner, the cells were treated by NIR laser irradiation and analyzed by live/dead assay.

[0067] The live/dead assay was carried out through the following method. The breast cancer cells and glioblastoma cells were injected at 110.sup.5 into the 96-well plate and the microchambers. One day after cell injection, the cell culture medium was exchanged with a cell culture medium containing 15 v/v %, and placed for about 6 hours in a cell incubator. Then, NIR was irradiated to the chambers and the 96-well plate. Resultantly, the cell viability was analyzed by CCK-8 (cell-counting kit-8, USA) in the 96-well plate (FIG. 4b), and analyzed through fluorescence using a confocal microscope by live/dead assay (invitrogen, USA) in the microchambers (FIG. 4c).

Results and Discussion

Fabrication of GelMA Hydrogel-Based 3D Microfluidic Co-Culture Device

[0068] We developed the photo-crosslinkable GelMA hydrogel-based 3D microfluidic culture device (FIG. 1). The GelMA hydrogel-based 3D microfluidic device was fabricated by a two-step photolithography process to be composed of four microchambers and a cruciform microfluidic channel connected to bridge microchannels (FIG. 1C). The four microchambers (250 m in thickness) were connected by microgrooved bridge microchannels (40 m in thickness) (FIG. 1C).

[0069] The 250 m-thick microchambers were filled with vascular endothelial cell-encapsulated GelMA hydrogels, breast cancer calls, and glioblastoma cells. The 40 m-thick microgrooved bridge channels increased the fluidic resistance. GelMA hydrogels were photo-crosslinked via UV in the cruciform microchannel. The cruciform photo-crosslinked GelMA hydrogels in the microchamber function as a physical barrier to inhibit the molecular diffusion across bridge microchannels, thereby allowing culture of vascular endothelial cells. Then, breast cancer cells and glioblastoma cells were injected while crossing each other. This multi-compartment microfluidic culture device has many advantages in cellular interaction and high-throughput drug screening, but in the previous microfluidic co-culture device, the photothermal therapy and the photo-crosslinkable hydrogel-based 3D microfluidic device for co-culture of cancer cells were not considered.

Effects of GelMA Hydrogel Concentration on Porosity and Molecular Diffusion

[0070] As a result of verifying the effect of GelMA hydrogel concentration on the porosity, the pore size was inversely proportional to GelMA hydrogel concentration (FIG. 2). SEM images indicate that the porosity of 25 w/v % GelMA hydrogels showed uniform sizes and shapes compared to 5 w/v % GelMA hydrogels (FIGS. 2a to 2c). The pore size of 5 w/v % GelMA hydrogels was 34 m, whereas the pore size of 25 w/v % GelMA hydrogels was 4 m (FIG. 3A). The porosity of 25 w/v % GelMA hydrogels showed circular shapes (aspect ratio=1), whereas 5 w/v % GelMA hydrogels showed elliptical shapes (aspect ratio=1.9, FIG. 3B). Furthermore, as a result of investigating the effect of GelMA hydrogel concentration on the molecular diffusion, the molecular diffusion easily occurred in 5 w/v % GelMA hydrogels, whereas 25 w/v % GelMA hydrogels completely inhibited the molecular diffusion. Therefore, it was determined that 5 w/v % GelMA hydrogels could not be used as a barrier. In contrast, 15 w/v % GelMA hydrogels were determined to be unfavorable since they may be used as a barrier but the pore size thereof is too small to encapsulate cells. In the present invention, it was determined that the suitable concentration of GelMA hydrogels was 10 w/v % GelMA for the use as a barrier of the microfluidic chip and for cell encapsulation.

Analysis of Photothermal Therapy Effect

[0071] As a result of analyzing the temperature increase after 20, 30, and 40 l of gold nanorods were mixed with 200 l of cell culture medium and NIR laser was irradiated, the temperature increase was dependent on the concentration of gold nanorods (FIG. 6A). It was verified that, in the solution (20 v/v %), in which 30 l of gold nanorods were mixed in 200 l of the cell culture medium, the cells were killed by the photothermal effect while the shape of the cells was not influenced.

[0072] From the preliminary test results, when treated with 200 l+40 l of gold nanorods solution, the cells became unhealthy before the photothermal treatment (data not shown). Generally, the photothermal treatment at 45 C. or higher may damage tissues as well as cells. Therefore, the 200 l+40 l of gold nanorods were determined to be inappropriate in optimizing photothermal conditions.

[0073] Meanwhile, the glioblastoma cells and breast cancer cells were cultured in the chip for 1 day, and, in a similar manner, the cells were irradiated with NIR laser and analyzed by the live/dead assay, and as a result, most cells were dead by the photothermal effect.

Co-Culture of Cancer Cells in 3D Microfluidic Device

[0074] The glioblastoma cells and breast cancer cells were injected into different microchambers and co-cultured. The vascular endothelial cells were encapsulated in gelatin methacrylate hydrogels and injected into the cruciform microfluidic channel. The GelMA hydrogel injected into the microfluidic channel became a physical barrier, and the cross-contamination of the respective cancer cells and the culture media thereof did not occur. The vascular endothelial cell culture medium containing VEGF was allowed to flow through the microfluidic channel, and as a result, it was verified that the cancer cells (U87MG) migrated toward the vascular endothelial cells (FIG. 7).

[0075] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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