KR101647095B1 - Microfluidic system, manufacturing method thereof and method of cell encapsulation in hydrogel - Google Patents

Abstract

The present invention relates to a technique of supporting cells on a hydrogel, and more particularly, to a microfluidic device in which fine cells are dispersed in a single cell while passing through a fine columnar array zone and a focusing zone and supported on a hydrogel, To a method of supporting cells on a gel. By applying the cell carrying method using the microfluidic device according to the present invention, microorganism cells that are not spherical aggregated are dispersed by using a structure called a micropillar array zone, and the droplets are carried in the form of single cells in hydrogel drops in a focusing zone, It is possible to solve the problem of cell aggregation due to the polymer and to carry the separated cells in the form of a single cell in a hydrogel.

Description

TECHNICAL FIELD The present invention relates to a microfluidic device, a manufacturing method of the device, and a method of supporting cells on a hydrogel.

The present invention relates to a technique of supporting cells on a hydrogel, and more particularly, to a microfluidic device in which fine cells are dispersed in a single cell while passing through a fine columnar array zone and a focusing zone and supported on a hydrogel, To a method of supporting cells on a gel.

A hydrogel generally refers to a polymer material having a three-dimensional hydrophilic network structure that may contain a large amount of water. The hydrogel is capable of absorbing at least 20% of the total weight of the water, and absorbing at least 95% of the water is called a highly absorbing hydrogel. The hydrogel is composed of a homopolymer or a copolymer and forms a structurally stable three-dimensional network structure with little fluidity due to external history. Such a structure may be formed by various factors such as covalent bonding, hydrogen bonding, van der Waals bonding, . It is thermodynamically stable after swelling in aqueous solution and has mechanical and physicochemical properties corresponding to the intermediate form of liquid and solid. The swelling degree of the hydrogel can be controlled according to the chemical structure, hydrophilicity and cross-linking degree between the polymer chains of the polymer. Thus, it is possible to produce a hydrogel having various shapes and properties depending on the constitution and manufacturing method.

Synthetic polymers have the advantage that they can be varied in variety and can vary widely in their physico-chemical surface properties or mechanical properties. A typical hydrogel used as a biomaterial is PHEMA. PHEMA has a hydrophilic hydroxyl group (-OH) on the side branch and can be easily hydrated. A representative method used for preparing a hydrogel using a synthetic polymer is a method of block copolymerizing hydrophilic polyethylene oxide with another hydrophobic polymer. PEO-PPOPEO, PEO-polyesters (PLA, PCL and PLGA) derivatives, and the like. In addition, poly-N-isopropylacrylamide (PNIPAAm) derivatives are also being studied as temperature-sensitive hydrogels. Hydrophilic monomers such as polyacrylamide (PAAm), polymethacrylic acid, polymaleic acid, polyvinyl alcohol (PVA), polyethylene oxide (PEO) and poly N-vinyl pyrrolidone (PVP) . Hydrogels composed of various copolymers such as poly (MMA-co-HEMA), poly (AN-aryl sulfonate) and P (GEMA-sulfate) have been developed.

Korean Patent Publication No. 1360942

In order to obtain a high value-added biomaterial, a technique of supporting cells on a hydrogel has been used. Particularly, the technique of carrying cells using a microfluidic device has been carried out by several groups, but most of them are limited to animal cells and spherical cells. However, since cells capable of obtaining a large number of biomaterials and biopharmaceuticals are microorganisms, techniques for targeting cells and microorganisms other than spherical cells are required. However, when the conventional microfluidic device is used to carry the microbial cells on the hydrogel, it is difficult to apply the microbial cells because the shape of the cells is not spherical, and the aggregation of the cells due to the high molecular substance such as hydrogel occurs Therefore, there remains a problem to be solved.

SUMMARY OF THE INVENTION The present invention provides a microfluidic device for supporting cells on a hydrogel, comprising: a sample inlet for injecting a microorganism as a host of cells to be supported on the hydrogel; A microchannel array zone in which a plurality of fine columns are formed on the first microchannel, a dispersion medium in which a surfactant is added to the oil is formed on the left and right sides of the first microchannel, A second microchannel formed from the oil injection port to meet with the first microchannel, a focusing zone where the first microchannel is formed in the vicinity of the second microchannel and the width of the channel is significantly reduced, A third microchannel formed from the focusing zone to the sample outlet, and a third microchannel formed from the foil And a sample outlet through which the hydrogel-bearing cells formed in the dome are flowed out. The fine columnar array zone is arranged in the order of the cylinder-shaped fine columns from the sample injection port, The fine pillar array zone is divided into n sections (2 ≤ n ≤ 5) by the same column spacing or the same diameter, and the first micro column array zone A fine pillar array zone is formed and a curved portion for changing the direction of the flow path is formed between the n-th minute pillar array zone sections, and a total of n-1 curved portions are provided in the first micro flow path. The present invention provides a microfluidic device for supporting cells on a hydrogel.

The present invention also provides a method for supporting a cell using a microfluidic device for supporting the above-described cells on a hydrogel, comprising the steps of preparing a microorganism to be a host of a cell to be analyzed, Preparing a cell solution by adding PEGDA: Polyethylene Glycol Diacrylate), preparing a dispersion medium containing a surfactant in oil, injecting the cell solution and the dispersion medium into a sample inlet and an oil inlet of a microfluidic device, respectively Said cell solution passing through a microporous array of microfluidic devices and dispersed in a single cell body, contacting said dispersed single cell body with said dispersion medium, said single cell body comprising a microfluidic device Passing through the focusing zone of the device, Step is included to form a hydrogel droplet, and provides a cell supporting method comprising the step of reaching and collecting in the hydrogel droplets sample outlet.

The microfluidic device for supporting the cells of the present invention on a hydrogel and the cell supporting method using the microfluidic device can be used as a medium without diluting the cells. By dispersing non-spherical microbial cells and carrying them in the form of single cells in droplets in the focusing zone, it is possible to solve the problem of cell aggregation due to the polymer. Therefore, it can be the core of ingenious technology to acquire high value-added biomaterials.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a principal structural plan view of a microfluidic device for supporting cells of the present invention on a hydrogel; FIG.
FIG. 2 is an enlarged view of a micropillar array zone and a focusing zone as one embodiment of a microfluidic device for supporting cells of the present invention on a hydrogel.
FIG. 3 is a perspective view illustrating a sample of a microfluidic device for supporting cells of the present invention on a hydrogel, oil inflow, and hydrogel outflow method.
4 is a flowchart of a method of manufacturing a microfluidic device for supporting cells of the present invention on a hydrogel.
FIG. 5 is a flowchart of a method of supporting a cell using a microfluidic device for supporting cells of the present invention on a hydrogel.
FIG. 6 is an explanatory view for explaining the process of dispersing microorganisms as the host cells into single cells by the micropillar array zone of the microfluidic device of the present invention. FIG.
FIG. 7 is an explanatory view illustrating a process in which a microorganism serving as a host of cells is dispersed into a single cell by a micropillar array zone of a microfluidic device according to the present invention.
FIG. 8 is an explanatory view illustrating a process in which a single cell dispersed in a solution-state hydrogel by a micro-pillar array zone of a microfluidic device according to the present invention is passed through a focusing zone and supported on a gel droplet.
FIG. 9 is a photograph showing the distribution process of the cells passing through the micropillar array zone of the microfluidic device for carrying the cells of the present invention on the hydrogel in time sequence.
10 is a photograph showing a comparison of cell dispersing effects of microfluidic devices for supporting a cell of the present invention on a hydrogel, according to presence or absence of a micropillar array zone.
11 is a graph comparing cell dispersing effects of a microfluidic device for supporting a cell of the present invention on a hydrogel with or without a micropillar array zone.
FIG. 12 is a photograph of the number of cells supported on the hydrogel according to the cell concentration, and a photograph of the cell carried on the actual hydrogel.

The present invention relates to a technique of supporting cells on a hydrogel, and more particularly, to a microfluidic device in which fine cells are dispersed in a single cell while passing through a fine columnar array zone and a focusing zone and supported on a hydrogel, The present invention relates to a method for supporting cells on a gel, and the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is a plan view of a microfluidic device for supporting cells of the present invention on a hydrogel. The microfluidic device includes a sample inlet 10 for injecting a microorganism to be a mother of a target cell to be supported on the hydrogel, A microchannel array zone 30 in which a plurality of fine columns are formed on the first microchannel 20 and a second microchannel array zone 30 in which a plurality of microchannels are formed on the first microchannel 20 from the first microchannel 20 to the focusing zone 60, An oil injection port 40 formed on the left and right sides of the first microchannel 20 and a second microchannel 50 formed from the oil injection port 40 to meet with the first microchannel, And a third microchannel 70 formed from the focusing zone 60 to the sample outlet 80. The third microchannel 70 is formed in the vicinity of the third microchannel 50, 70) in the focusing zone 60 And a sample outlet 80 through which the formed hydrogel-bearing cells flow out.

Particularly, FIG. 2 is a partial enlarged view according to an embodiment of the present invention. As shown in FIG. 2, the fine pillar array zone 30 formed on the first microchannel 20 has cylindrical- (2 ≤ n ≤ 5) by the same column spacing or the same diameter so as to separate the columns from the sample inlet (10) The first fine pillar array zone to the n < th > minute pillar array zone are formed in the order of the interval or diameter. Also, n-1 curved portions 21 are formed in the first microchannel 20 between the n-th minute columnar array zone sections, so that the direction of the flow channel can be switched.

As shown in FIG. 2, a section where the fine pillar array zone 30 of the first microchannel is formed, a section from the n-th fine pillar array zone end of the first microchannel to the focusing zone 60 A focusing zone 60 and a focusing zone 60 of the third microchannel to the sample outlet 80. The microchannel array of the first microchannel 60 may include a plurality of microchannels, The width of the section where the zone 30 is formed is 500 to 700 占 퐉, the width of the section from the nth minute columnar array terminal end of the first microchannel to the focusing zone 60 is 160 to 200 占 퐉, The zone 60 is 40 to 60 mu m and the section from the focusing zone 60 of the third microchannel to the sample outlet 80 is 500 to 700 mu m. 2, the width of a section in which the micropillar array zone 30 of the first microchannel is formed is 600 mu m, and the width of the microchannel array zone 30 of the first microchannel A section from the focusing zone 60 to the sample outlet 80 of the third microfluidic channel is 580 占 퐉; the width of the section to the focusing zone 60 is 180 占 퐉; the section of the focusing zone 60 is 50 占 퐉; Respectively.

FIG. 3 is a perspective view illustrating a sample of a microfluidic device for supporting the cells of the present invention on a hydrogel, an oil inflow and a hydrogel outflow method, wherein the sample injection port 10 and the oil injection port 40 are connected to a syringe pump And shows that the cell solution 100 and the dispersion medium 200 are injected and the supported hydrogel is collected as a single cell.

FIG. 4 is a flowchart of a manufacturing method of a microfluidic device for supporting the above-described cells on a hydrogel. First, a master mold is manufactured by photolithography. At this time, the master mold is formed so as to have fine columns formed at an engraved surface on the micro flow path. The prepared master mold is filled with PDMS (Polydimethylsiloxane), and the master mold polymer filled with the PDMS is cured in an oven at 60 to 80 ° C for 1 to 3 hours. Then, the cured PDMS layer is removed from the master mold A sample inlet 10 and a sample outlet 80 are formed by a punching process on a PDMS layer formed by a master mold in which cylinder-shaped fine pores having different column intervals or diameters are formed in a positive angle, thereby manufacturing a microfluidic device. Then, the PDMS-based microfluidic device is washed with ethanol and distilled water to remove residues and dust, thereby completing the fabrication.

FIG. 5 is a flowchart of a method of carrying cells using a microfluidic device for carrying cells of the present invention on a hydrogel. First, microorganisms to be the host of cells to be analyzed are prepared, and then polyethylene glycol diarylate (PEGDA: Polyethylene Glycol Diacrylate) is added to prepare the cell solution 100. A surfactant is added to the oil to prepare a dispersion medium (200). The cell solution 100 and the dispersion medium 200 thus prepared are injected into a sample injection port 10 and an oil injection port 40 of the microfluidic device using a syringe pump.

The injected cell solution 100 passes through the micropillar array zone 30 of the microfluidic device along the first microchannel 20 and is dispersed into a single cell body. . 8, the dispersed single cell body is brought into contact with the dispersion medium 200 moved through the second microchannel 50 in the vicinity of the focusing zone 60, The hydrogel drops passing through the focusing zone 60 of the microfluidic device together with the dispersion medium 200 and containing the single cell body are formed and the hydrogel droplets thus formed are transported along the third microfluidic channel 70 to the sample outlet 80).

The cell solution (100) of the present invention is in the LB medium and contains 3 × 10 ⁸ to 8 × 10 ⁸ cells / mL of microorganism which is the host of the cell, 24.51 to 26.03 wt% of polyethylene glycol diacetate (PEGDA: Polyethylene Glycol Diacrylate, 0.78 to 1.23 wt% potassium persulfate (KPS), 0.25 to 0.31 wt% of D-sorbitol and 36.44 to 37.13 wt% of distilled water, (PEGDA), 0.3 g of polyethylene glycol diacrylate (PEGDA), 12.5 mg of potassium persulfate (KPS) and 12.5 mg of potassium persulfate - 4 mg of sorbitol (D-sorbitol) and 0.5 mL of distilled water.

FIG. 9 is a photograph showing the distribution process of the cells passing through the micropillar array zone of the microfluidic device for carrying the cells of the present invention on the hydrogel in time sequence. The aggregation phenomenon of E. coli applied as an embodiment of the present invention can be explained by van der Waals force and deflation force. The aggregated cells are separated step by step through the micropillar array zone and ultimately carried on the hydrogel as a single cell. The principle of dispersing the cell mass is that the cells in the cell solution 100 flow along the cell solution 100 in the direction of the microchannel, where the cell solution 100 hits the micropillar by the pulling force of the cells do. In the first place, large lumps of cells are broken into small lumps, and because of the narrowed sidewall by the micro-pillars, additional cell separation effects are shown due to the shear stress applied to the cells.

FIGS. 10 and 11 are photographs and graphs comparing cell dispersing effects of microfluidic devices for supporting cells of the present invention on a hydrogel, according to presence or absence of a micropillar array zone. 10 (a) and 10 (c) are structures without a micropillar array zone, while (b) and (d) are structures with the micropillar array zone of the present invention. In (a) and (c) without a microcolumn array zone, many aggregated cells were observed (a yellow circle Cell cluster), but (b) and (d) with a microcolumn array zone showed few aggregated cells . Also, according to FIG. 11, which is a graph showing the results of analyzing the number of cells appearing per unit area divided by red lines from 1 (single cell) to 5 (cell clustering result), a region having a micropillar array zone In microstructure, one single cell was found the most, whereas in the case of No Microstructure where no micropillar array zone was found, 5 cells were found most.

FIG. 12 is a graph showing the number of cells supported on the hydrogel according to the cell concentration and the shape of the cells supported on the actual hydrogel, and the cells dispersed due to the effect of the fluid flow change due to the presence of the fine column, And the dispersion medium 200 in which the surfactant is mixed with the oil flowing from both sides through the second micro flow path passes through the focusing zone which is a flow path whose width is remarkably reduced while compressing the cell solution 100 from both sides, Level stretching force, and with the aid of this force, dispersed cells can be contained in droplets of the hydrogel one at a time. The cells thus supported on the hydrogel are shown in Fig.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it should be understood that various changes and modifications will be apparent to those skilled in the art. Obviously, the invention is not limited to the embodiments described above. Accordingly, the scope of protection of the present invention should be construed according to the following claims, and all technical ideas which fall within the scope of equivalence by alteration, substitution, substitution, Range. In addition, it should be clarified that some configurations of the drawings are intended to explain the configuration more clearly and are provided in an exaggerated or reduced size than the actual configuration.

10. Sample inlet
20. First microchannel
21. Curvature portion
30. Fine Column Array Zone
31. First fine column array zone
32. Second fine column array zone
33. Third Fine Column Array Zone
40. Oil inlet
50. Second fine channel
60. Focusing zone
70. Third Fine Channel
80. Sample outlet
100. Cell solution
200. Distributions
300. Cell body
400. Hydrogel-supported cell bodies

Claims (11)

A microfluidic device for supporting cells in a hydrogel,
A sample inlet for injecting a microorganism, which is a host of cells to be supported on the hydrogel;
A first micro flow path formed from the sample injection port to the focusing zone;
A micropillar array zone in which a plurality of fine columns are formed on the first microchannel;
An oil injection port formed on the left and right sides of the first micro flow path to inject a dispersion medium containing a surfactant into the oil;
A second micro flow path formed from the oil injection port to meet the first micro flow path;
A focusing zone in which the first microchannel is formed near the second microchannel and the width of the channel is significantly reduced;
A third micro flow path formed from the focusing zone to the sample outlet; And
A sample outlet through which the hydrogel-bearing cells formed in the focusing zone flow out as the third fine channel-terminating portion;
And,
The fine pillar array zones are arranged in the order of the cylinder-shaped fine pillar having a smaller value from the sample inlet to the column spacing or diameter,
The fine pillar array zone is divided into n sections (2 ≤ n ≤ 5) by the same column spacing or the same diameter, and the first micro column array zone A fine pillar array zone is formed,
And one curved portion for changing the direction of the flow path is provided between the n-th minute columnar array zone sections, and a total of n-1 curved portions are provided in the first micro flow path. A microfluidic device.
delete delete The method according to claim 1,
A section from the n-th minute columnar array zone end of the first microchannel to the focusing zone, a focusing zone section, and a focusing zone of the third microchannel from the focusing zone of the first microchannel, Wherein the flow path width of the section is different from that of the sample outlet to the sample outlet.
5. The method of claim 4,
Wherein a width of a section where the fine pillar array zone of the first microchannel is formed is 500 to 700 mu m and a width of a section from the end of the nth minute pillar array zone of the first microchannel to the focusing zone is 160 to 200 mu m , The width of the focusing zone section is 40 to 60 占 퐉, and the width of the section from the focusing zone of the third microchannel to the sample outlet is 500 to 700 占 퐉. The microfluid Dick device.
A method of manufacturing a microfluidic device for supporting a cell according to claim 1 on a hydrogel,
I) fabricating a master mold by photolithography;
Ii) filling PDMS (Polydimethylsiloxane) on the prepared master mold;
Iii) curing the master mold polymer filled with the PDMS in an oven at a temperature of 60 to 80 ° C for 1 to 3 hours;
Iv) removing the cured PDMS layer from the master mold;
V) forming a microfluidic device by forming a sample inlet and a sample outlet in the removed PDMS layer; And
Vi) washing the PDMS-based microfluidic device with ethanol and distilled water;
/ RTI >
In the step (iv), the fine columns of the PDMS layer removed from the master mold are formed on the first microchannel more than one in the form of an array, the cylinders having different column spacings or diameters, the fine columns are formed in a convex shape,
The fine columnar array zone in which the fine columns are formed is arranged in the order of the cylinder-shaped fine columns having a smaller value from the sample injection port to the column spacing or diameter,
The fine pillar array zone is divided into n sections (2 ≤ n ≤ 5) by the same column spacing or the same diameter, and the first micro column array zone A fine pillar array zone is formed,
And one curved portion for changing the direction of the flow path is provided between the n-th minute columnar array zone sections, and a total of n-1 curved portions are provided in the first micro flow path. Wherein the microfluidic device is a microfluidic device.
delete A method for supporting a cell using a microfluidic device for supporting a cell according to claim 1 on a hydrogel,
(I) preparing a microorganism to be the host of the cell to be analyzed;
II) preparing a cell solution by adding polyethylene glycol diacrylate (PEGDA) to the microorganism;
III) preparing a dispersion medium in which a surfactant is added to the oil;
IV) injecting the cell solution and the dispersion medium into a sample inlet and an oil inlet of a microfluidic device, respectively;
V) passing the cell solution through a micropillar array zone of the microfluidic device and dispersing into a single cell body;
VI) contacting the dispersed single cell body with the dispersion medium;
VII) forming a hydrogel droplet containing the single cell body through the focusing cell of the microfluidic device together with the dispersion medium; And
VIII) reaching and collecting the hydrogel droplets at the sample outlet;
Wherein the cell-supporting method comprises the steps of:
9. The method of claim 8,
The cell solution in the step (II) is a microbial cell of 3 × 10 ⁸ to 8 × 10 ⁸ cells / mL which is the matrix of the LB medium, a polyethylene glycol diacrylate (PEGDA) of 24.51 to 26.03 wt% , 0.78 to 1.23 wt% potassium persulfate (KPS), 0.25 to 0.31 wt% of D-sorbitol and 36.44 to 37.13 wt% of distilled water.
9. The method of claim 8,
Wherein the cell solution and the dispersion medium of step (IV) are injected using a syringe pump.
delete

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