KR20150135669A - Hydrogel-based microfluidic co-culture device - Google Patents

Abstract

The present invention relates to a hydrogel-based three-dimensional cell co-culturing microfluidic device. The hydrogel-based three-dimensional cell co-culturing microfluidic device comprises (a) a barrier formed with a hydrogel having gelatin and an acryl polymer mixed; and (b) a scaffold formed with a collagen hydrogel. According to the present invention, the hydrogel-based three-dimensional cell co-culturing microfluidic device is used as an important tool for division environment control and co-culture of a cell.

Description

Hydrogel-based microfluidic co-culture device for cell co-

The present invention relates to a hydrogel based microfluidic chip for cell co-culture.

Neural stem cells (NSCs) are a very important cell source for studying neurogenic metabolic diseases and spinal cord trauma. ¹ NSCs are known to differentiate into three major cell lines: neurons, astrocytes, and oligodendrocytes. ^{2 In} general, NCS differentiates into astrocytes and glial cells after being differentiated into neurons. ³ NSC-derived neurogenesis was studied using an extracellular matrix (ECM) -based hydrogel. For example, NSCs isolated from the cortical nerve epithelium of the embryo were suspended in collagen type I gel to produce a three-dimensional nerve circuit. NSCs encapsulated in collagen will grow into neurons. On the other hand, GFAP (Glial Fibrillary Acidic Protein) -based astrocytes and O4-positive glial cells were not observed until day 10. Electrophysiological analysis confirmed that the NSCs cultured in 3D collagen changed voltage-gated K ⁺ and Na ⁺ ion currents when differentiated into Tuj1-positive neurons. However, in the undifferentiated NSC, the voltage-switching Na ⁺ ion current was not large. In addition, it was confirmed that encapsulated NSC-derived neurons in the collagen gel exhibited synaptic vesicle recycling. Furthermore, the 3D collagen type I gel-hyaluronan substrate induced NSC-derived neural differentiation. ^5- embryonic brain-derived precursor cells grew faster than mature cells. NSCs cultured in 3D collagen type I gel-hyaluronan matrix for 6 days were mainly differentiated into neurons (-70%). On the other hand, on day 6 only 14% NSC-derived neurons were observed on the fibronectin-coated glass. This means that a large number of [beta] < 11 > -tubulin-positive cells express mature neurogenic differentiation through the expression of glutamate, [gamma] -aminobutyric acid and cytosine I.

Hydrogels have attracted much attention for their application to implantable materials and regenerative tissues. Hydrogels containing water have complex polymeric bonds in 3D. ^{6,7 In} particular, natural hydrogels (e.g., gelatin, collagen and hyaluronate) have been widely used as alternative materials to replace tissue supports due to the chemical and mechanical properties of biodegradable hydrogels similar to those of human tissues. ^8-10 hydrogel-based 3D culture has been widely used in stem cell-based tissue engineering. ^11,12 In recent years, photo-crosslinkable hydrogels have been used to control the differentiation of stem cells. For example, a cooper-free click chemistry-based cell compatible hydrogel was used to spatially encapsulate and pattern cells. ¹³ showed cell-encapsulated hydrogels prepared by poly (ethylene glycol) tetra-azide and self-quenched collagenasesensitive detection peptides using a bis (cyclooctyne) -functinalized peptide crosslinker. Spatially formed fibroblasts in the hydrogel containing the Arg-Gly-Asp (RGD) peptide were observed. Such a method may be useful for photosensitive preparation of biologically functional hydrogels. Porous photo-crosslinking methacrylamide chitosan has been used to induce NSC differentiation. ¹⁴ To control porosity, D-mannitol crystals were mixed with methacrylamide chitosan. D-mannitol crystals increased the average size of the pores and increased the oxygen diffusion rate. Growth of NSCs cultured in porous methacrylamide chitosan was inversely proportional to porosity. On the other hand, porous methacrylamide chitosan supports induced differentiation of NSCs. A methacrylate-modified hyaluronic acid-based hydrogel was used to control the differentiation of neural progenitor cells. The effect of methacrylate-modified hyaluronic acid-based hydrogel concentration on compression factor ¹⁵ was investigated and the compressive modulus increased with hydrogel concentration. Neural progenitor cells were light-encapsulated in hyaluronic acid hydrogel as nerve differentiation. Interestingly, most of the cultured neural progenitor cells in hard hydrogels differentiated into GFAP-positive astrocytes. It was confirmed that the differentiation of neural progenitor cells was significantly influenced by the mechanical characteristics of hydrogel similar to brain tissue. Photocrosslinked RGD alginate gels have been used directly in lipid precursor cells. Mechanical particular hydrogel ¹⁶ was controlled by the attached peptide density and Ca ^{2 +} concentrations. Cells in the rigid hydrogel were more proliferated than in the hydrogel, and most pre - adipocytes cultured in the rigid hydrogel secreted VEGF (Vascular Endothelial Growth Factors). This implies that adipocyte-derived differentiation is limited to the mechanical strength of the hydrogel substrate.

Differentiation of stem cells is affected by various extracellular microenvironment. ¹⁷ To control the fate of stem cells, a variety of multifunctional microfluidic devices have recently been developed. ^18-22 For example, a microfluidic culture apparatus having a compartment for inducing neural differentiation from mouse embryonic stem cells has been developed. ^The embryonic body, which was loaded into the microfluidic culture device, differentiated into neurons and expanded the axon proliferation via the bridge microcavity to form an array of axon protrusions. The use of a 3D gel substrate as a support can be achieved by using in- vivo tissue environment, which has a significant advantage over 2D culture. In previous studies, NSCs were cultured in ECM-based 3D microfluidic devices. ²⁰ NSC and ECM hydrogels (for example, collagen type I, martriel and a mixture of collagen type I and matrigel) were loaded at the center of the microcavity, and growth media containing various growth factors were placed in the side passageway. The effects of three ECM hydrogels on NSC differentiation in microfluidic devices were investigated. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that NSCs cultured in 3D matrigel-based hydrogel were well differentiated into neuron and glomeruli cells compared to cells cultured in collagen gel . This is because laminin is a major constituent of matrigel. On the other hand, qRT-PCR analysis showed that NSCs cultured in Matrigel or 3D ECM hydrogels did not differentiate into mostly glial cells. Furthermore, 3D microfluidic devices have been applied to co-culture of mouse embryonic stem cells, breast cancer cells and hepatocytes. ²⁴ Two-layered microfluidic device with embedded macroscopic membrane was used for co-cultivation of Janus pattern of 3D ellipsoid. The ellipsoid, which consists of several different species, is a fluid mechanics device of microfluidics that regulates the shape of mechanical cell micropatterning by the geometry of the microchannels. (MDA-MB-231) or hepatocellular carcinoma cells (HepG2) are located outside the ellipsoid, suggesting that the JANUS 3D cell ellipsoid regulates spatial differentiation of mouse embryonic stem cells . Although prior 3D microfluidic devices have been used in cell-based neural differentiation or co-culture systems, the effects of photo-crosslinking hydrogels on molecular diffusion and cross-contamination of 3D microfluidic co- I did not consider it.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a 3D cell co-culture microfluidic device capable of regulating the differentiation environment of cells. As a result, the molecular diffusion of the culture medium according to the gelatin hydrogel concentration of the barrier composed of gelatin hydrogel was controlled, and the neural stem cells and cancer cells encapsulated in the collagen hydrogel were co-cultured to form neural stem cells By confirming differentiation into neurons, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a hydrogel-based 3D cell co-culture microfluidic device.

It is another object of the present invention to provide a method of manufacturing a hydrogel-based 3D cell co-culture microfluidic device.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition comprising: (a) a barrier composed of a gelatin hydrogel; And (b) a cell-based scaffold composed of collagen hydrogel. The present invention provides a hydrogel-based 3D cell co-culture microfluidic device.

The present inventors have tried to develop a 3D cell co-culture microfluidic device capable of regulating the differentiation environment of cells. As a result, the molecular diffusion of the culture medium according to the gelatin hydrogel concentration of the barrier composed of gelatin hydrogel was controlled, and the neural stem cells and the cancer cells encapsulated in the collagen hydrogel were co-cultured, .

The main feature of the present invention is to control the diffusion of molecules between cells co-cultivating a barrier composed of hydrogel in a microfluidic device for cell co-culture.

The hydrogel-based 3D cell co-culture microfluidic device of the present invention comprises a barrier composed of hydrogel mixed with gelatin and acrylic polymer.

According to an embodiment of the present invention, the acrylic polymer may be a methacrylic acid copolymer, a methyl methacrylic acid copolymer, an acrylic acid and a methacrylic acid copolymer, an ethoxyethyl methacrylic acid copolymer, a cyanoethyl methacrylic acid copolymer Wherein the acrylic polymer is an acrylic polymer selected from the group consisting of poly (meth) acrylic acid copolymers, aminoalkyl methacrylic acid copolymers, poly (acrylic acid) copolymers, polyacrylamide copolymers, glycidyl methacrylic acid copolymers, , The acrylic polymer is an acrylic polymer selected from the group consisting of a methacrylic acid copolymer, a methyl methacrylic acid copolymer, an acrylic acid and a methacrylic acid copolymer, and a mixture thereof. According to a specific embodiment of the present invention, The acrylic polymer is a methacrylic acid copolymer.

The hydrogel mixed with the gelatin and the acrylic polymer of the present invention can control the molecular diffusion of the cell co-culture by controlling the concentration. The hydrogel in which the gelatin and the acrylic polymer are mixed can be prepared according to various methods known in the art. For example, gelatin was mixed with PBS (Phosphate Buffered Saline) at 50 ° C until completely dissolved, mixed with methacrylic anhydride at a rate of 0.5 ml / min, and gelatin methacylate hydrogel .

According to one embodiment of the present invention, the hydrogel mixed with the gelatin and the acrylic polymer has a concentration of 1-50 wt%.

The molecular diffusion of the cell co-culture can be controlled by adjusting the concentration of the hydrogel in which the gelatin and the acrylic polymer are mixed according to the present invention. When the concentration of the hydrogel mixed with the gelatin and the acrylic polymer is 1-20% by weight, molecular diffusion proceeds and co-cultured cells interact with each other. When the concentration of the hydrogel mixed with the gelatin and the acrylic polymer is in the range of 21-50 wt%, the molecular diffusion is inhibited and the interaction of the co-cultured cells is blocked.

The hydrogel mixed with the gelatin and acrylic polymer of the hydrogel-based 3D cell co-culture microfluidic device of the present invention photo-crosslinks.

As used herein, the term " photocrosslinking " refers to a process in which light is irradiated in the presence of a photoinitiator to form covalent and physical crosslinks to polymerize. The photoinitiator is a chemical that initiates polymerization and / or radical crosslinking by light.

The photocrosslinking of gelatin and acrylic polymer mixed hydrogel of the present invention can be carried out by mixing GelMA hydrogel with PBS and 2-hydroxy-1- (4- (hydroxyethoxy) phenyl (2-hydroxy-1- (4- (hydroxyethoxy) phenyl) -2-methyl-1-propanone), injected into a chamber, Wavelength) to induce photo-crosslinking.

The hydrogel-based 3D cell co-culture microfluidic device of the present invention includes collagen hydrogel.

According to one embodiment of the present invention, the collagen is a collagen selected from the group consisting of Collagen Type I, Collagen Type II, Collagen Type III, Collagen Type IV, Collagen Type V and mixtures thereof, and in another embodiment of the present invention According to a specific embodiment of the present invention, the collagen is collagen type I, collagen type II, collagen type III and mixtures thereof.

The collagen hydrogel of the present invention is used as a scaffold for cell co-culture. The term " scaffold " as used herein has the same meaning as " scaffold " or " cell carrier ".

According to one embodiment of the present invention, the collagen hydrogel is encapsulated by cells.

As used herein, the term " encapsulation " refers to the encapsulation of a semi-permeable gel (e.g., a gelatinized starch) that is capable of bi-directional diffusion of molecules, such as inflow of essential oxygen, nutrients, growth factors, Or membrane) is immobilized. The main motivation for cell encapsulation technology is to overcome the problems existing in graft rejection in tissue engineering applications to reduce the long-term use of immunosuppressive drugs to combat side effects after organ transplantation .

The collagen hydrogel in which the cells are encapsulated enables 3D culture of the cells.

The term " 3D cell culture " in this specification means culturing in an artificially created environment such that biological cells can grow and interact with the surrounding environment in three dimensions.

According to another aspect of the present invention, there is provided a method of making a hydrogel-based 3D cell co-cultured microfluidic device comprising the steps of:

(a) fabricating a microfluidic device including a chamber consisting of five to five chambers and a bridge channel connecting the chambers;

(b) injecting a hydrogel mixed with gelatin and acrylic polymer into the third chamber;

(c) photo-crosslinking the hydrogel mixed with the gelatin and the acrylic polymer;

(d) encapsulating the two kinds of cells with a collagen hydrogel, respectively;

(e) injecting the result of step (d) into the second and fourth chambers, respectively; And

(f) co-culturing the cells.

In order to manufacture the hydrogel-based 3D cell co-culture microfluidic device of the present invention, first, a chamber composed of five to five chambers and a bridge channel connecting the chambers are formed To produce a microfluidic device.

According to an embodiment of the present invention, the microfluidic device manufactures chamber and bridge channels in a two-step photolithography process using known methods. Briefly, to fabricate 3D microfluidic co-culture devices, design chamber and bridge channels with the AutoCAD program. SU-8 25 photoresist was spin-coated (1000 rpm, 60 seconds and 40 Gm in thickness) on silicon wafers and SU-8 100 was coated with SU -8 8 Spin-coat (1000 rpm, 60 seconds and 250 Gm in thickness) on the photoresist-patterned substrate. PDMS [poly (dimethylsiloxane)] precursor solution is viewed as a photoresist-patterned silicon wafer, and a PDMS-based 3D microfluidic co-culture device is bonded to a glass slide by oxygen plasma treatment.

Next, a hydrogel mixed with gelatin and acrylic polymer is injected into the third chamber of the microfluidic device.

The hydrogel in which the gelatin and the acrylic polymer are mixed acts as a barrier in the microfluidic device. The first to fifth chambers constituting the microfluidic device of the present invention refer to the chambers a to e of FIG. 1A. The third chamber located at the center, that is, the c chamber, is filled with the hydrogel mixed with the gelatin and the acrylic polymer.

The hydrogel in which the gelatin and the acrylic polymer are mixed should be photo-crosslinked. Therefore, the photo-initiator includes a photoinitiator. After injecting the chamber, UV is irradiated to induce photo-crosslinking.

Next, for cell co-culture, the two types of cells are each encapsulated with collagen hydrogel and injected into the second and fourth chambers, respectively. The second and fourth chambers correspond to the chambers b and d of FIG. 1A.

For culturing the encapsulated cells, the culture medium is injected into the first and fifth chambers. The first and fifth chambers correspond to a and e chambers in FIG. 1A, respectively. The culture medium may be injected with any culture medium known in the art, for example, one or more culture medium selected from the group consisting of DMEM (Dulbecco ' s Modified Eagle Medium), F12, RPMI1640 and mixtures thereof as a basic culture medium And may additionally include various supplements known in the art. For example, B27 supplement, L-glutamine, gentamycin, heparin, FBS (Fetal Bovine Serum) and penicillin-treptomycin.

Finally, cells are co-cultured.

According to one embodiment of the present invention, the cell is a neural stem cell, an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a haematopoietic stem cell One or more stem cells selected from the group consisting of HSC, haemopoietic precursor cells (HPC), lymphoid progenitor cells, and thymic progenitor cells; And cancer cells, breast cancer cells, prostate cancer cells, rectal cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, bladder cancer cells, endometrial cancer cells, cervical cancer cells, liver cancer cells, kidney cancer cells, thyroid cancer cells, bone cancer cells, lymphoma cancer cells and skin cancer cells Lt; / RTI > cancer cells. According to another embodiment of the present invention, the cell comprises at least one stem cell selected from the group consisting of neural stem cells, embryonic stem cells, and induced pluripotent stem cells; And cancer cells selected from the group consisting of breast cancer cells, prostate cancer cells, rectal cancer cells and lung cancer cells. According to a particular embodiment of the invention, the cell is a neural stem cell and a breast cancer cell.

The method of the present invention is a method for manufacturing the hydrogel-based 3D-cell co-cultured microfluidic device, the description of which is common between the two is omitted in order to avoid the excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a hydrogel-based 3D cell co-culture microfluidic device and a method of manufacturing the same.

(b) The present invention can be used as an important tool for controlling the cell differentiation environment and co-culturing.

(c) The present invention is excellent in biocompatibility, mechanical properties, and economy.

Figures 1A-1C show a photocrosslinked hydrogel-based 3D microfluidic co-culture device. Figure 1 a shows a schematic of a photocrosslinking hydrogel-based 3D microfluidic co-culture device consisting of five chambers and four bridge channels. Figure IB shows a cross-section of a photocrosslinked hydrogel-based 3D microfluidic co-culture device. Figure 1c shows a photograph of a photocrosslinked hydrogel-based 3D microfluidic co-culture device.
Figures 2A-2C show SEM images according to 5 w / v%, 15 w / v% and 25 w / v% of photo-crosslinked GelMA hydrogel. The scale bar represents 20 mu m.
Figures 3a-3b show the effect of GelMA hydrogel concentration (5-25 w / v%). 3A and 3B show the hole size and the aspect ratio, respectively. The aspect ratio means the length of the hole divided by the width of the hole (* p <0.05, ** p <0.01).
Figures 4a-4d depict an analysis of the molecular diffusion of a photocrosslinked GelMA hydrogel-loaded 3D microfluidic co-culture device. 4A shows a fluorescence image of a GelMA hydrogel-loading c chamber, and a collagen type I gel-loading b and d chamber. Figure 4b shows a fluorescence image of a green fluorescent dye-loaded ab chamber, a 25 w / v% GelMA hydrogel-loading c chamber and a red fluorescence die-loading de chamber. Figures 4c and 4d show the results of 1 day and 6 days for molecular diffusion of the GelMA hydrogel-loading c chamber, respectively (* p < 0.05, ** p < 0.01). The FITC-dextran (20 kDa) solution was loaded into the a chamber. Fluorescent molecules were diffused from the b chamber to the d chamber. The scale bar represents 1 mm.
Figures 5a to 5d show 2D surfaces and 3D collagen gels in which NSCs and cancer cells have been cultured. Figure 5a shows a confocal microscope image of NSC-derived neurons cultured for 6 days on a 2D surface. FIG. 5B shows a confocal microscope image of cancer cells cultured on a 2D surface for 6 days. FIG. Figure 5c shows a confocal microscopic image of neural differentiation of NSC cultured in 3D collagen gel for 6 days. 5D shows a confocal microscope image of cancer cells cultured in 3D collagen gel for 6 days. The scale bar represents 50 占 퐉.
Figures 6a through 6e show NSC and cancer cell co-cultures of a hydrogel-based 3D microfluidic device. Figure 6a shows a confocal microscopic image of neuronal differentiation from NSCs cultured for 6 days in a hydrogel-based 3D microfluidic co-culture device. 6B shows a confocal microscope image of cancer cells cultured for 6 days in a hydrogel-based 3D microfluidic co-culture device. Figure 6c shows a high magnification confocal microscope image of the white dotted box of Figure 6a. Figure 6d shows a high magnification confocal microscope image of the white dotted box of Figure 6b. Figure 6E shows the results of analysis of NSC-derived neuronal differentiation of a photo-crosslinked hydrogel-based 3D microfluidic co-culture device. The scale bar represents 50 占 퐉.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Manufacture of 3D microfluidic co-culture apparatus

Chamber and bridge channels were fabricated by a two-step photolithography method using known methods. ²⁵ 3D microfluidic co-cultured to prepare the device, the chamber was designed and bridge channel as AutoCAD program. To fabricate the bridge channel, SU-8 25 photoresist was spin-coated (1000 rpm, 60 seconds and 40 Gm in thickness) on a silicon wafer. To prepare the chamber, SU-8100 was spin-coated (1000 rpm, 60 seconds and 250 Gm in thickness) on SU-8 25 photoresist-patterned substrate. The PDMS [poly (dimethylsiloxane)] precursor solution was viewed as a photoresist-patterned silicon wafer and the PDMS-based 3D microfluidic co-culture device was bonded to the glass slide by oxygen plasma treatment (Femto Scientific, Korea).

GelMA Hydrogel Synthesis

A photocrosslinked GelMA hydrogel was synthesized according to a known method. ^{26 and 27} briefly, the type A pork skin gelatin and stirred at 50 ℃, and mixed with a PBS (Phosphate Buffered Saline, GIBCO, USA) until completely dissolved. Methacrylic anhydride was added at a rate of 0.5 ml / min under stirring conditions for 2 hours. The mixture was placed in a 12-14 kDa cut-off dialysis tube and dialyzed against distilled water at 40 ° C for 3-4 days to remove salts and methacrylic acid. The solution was lyophilized for one week and stored at -80 ° C.

Scanning electron microscope

The structure of GelMA Hyde 99b roseline was analyzed using Scanning Electron Microscope (SEM). The expanded hydrogel was frozen and lyophilized. The lyophilized sample was cut and plated with platinum using a turbo sprayer (EMITECH, K575X). SEM images were obtained at 20 kV high pressure. Image J software was used to analyze porosity and aspect.

Culture of NSC and cancer cells

(DMEM: F12) supplemented with 2% B27 supplement, 1% L-glutamine, 10 ug / ml gentamycin (Invitrogen, Auckland, New Zealand) and 10 units / ml heparin (Sigma, MO, USA) in a laminin- Human NSC (ReN VM cell line) was cultured in an incubator under the conditions of 5% CO ₂ and 37 ° C with Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 medium. Accutase was used to remove the cells attached to the culture flask. In addition, human breast cancer cell line (MCF7) was cultured. And cultured in DMEM supplemented with 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin.

GelMA Hydrogel and Cell-Loading of Encapsulated Collagen Gel

The lyophilized 25 w / v% GelMA hydrogel was dissolved in PBS and a photo-initiator at a concentration of 0.5 w / v% 2-hydroxy-1- (4- (hydroxyethoxy) phenyl) -2- 2-methyl-1-propanone, Irgacure 2959, CIBA Chemicals). 8 [mu] l GelMA hydrogel solution was injected into the c chamber inlet of the 3D microfluidic co-culture apparatus (Fig. 1A) and photo-crosslinked by irradiation with ultraviolet light (360-480 nm wavelength) for 20 seconds. To culture cells in 3D, 2 x ¹⁰⁶ cells / ml NSC and cancer cells on ice were encapsulated in 2 mg / ml collagen type I gel. 6 [mu] l cell-encapsulated collagen gel was injected into the b-chamber and the d-chamber of the 3D microfluidic cavity-culture apparatus, respectively (Fig. The cells were incubated at 37 [deg.] C for 30-40 minutes.

Immunocytochemistry

After 6 days of incubation in a GelMA hydrogel-based 3D microfluidic co-culture device, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS, the cells were treated with 1% Triton X-100 (in PBS) for 30 minutes to give permeability and blocked with 1% BSA (bovine serum albumin, Sigma, USA) for 1 hour. Cells were immunostained overnight with primary antibody (anti-nerve class Ⅲ β-tubulin (Tuj1), Stem Cell Technology, Canada) and incubated with secondary antibody (Alexa Fluor 488 goto anti-mouse IgG and Alexa fluor 594 paloidine, Invitrogen, USA) at room temperature for 6 hours. Cell nuclei were also stained with DAPI (0.1 [mu] g / ml) for 30 minutes at room temperature.

Results and Discussion

Come Hydrogel -Based 3D microfluidic co-culture apparatus

A photocrosslinkable GelMA hydrogel-based 3D microfluidic co-culture device was developed (Fig. 1). The GelMA hydrogel-based 3D microfluidic device is fabricated by a two-step photolithography process to consist of five chambers (a to e chambers) and four breech channels (Fig. 1A). Five chambers (250 占 퐉 thickness) are connected by a fine groove bridge channel (40 占 퐉 thickness) (Fig. 1b). A chamber of 250 μm thickness is filled with a gelation of GelMA hydrogel and cell-encapsulated collagen type I gel. A channel of fine grooves of 40 μm thickness increases the resistance of the fluid. The Gel chamber hydrogel of the c chamber was photocrosslinked through the mask film with UV, and the GelMA hydrogel inside the chamber c was selectively photo-patterned. After photo-crosslinking of the GelMA hydrogel, a sealing tape was used to seal the inlet and outlet of the chamber c to block the evaporation of solution in the inlet and outlet of the chamber c. The photo-crosslinked GelMA hydrogel in the c chamber inhibits molecular diffusion through the bridge channel to the physical barrier. Cell-encapsulated collagen type I was loaded into b-chamber and d-chamber. Neurons and coliform cells were previously co-cultured in multi-section microfluidic devices. ²⁸ The mass loss of the central nervous system (CNS) was isolated from the microchannels of the six peripheral zones. The effect of co-culture on astrocytes and glial progenitor precursor cells was confirmed by the separated axon layer. This is because the axon was grown above the astrocytes, and the astrocytes thrust out the axon, resulting in the division of the axon by the astrocytes. On the other hand, while isolated axons were co-cultured, mature myelin protein was highly expressed in spindle-cell progenitor precursor cells. Therefore, multi-compartment microfluidic culturing devices have many advantages in fastone-glare interaction and high-speed mass drug screening. Neurons and astrocytes were co-cultured in a microfluidic device similar to proximal lateral sclerosis. ²⁹ Neuronal cells were co-cultured with infected astrocytes without cell-cell connections directly in the microfluidic device to produce cell-cell metabolic gradients. Indicating that the density of co-cultured infectious astrocytes with neurons is greatly reduced. Glutamate changes were produced in neuron-astrocytic co-cultured microfluidic devices, and proper treatment of glutamate did not affect neuronal death. Such microfluidic co-culture devices that examined these previous neuron-cell interactions did not consider the photo-crosslinked hydrogel-based 3D microfluidic bodies for co-culture of neural stem cells and cancer cells.

For Porosity and Molecular Diffusion Come Hydrogel  Effect of Concentration

Examination of the effect of GelMA hydrogel concentration on porosity showed that the pore size was inversely proportional to GelMA hydrogel concentration (FIG. 2). SEM images show the uniform size and morphology compared to 5 w / v% hydrogels with porosity of 25 w / v% GelMA hydrogel (Figs. 2a-2c). The pore size of the 5 w / v% GelMA hydrogel was 34 microns whereas that of 25 w / v% GelMA hydrogel was 4 microns (Figure 3a). The porosity of the 25 w / v% GelMA hydrogel is circular (aspect ratio = 1) while the 5 w / v% GelMA hydrogel is elliptical (aspect ratio = 1.9, FIG. In addition, the effect of GelMA hydrogel concentration on molecular diffusion was investigated (Figure 4). FITC (fluorescein isothiocyanate) -dextran (20 kDa) was loaded into the b-chamber and photo-patterned with 5-25 w / v% Gel in the c-chamber. For 6 days, the 25 w / v% GelMA hydrogel completely inhibited the molecular diffusion, which means that the 25 w / v% GelMA hydrogel can be used as a physical barrier in the c chamber (Fig. 4b). In contrast, the FITC-dextran solution in the 5-15 w / v% hydrogel-based microfluidic chamber was easily diffused. Interestingly, when 5 w / v% GelMA hydrogel (34 urn porosity) was injected into the c chamber, the FITC-dextran solution in the b chamber nearly spread to the d chamber in one day. In contrast, when 25 w / v% GelMA hydrogel (4 urn porosity) was injected into the c chamber, the FITC-dextran solution of b-chamber for 6 days hardly diffused into the d-chamber. As a result, a 210 [mu] m thick gap between the 4 [mu] m small pore and chap (40 [mu] m thickness of 25 w / v% GelMA hydrogel and the bridge channel (250 [mu] m thickness) inhibited molecular diffusion, - No cross-contamination. The effect of GelMA hydrogel concentration on degree of expansion was previously investigated. ²⁶ It is explained that the mass expansion ratio is inversely proportional to the concentration of GelMA hydrogel, and the expansion ratio of 15 w / v% GelMA hydrogel is reduced by 50% as compared with 5 w / v% GelMA hydrogel. This means that the high concentration GelMA hydrogel suppresses molecular diffusion. GelMA hydrogel was used to control cell-cell interactions in a two-layer microfluidic device. ³⁰ membrane endothelial cells were cultured in a porous membrane and encapsulated intercellular cells in GelMA hydrogel used as an intradermal 3D substrate were cultured on the porous membrane. GelMA hydrogels exhibited high physical stability and physiologically-related elastic modulus. The effect of shear stress on the paracrine interactions between the intercellular and the endothelial cells of the valve prevents the differentiation of endothelial cells into myofibroblasts by intercellular disease when shear stress is applied . However, this study did not consider GelMA hydrogel as a physical barrier in the microfluidic device.

In 3D microfluidic device NSC  And co-culture of cancer cells

Co-culture of NSCs and cancer cells for application to regenerated tissue structures has received much attention. NSC and cancer cells were co-cultured on 2D surface and 3D collagen gel for 6 days (Figure 5). The confocal microscope image shows the difference between cultured NSCs and cancer cells in 3D collagen gel and NSC and cancer cells cultured on 2D surface. NSCs and cancer cells cultured on a 2D surface were relatively polarized (Figures 5a and 5b), while NSCs and cancer cells cultured on 3D collagen gels were circular (Figures 5c and 5d). At the same time, the effect of 3D collagen gel on the response of NSCs and cancer cells in a 3D microfluidic co-culture device was investigated (Figure 6). NSC and cancer cells encapsulated in collagen type I gel were loaded into b and d chambers, respectively. To block the diffusion of the medium into the bridge channel, the c chamber was loaded with 25 w / v% GelMA hydrogel. Confocal microscope images show highly expressed Tuj1-positive neurons in collagen gel-loading b chamber and Paloidin-positive cancer cells co-cultured in collagen-gel loading d chamber (Figs. 6a and 6b). As a result, since the 25 w / v% GelMA hydrogel was used as a physical barrier, the culture medium of NSC and cancer cells did not cross-contaminate. Neurite outgrowth of Tujl-positive neurons from cultured NSCs in collagen gel-loading b chamber was confirmed (Figure 6c). Immunocytochemical analysis revealed that 66% of the NSCs differentiated into Tuj1-positive neurons in the hydrogel-based 3D microfluidic co-culture device (Fig. 6E). To study cancer therapy, previous interactions between breast cancer cells and nerve cells or NSCs were investigated. ^31,32 For example, the effects of RhoA subfamily-based low molecular weight on dysfunction of neuronal and breast cancer cells have been studied. ³¹ Rho-specific inhibitor rhosin inhibited the formation of MCF7-derived mammalian mammosphere in breast cancer cells and showed dysfunction leading to apoptosis of breast cancer cells. This implies that rosin inhibits the migration, invasion and metastasis of breast cancer cells by inhibiting the activity of RhoA or RhoC, which is directly related to cancer metastasis. In contrast, rosin enhances neurite outgrowth of PC21 neuronal cells in the presence of NGF (Nerve Growth Factor). Human induced pluripotent stem cell-derived NSCs have been used for application to the genetic therapy of breast cancer cells. NSC derived from human fibroblast-derived human induced pluripotent stem cells was prepared using ³² lentiviral transgenic lines. For cancer gene therapy, human induced pluripotent stem cell-derived NSCs were transfected with baculovirus vectors harboring a kinase suicide gene. This has successfully demonstrated that NSCs migrate to mouse breast cancer and inhibit the proliferation and metastasis of breast cancer.

conclusion

We have developed a photo-crosslinked hydrogel-based 3D microfluidic device for co-culture of NSCs and cancer cells. Investigation of the effect of photocrosslinking GelMA hydrogel concentration on porosity confirmed a 4 μm diameter circular form of 25 w / v% GelMA hydrogel. In 3D microfluidic devices, 25 w / v% GelMA hydrogel was used as a physical barrier to inhibit molecular diffusion and cross-contamination, so 66% NSCs differentiated into Tuj1-positive neurons by co-culture of cancer cells for 6 days . Thus, a photocrosslinked hydrogel-based 3D microfluidic co-culture device will contribute to the study of nerve regeneration of the nervous system damaged by cancer cells.

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While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (10)

(a) a barrier composed of a hydrogel mixed with gelatin and an acrylic polymer; And (b) a scaffold composed of a collagen hydrogel. The co-culture microfluidic device is a hydrogel-based 3D cell co-culture device.
[2] The method of claim 1, wherein the acrylic polymer is selected from the group consisting of acrylic acid and methacrylic acid copolymer, methacrylic acid copolymer, methyl methacrylic acid copolymer, ethoxyethyl methacrylic acid copolymer, cyanoethyl methacrylic acid copolymer, amino Wherein the polymer is an acrylic polymer selected from the group consisting of alkyl methacrylic acid copolymers, poly (acrylic acid) copolymers, polyacrylamide copolymers, glycidyl methacrylic acid copolymers, and mixtures thereof.
The apparatus of claim 1, wherein the hydrogel mixed with the gelatin and the acrylic polymer has a concentration of 1-50 wt%.
The apparatus of claim 1, wherein the hydrogel in which the gelatin and the acrylic polymer are mixed is photo-crosslinked.
The device according to claim 1, wherein the collagen is a collagen selected from the group consisting of Collagen Type I, Collagen Type II, Collagen Type III, Collagen Type IV, Collagen Type V and mixtures thereof.
2. The device of claim 1, wherein the collagel hydrogel is encapsulated by cells.
A method of preparing a hydrogel-based 3D cell co-cultured microfluidic device comprising the steps of:
(a) fabricating a microfluidic device including a chamber consisting of five to five chambers and a bridge channel connecting the chambers;
(b) injecting a hydrogel mixed with gelatin and acrylic polymer into the third chamber;
(c) photo-crosslinking the hydrogel mixed with the gelatin and the acrylic polymer;
(d) encapsulating the two kinds of cells with a collagen hydrogel, respectively;
(e) injecting the result of step (d) into the second and fourth chambers, respectively;
(f) injecting the culture medium into the first and fifth chambers, respectively; And
(g) co-culturing the cells.
[8] The method of claim 7, wherein the acrylic polymer is selected from the group consisting of acrylic acid and methacrylic acid copolymer, methacrylic acid copolymer, methylmethacrylic acid copolymer, ethoxyethylmethacrylic acid copolymer, cyanoethylmethacrylic acid copolymer, amino Wherein the polymer is an acrylic polymer selected from the group consisting of alkyl methacrylate copolymers, poly (acrylic acid) copolymers, polyacrylamide copolymers, glycidyl methacrylic acid copolymers, and mixtures thereof.
The method according to claim 7, wherein the hydrogel mixed with the gelatin and the acrylic polymer has a concentration of 1-50 wt%.
8. The method of claim 7, wherein the collagen is collagen selected from the group consisting of Collagen Type I, Collagen Type II, Collagen Type III, Collagen Type IV, Collagen Type V and mixtures thereof.

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