KR101544391B1 - Preparation method of micro hemisphere array plate, microfluidics chip comprising micro hemisphere array plate and culture method of cell spheroid using the same - Google Patents

Abstract

The present invention relates to a method of manufacturing a microsphere array plate, a microfluidic device including a microsphere array plate, and a method of culturing cell aggregates using the same.
When the cells are three-dimensionally cultured using the microfluidic array plate according to the present invention, the microfluidic device including the microsphere array plate, and the method of culturing the cell aggregate using the microfluidic array plate, Lt; / RTI > In particular, since the cells are cultured under similar conditions to the environment in the human body, it is possible to form the cell aggregate in a better state than the existing cell culture methods.
The cell aggregate cultured through the present invention can be directly used for cell therapy and can be obtained by culturing cells that are difficult to obtain artificially. In addition, since it is cultured by providing conditions similar to human body, it is possible to form cell aggregates of cells which are difficult to be aggregated in three dimensions. In addition, it is possible to co-culture two or more cells, and at this time, it is possible to form a cell cluster of excellent quality because it is cultured under conditions similar to human body. CONCLUSION Through the present invention, dramatic improvements in drug screening, cytotoxicity, and various tests can be achieved.

Description

The present invention relates to a microfluidic device, a microfluidic device, a microfluidic device, a microfluidic device, a microfluidic device, and a method for manufacturing a microfluidic device. using the same}

A microfluidic device including the microfluidic array plate, and a method for culturing the cell aggregate using the microfluidic device.

Cells in the body form aggregates in three dimensions through interaction with surrounding cells and extracellular matrix. These three - dimensional shapes play a very important role in biochemistry and mechanically in cell physiology. Particularly, the cell aggregation formed in a three-dimensional shape can be used for the clinical development of new drugs or for the differentiation using stem cells in the study on cells constituting general tissues, cells constituting organs, cancer cells and stem cells And is playing a very important role in

However, in general, it is very difficult to cultivate the cells in a three-dimensional shape. In particular, it is more difficult to cultivate human primary cells three-dimensionally. Due to these problems, it is generally cultured in 2D (2D) and used for drug screening and various experiments. However, when the two-dimensional culture is carried out, it is in a very different environment from that in the living body. Therefore, the characteristics of the cell itself or the tissue specificity of the cell used in the experiment are lost and as a result, There is a problem that it is very difficult.

Therefore, it is very important to cultivate cells in 3D (3D) shape in vitro, and many studies on this are underway. These three-dimensional culture methods include haning-drop culture, nonadhesive surface, spinner flask, and fotary system. However, these methods are not simple and difficult to mass-produce, and it is difficult to appropriately control the shape, .

Korean Patent Laid-Open No. 10-2013-0013537 (Patent Document 1) discloses a prior art document for the improvement of these problems on the manufacture of hemispherical microwells using surface tension and the formation of cell aggregates using the same. However, in Patent Document 1, since the micro hemisphere is not a precise manufacturing method, it can not achieve a perfect hemispherical shape. Therefore, even if a cell aggregate is formed, separation in microwell is not perfect in the collecting process, Are separated from the microwell, and the shape of the cell and cell aggregate is collected while being destroyed. In addition, since the cells are cultured without forming an environment similar to the fluid flow in the human body, the state of the formed cell aggregate is not excellent.

Patent Document 1. Korean Patent Publication No. 10-2013-0013537

SUMMARY OF THE INVENTION It is an object of the present invention to provide a microfluidic device and a microfluidic device, including a microsphere array plate and a microsphere array plate, which enable a cell aggregate to form an aggregate in a better state. . In particular, in the case of a microfluidic device including a microsphere array plate, there is a flow of a constant flow to form an environment similar to the environment in the human body, thereby culturing the cells to form a better cell aggregate, Lt; RTI ID = 0.0 > cell < / RTI >

According to an aspect of the present invention, there is provided a method of manufacturing a microsphere array plate,

1) attaching a photosensitive photoresist on a silicon substrate;

2) adjusting the height of the photosensitive photoresist through spin coating;

3) hemispherically etching the photoresist through overcuring;

4) depositing a primary metal layer on the etched surface after step 3);

5) depositing a secondary metal layer on the primary metal layer after deposition;

6) forming a mold core layer on the secondary metal layer;

7) planarizing the upper surface of the mold core layer after the step 6);

8) separating the mold core layer after step 7);

9) injection molding the microsphere array plate using the separated mold core layer as a mold; And

10) imparting hydrophilicity or hydrophobicity to the surface of the microsphere array plate;

.

A microfluidic device including a microsphere array plate according to another aspect of the present invention includes:

A sample injection unit injecting a sample containing a single cell or a plurality of cells, a cell culture liquid, and injecting the sample through a single or a plurality of channels;

Wherein the sample is moved through a single or a plurality of channels, the single or plural channels are in a zigzag shape, and the single or plural channels are connected to the pyramid Wherein the plurality of steps include a flow channel connecting the plurality of steps and including at least one channel higher than the upper step, and the plurality of steps includes a flow channel connecting the plurality of steps. And

A plurality of microspheres, which are connected to the sample mixing unit and are connected to channels constituting the lowest stage among the plurality of steps, and in which a plurality of single or mixed cells in the mixed sample are cultured in three dimensions to form a cell aggregate A cell cluster forming section including an array plate;

.

When the cells are three-dimensionally cultured using the microfluidic array plate according to the present invention, the microfluidic device including the microsphere array plate, and the method of culturing the cell aggregate using the microfluidic array plate, Lt; / RTI > In particular, since the cells are cultured under similar conditions to the environment in the human body, it is possible to form the cell aggregate in a better state than the existing cell culture methods. The cell aggregate cultured through the present invention can be directly used for cell therapy and can be obtained by culturing cells that are difficult to obtain artificially. In addition, since it is cultured by providing conditions similar to human body, it is possible to form cell aggregates of cells which are difficult to be aggregated in three dimensions. In addition, it is possible to co-culture two or more cells, and at this time, it is possible to form a cell cluster of excellent quality because it is cultured under conditions similar to human body. As a result, the present invention can achieve dramatic improvements in drug screening, cytotoxicity, and various tests.

FIG. 1 is a diagram illustrating a manufacturing process of the microsphere array plate according to the first embodiment. FIG.
2 is a photograph showing a microsphere array plate fabricated according to Example 1. Fig.
FIG. 3 is a schematic view showing a process of culturing hADSCs through a microsphere array plate fabricated according to Example 1. FIG.
FIG. 4 is a schematic view showing a three-dimensional co-culture through a microsphere array plate fabricated according to Example 1. FIG.
5 is a cross-sectional view of a microfluidic device including a microsphere array plate according to a second embodiment.
6 is a photograph of a microfluidic device including a microsphere array plate according to the second embodiment.
FIG. 7 is a photograph showing a three-dimensional culture of human cells forming a cell aggregate through a microsphere array plate fabricated according to Example 1. FIG.
Fig. 8 is a photograph showing cell aggregation formation in Comparative Example 1 and Example 1; Fig.
9 is a photograph comprehensively comparing the cases of Comparative Example 2 and Example 1. Fig.
FIG. 10 is a photograph showing the formation of cell aggregates using human hepatocytes in the case of fluid flow as in Example 2 and in the absence of fluid flow.
FIG. 11 is a photograph showing the results of culturing human primary hepatocytes with and without fluid flow as in Example 2. FIG.
12 is a photograph comprehensively comparing the cases of Comparative Example 2 and Example 2. Fig.
13 is a photograph showing the result of three-dimensional co-culture of human liver cells and hADSCs through a microsphere sphere array plate prepared according to Example 1. Fig.
FIG. 14 is a graph and a photograph showing the result of the functional measurement test when human liver cells and hADSC are three-dimensionally co-cultured through the microsphere sphere array plate prepared according to Example 1. FIG.
FIG. 15 is a TEM photograph of the inside of a three-dimensionally co-cultured cell cluster through a microsphere sphere array plate prepared according to Example 1. FIG.
16 is a photograph showing that a three-dimensional co-cultured cell aggregate is taken out from a hemisphere through a microsphere sphere array plate prepared according to Example 1, and shows excellent state.
17 is a photograph showing the result of co-culturing human primary hepatocytes and hADSC in 2D.
18 is a photograph showing the result of co-culturing human primary hepatocytes and hADSCs in 2D and then staining them.
FIG. 19 is a photograph showing albumin secretion when human primary hepatocytes and hADSCs were co-cultured on 3D in Example 1; FIG.
FIG. 20 is a photograph comprehensively comparing the cases where human primary hepatocytes and hADSC were co-cultured in 2D and 3D.

Accordingly, the present inventors have developed a microfluidic array plate manufacturing method capable of forming a cell aggregate in a better state while providing culture conditions in an environment similar to a human body, and a microfluidic device including a microsphere array plate As a result of intensive studies, the present inventors have discovered a microfluidic device array plate manufacturing method, a microfluidic device including a microsphere array plate, and a method of culturing a cell aggregate using the same.

Specifically, a method of manufacturing a microsphere array plate according to the present invention includes:

1) attaching a photosensitive photoresist on a silicon substrate;

2) adjusting the height of the photosensitive photoresist through spin coating;

3) hemispherically etching the photoresist through overcuring;

4) depositing a primary metal layer on the etched surface after step 3);

5) depositing a secondary metal layer on the primary metal layer after deposition;

6) forming a mold core layer on the secondary metal layer;

7) planarizing the upper surface of the mold core layer after the step 6);

8) separating the mold core layer after step 7);

9) injection molding the microsphere array plate using the separated mold core layer as a mold; And

10) imparting hydrophilicity or hydrophobicity to the surface of the microsphere array plate;

.

In the step 1), the silicon substrate is easy to adhere the photosensitive photoresist.

The photosensitive photoresist is generally referred to as a positive series in which a light-receiving portion is cross-linked when exposed to ultraviolet light (UV, 350 to 400 nm) is a negative series and a light-receiving portion is developed, It is easy to form a hemisphere, and both negative and positive types can be included.

The photosensitive photoresist to be attached in the step 1) preferably has a length of 100-1,000 mu m. When the length of the photosensitive photoresist is less than 100 탆, the diameter and depth are too small to form a hemisphere after etching. If the length of the photosensitive photoresist exceeds 1,000 탆, It is not preferable that the cells to be grown are difficult to aggregate and maximize the original substrate of the cells. Also, the length of the photosensitive photoresist should fall within the above-mentioned range, so that the cell aggregate is formed in an optimum condition to be excellent. It is also possible to adjust the depth of the hemisphere by adjusting the coating height and temperature condition of the photosensitive photoresist within the length range.

If the spin coating is performed as in step 2), it is possible to control the height of the photosensitive photoresist.

The photosensitive photoresist can form a hemisphere by etching the photosensitive photoresist through overcuring as in step 3). In the overcuring performed at this time, when the temperature or more suitable for the photosensitive photoresist is heated, the edges of the edge portions are rounded to form a curved surface. When the hemisphere is formed through the overcuring, the hemispheres can be more precisely formed as compared with the conventional hemispherical microwell manufacturing method, so that the cell aggregate can be formed well, and even after the culturing, Can be separated from the microsphere array plate.

In the step 4), the primary metal layer is deposited on the surface of the etched photosensitive photoresist. Although there is no particular limitation on the method of the deposition, the deposition is preferably performed using chemical vapor deposition, physical vapor deposition, can do. Preferably, the primary metal layer to be deposited is one or more selected from the group consisting of Cr, Ti, Au, Ni, Cu, Al, and Fe for easier separation of the mold core layer. When the height of the primary metal layer is less than 100 ANGSTROM, it is not preferable to increase the secondary metal layer by decreasing the degree of adhesion of the secondary metal layer, If the height of the secondary metal layer exceeds 500 ANGSTROM, peeling phenomenon in which the seed metal layer occurs may be undesirable.

After the deposition of the primary metal layer, it is preferable to deposit the secondary metal layer on the secondary metal layer. The secondary metal layer is for facilitating the electroplating of the metal core, and may be made of the same material as the metal core, It is preferably at least one selected from the group consisting of Au, Ag, Pt, Ni and Cu. The reason for depositing the primary metal layer and the secondary metal layer separately is that it is difficult to raise the mold core up to a desired height in the electroplating process because the secondary metal layer itself deteriorates the thin film adhesion. There is no particular limitation on the method of depositing the secondary metal layer, but the deposition can be preferably performed by chemical vapor deposition, physical vapor deposition, or the like. When the deposition height of the secondary metal layer is less than 1,000 ANGSTROM, the stress of the thin film itself is weak, which is not preferable for plating, and the height of the secondary metal layer is preferably not less than 1,000 ANGSTROM If it exceeds 2,000 A, the surface roughness (RMS) value becomes high, which is undesirable because it may affect the uniformity in forming the mold core layer in step 6).

In the step 6), a mold core layer is formed on the secondary metal layer. At this time, a preferable method of forming the mold core layer is a method of electroplating because the metal layer can be raised to a high level. The material of the mold core layer is preferably at least one selected from the group consisting of nickel, titanium and aluminum, and they are preferable because they have a strength enough to be used as a mold core.

In the step 7), the upper surface of the mold core layer formed by the step 6) is planarized. By flattening the mold core layer, the microsphere array plate injected by the step 9) can be mounted flat on the mold, and further, the injection moldability of the microsphere array plate to be manufactured can be improved. The planarization can be used without limitation as long as it is a method of planarizing the mold core layer so as to achieve the level of the microsphere array plate. Preferably, the planarization is performed by chemical mechanical planarization (CMP), bright dipping, Tumbling barreling, buffing, belt sanding, picking, and the like.

In the step 8), the mold core layer is separated. The method of separating the mold core layer is not particularly limited. Preferably, the silicon substrate is melted and removed with KOH, TMAH, Is removed with an etching solution and separated. Also, although not particularly limited in this process, if the secondary metal layer is different from the mold core, the secondary metal layer can be also separated.

In the step 9), the microsphere array plate is injection-molded using the mold core layer as a mold. The injection molding method can be used without particular limitation, as long as it is suitable for injection molding of the microsphere array plate.

The material for the injection molding may be any material that can be injection-molded, and is preferably selected from the group consisting of polycarbonate (PC), polymethylmethacrylate (PMMA), polystyrene (PS), and cyclic olefin copolymer It can be any one or more.

In step 10), the surface of the microsphere array plate may be hydrophilized or hydrophobically imparted. There is no particular limitation on the method of imparting hydrophilicity or hydrophobicity to the surface of the microsphere array plate. Preferably, And the degree of hydrophobicity and hydrophobicity of the hydrophilic polymer. Also, it is preferable to minimize the occurrence of air bubbles in the hemisphere when the cell aggregate is cultured through the microsphere array plate which is finally produced through such surface modification, thereby maximizing the formation of a three-dimensional aggregate of cells.

The hemisphere of the microsphere array plate manufactured by the above-described method preferably has a diameter of 100-1000 μm, and it is preferable to allow the formation of a three-dimensional cell cluster in the above range.

The method of manufacturing the microsphere array plate according to the present invention forms a more hemispherical and semi-spherical array shape than the conventional hemispherical microwell manufacturing method. Therefore, it forms a cell cluster closer to three dimensions.

Also, when the cell aggregate is cultured using the microsphere array plate manufactured by the method of manufacturing the microsphere sphere array plate according to the present invention, the formation of the cell aggregate is superior to the conventional method. Also, separation of the formed cell aggregate from the microsphere array plate can be performed without damage to the cell aggregate. This corresponds to a much better formation of the cell aggregate than the conventional two-dimensional culture method.

A microfluidic device including a microsphere array plate according to another aspect of the present invention includes:

A sample injection unit 1 for injecting a sample containing a single cell or a plurality of cells, a cell culture liquid, and injecting the sample through a single or a plurality of channels 4;

Wherein the sample is moved through a single or a plurality of channels, the single or plural channels are in a zigzag shape, and the single or plural channels are connected to the pyramid And a flow channel (5) connecting the plurality of steps, wherein the plurality of steps further include one or more channels higher than the upper step in a lower step, (2); And

A plurality of microspheres, which are connected to the sample mixing unit and are connected to channels constituting the lowest stage among the plurality of steps, and in which a plurality of single or mixed cells in the mixed sample are cultured in three dimensions to form a cell aggregate A cell cluster forming section 3 including an array plate;

.

The microfluidic device according to the present invention enables a single cell or a plurality of cells to form a cell cluster in a three-dimensional manner, and when the cell cluster is formed, a fluid is passed under conditions similar to the environment in the human body to form a cell cluster . As the most important constituent of the human body, about 60% of adults are water, so the cells in the body form aggregates in the presence of fluid motion such as blood flow. Therefore, the present invention provides conditions similar to those in the human body to form a cell cluster of higher quality.

The sample and the cell culture liquid may be injected into the sample injection unit. The cells may be single or multiple cells, and the injected cells form a cell aggregate in the microsphere array plate. When injecting the sample including the cell and the cell culture liquid into the sample injection unit, it is preferable to inject the sample at a flow rate in the range of 10 nL / min to 10 uL / min. Injecting the sample at the flow rate in the above- In particular, when injected at a rate of less than 10 nL / min, it is not desirable to remove unnecessary cells around the hemisphere at the same time, and it is difficult to remove unnecessary cells around the hemisphere. The cells are difficult to sink into the hemisphere of the hemispherical array plate, which is not preferable. The cell culture fluid injected into the sample injecting section can be used without any particular limitation as long as it is a substance capable of culturing cells and flowing as a fluid. In addition, the inlet for injecting the sample in the sample injecting unit may be a single channel or a plurality of channels through which the sample can be injected in a single or multiple paths.

Meanwhile, the sample mixing portion is a portion where the sample is mixed while moving. At this time, the movement of the sample is performed through a single channel or a plurality of channels because the sample can be more easily mixed. If the diameter of the channel is less than 500 μm, the number of microspherical arrays becomes small and the fluid pressure of the channel becomes high, which is undesirable. When the diameter of the channel is 2.0 mm , It is difficult to move the sample under conditions similar to the human body, and it is not preferable to control the microsphere array. In order to more actively mix the samples, the single or plural channels are preferably zigzag. The single or plural channels in the zigzag form are repeatedly placed in the form of a pyramid through a plurality of steps. If the plural steps are repeated in the form of pyramid, the mixing of the sample and concentration gradient depending on the chamber can be actively performed. In addition, in order to implement the pyramid shape, the plurality of steps preferably further include one or more channels in a higher level as it goes to a lower level. And the plurality of steps are all connected by a flow channel connecting them. Such a sample mixing section achieves excellent effects of mixing samples through the above-described structure.

Meanwhile, the cell aggregate forming unit is connected to the lowest mixing stage among the plurality of stages while being connected to the sample mixing unit. A plurality of single or mixed cells in the mixed sample are cultured three-dimensionally on a plurality of microsphere array plates to form a cell aggregate. It is preferable that a plurality of the microsphere array plates have a similar microbial sphere array plate so as to provide a similar environment to the environment in the human body without lowering the flow rate of the culture liquid to form a good cell aggregate. Also, although not particularly limited, the number of microsphere array plates is preferably equal to the number of channels included in the lowest stage among the plurality of steps. This is because the formation of the cell aggregate by directly connecting the microsphere array plates to each channel constituting the lowest stage does not interfere with the sample movement up to the previous stage, As shown in FIG.

Although the microsphere array plate has no particular limitation, the microsphere array plate manufactured by the method of fabricating the microsphere array plate according to another aspect of the present invention can form a cell aggregate of higher quality And the reproducibility of the hemispherical formation is much higher than the conventional method. Also, separation of the formed cell aggregate from the microsphere array plate can be performed without damage to the cell aggregate. This corresponds to a much better formation of the cell aggregate than the conventional two-dimensional culture method.

Meanwhile, a plurality of single cells or mixed cells are submerged to form a cell aggregate within the hemispheres of the microsphere array plate. The impurities and unnecessary cells around the hemispheres of the microsphere array plate are separated from the hemisphere It is removed by the flow rate of the sample. This process is repeated several times to form and cultivate the cell aggregate within the hemisphere of the microsphere sphere array plate.

The microfluidic device according to the present invention includes three types of microfluidic devices including a microfluidic array plate. First, a sample containing cells and cell culture fluids is put in the microfluidic device, And secondly, it is capable of injecting two or more samples together including a functional culture solution and mixing them at the same time. Thirdly, a plurality of single or mixed cells in a hemisphere of a microsphere array plate can be cultured while forming a cell aggregate, and various cells can be made into a three-dimensional spherical shape under conditions similar to the environment in the human body.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example

Example  1: Micro Hemisphere  Fabrication of array plates and incubation of cell aggregates

<Micro Hemisphere  Fabrication of array plate>

A photosensitive photoresist was used in order to realize a microsphere pattern having a size of 500 .mu.m. Negative and positive photosensitive photoresists were used.

The height of the microsphere can be adjusted by spin-coating the photosensitive photoresist in the region of 300 μm, and the coated photoresist can be formed into a microsphere by over curing at a temperature of 150 ° C.

A seed metal layer was deposited on a microsphere array pattern formed on a silicon substrate using a thin film deposition apparatus. Titanium was used as a seed metal, and the height of the seed metal was increased by 300 Å. The secondary metal layer was made of Nickel and its height was raised to 1,500 Å. E-beam evaporator and D.C magnetic sputter were used for deposition of primary and secondary metal films.

The nickel layer was raised to a height of 0.8 mm by electro-plating on the secondary metal thin film layer. After the electroplating was completed, the rear surface was subjected to a CMP (Chemical Mechanical Planarization) process And polished so as to have a uniform flatness.

The nickel layer including the polished secondary metal thin film layer was separated and used as a mold core. The mold was mounted on a mold so as to perform injection molding.

The plastic material used for injection molding was injection-molded using P.S. (Polystyrene).

The completed microsphere plate controlled the hydrophilicity and hydrophobicity of the surface through oxygen plasma treatment and chemical surface treatment. Through such surface modification, the phenomenon of air bubbles in the microsphere array plate and the microsphere array microfluidic device was minimized And maximized the formation of three-dimensional hemispheres of the cells.

FIG. 1 is a schematic view showing a manufacturing process of the microsphere array plate. FIG. 2A is a photograph showing the microsphere array thus manufactured, and FIG. 2B is a microsphere array plate .

<Micro Hemisphere  Culture of cell aggregates using array plates>

1) Isolation of hADSC

The hADSC is isolated from adipose tissue removed from patients who underwent plastic surgery or liposuction. Isolation of hADSC first removes the blood fraction from the separated adipose tissue. Using a clean PBS solution, wash the cells repeatedly until the blood fraction is clear. After that, Type1 collagenase is dissolved in PBS at 0.2% and mixed with the washed cells, thereby breaking the intercellular junction and separating the tissue into cells. Mix the prepared collagenase solution with the washed fat tissue and shake for one hour. Collect the emulsified tissues, centrifuge at 600 g for 10 min, collect only the pellet, and filter them on 100 μm strainer. The filtered cells were washed three times in a T-75 flask. After passage 3-4, the cells were removed and used for 3-D culture.

2) hADSC culture

For three-dimensional culture, the hADSC cells were uncoated in the medium, and the cell solution was seeded on the microsphere array plate. After the cells had settled in the hemisphere, the remaining cells on the surface were removed by washing with the medium, and the new medium was re-inserted and cultured in an incubator in three dimensions. Fig. 3 is a schematic diagram showing such a culturing process.

3) Three-dimensional co-cultivation

4 is a schematic diagram showing a state in which a plurality of cells are co-cultured to form a three-dimensional cell cluster. In other words, after mixing two kinds of cells at the desired ratio, the cells are cultured in the hemisphere as in the previous procedure. After one day of incubation, the two cells are closely connected and integrated into a single sphere, resulting in a fully coupled, three-dimensional co-culture model.

Example  2: Micro Hemisphere  Culture of cell aggregates using microfluidic devices including array plates

<Micro Hemisphere  Development of a microfluidic device including an array plate &gt;

A microfluidic device including the microsphere array plate manufactured by the method of Example 1 was fabricated. Such a microfluidic device may be divided into a cell aggregation section including a sample injection section, a sample mixing section, and a microsphere array plate. 5 is a cross-sectional view thereof. 6 is a photograph of a microfluidic device including the microsphere array plate thus fabricated.

1) Isolation of human-derived hepatocytes

Human hepatocytes were isolated from hepatic tissue removed from patients who underwent hepatic resection using the conventional collagenase-two-step method. Briefly, the isolated liver tissue was first removed from the blood through EGTA perfusion, and then perfused with a type 2 collagenase solution to emulsify the collagenase into liver tissues. Thereafter, the hepatocytes were separated from the tissue through two washing steps, and the separated hepatocytes were immediately used immediately after separation.

2) Cell culture in microfluidic array plate microfluidic device

The hepatocytes were mixed with the medium and the cells were allowed to slowly pass through the chip at a flow rate of 1 uL / min in the microfluidic device so that the cells were submerged in the hemisphere of the microsphere array plate, Cells around the microsphere array were also effectively removed. This process was repeated several times to cultivate human-derived mammalian cells in a microfluidic device including a microsphere array plate so that the cells could grow into a three-dimensional sphere in the microfluidic device.

3) Three-dimensional co-cultivation

In the same way as described above, the cells are loaded, but two or more cells can be mixed with the medium in the desired ratio. In this way, three-dimensional co-cultivation can be performed in a microfluidic device having a flow rate.

On the other hand, the main functions of the microfluidic device including such a microsphere array plate are as follows.

A concentration gradient function capable of flowing into each chamber by concentration of a culture medium capable of culturing the first cell, a sample capable of inducing cell differentiation and characteristic transformation or function of the cell,

Since the second culture medium and the functional sample must be put together, a micromixer function capable of uniformly mixing two or more solutions,

The third is the ability to integrate various hemocytes into the hemispheres of the microsphere sphere plate to make a three-dimensional cell spheroid.

Thus, a fluid of 10 nL / min to 10 uL / min can be freely changed in the microchannel to provide an optimized microfluidic environment for three-dimensional cell culture.

Comparative Example

Comparative Example  One

Cells were cultured in the same manner as in Example 1, except that cells were cultured in hemispherical microwells prepared by existing methods.

Comparative Example  2

The cell aggregate was cultured in a two-dimensional (2D) manner by the existing method without injecting and moving the sample.

Experimental Example

Experimental Example  1: Micro Hemisphere  Observation of cell aggregates cultured on array plates

The cell aggregate cultured in the microsphere sphere array plate of Example 1 and the cell aggregate cultured by Comparative Example 1 were observed. The results are shown in FIGS. 7 and 8.

In FIG. 7A, it can be confirmed that the rounded cell spheres were well formed within one day after placing the hADSC in the microsphere array plate according to Example 1 above. In addition, when viability was observed by the Live / Dead assay after incubating the cells prepared in Fig. 7B for 9 days, it was confirmed that most of the cells were alive and well. In addition, when the microsphere was collected on the 9th day by collecting the cell sphere formed in C in FIG. 7, it was confirmed that microvilli, which is a characteristic of hADSC, appeared well, and several cells gathered to form a complete sphere . Since such a microsphere array plate can be produced in a desired number of areas and in a large area, it is easy to mass-produce, and the method is also very easy, and thus it is confirmed that it is very suitable for mass production of hADSC cells in a healthy state quickly and easily.

On the other hand, when the hADSC is raised as shown in FIG. 8A to Comparative Example 1, since the hADSC becomes an adherent cell adhering to the surface after 10 days from the generation of the cell spheres, It was confirmed that it spreads on the floor. However, in the case of Example 1, as shown in FIG. 8B, since the depth of the film can be adjusted in the case of Example 1, the cell structure remains intact even after the elapse of time in a chip made deeper, Long-term storage is possible. This shows that cells that do not have the aggregating properties can clump well and can regulate the depth of cells for long periods of time.

On the other hand, FIG. 9 is a graph showing the relationship between the 2D (FIGS. 9A and 9B) and the 3D (FIGS. 9C and 9D) 9 B, D) and SEM (Fig. 9E).

Experimental Example  2: Micro Hemisphere  Observation of cell aggregates cultured in microfluidic devices including array plates

Humam liver cells and samples were injected through the microfluidic device of Example 2 and cultured in a microfluidic array plate with no flow of fluid with the cell cluster (Figure 10B) in the flow of fluid Experiments were conducted to observe the cell aggregate (Fig. 10A).

In the case of human liver, aggregation does not work well in some ways, since the cell state is not very good when cells are obtained from partial hepatectomy.

As can be seen from FIG. 10A, aggregation did not occur when a human liver cell aggregate was formed in a state where there was no flow rate similar to a human body.

On the other hand, as can be seen from FIG. 10B, when a fluid flow is used through the microfluidic device of Example 2, cells having poor quality can be aggregated. Therefore, in a microfluidic device including a microsphere array plate, human primary cells, which are difficult to obtain in a good state, can be effectively cultured in three dimensions. In addition, Can give a good effect. By creating an environment similar to that of the human body, it not only maximizes the normal function of the cells, but also provides a continuous supply of fresh media through continuous fluid flow. By utilizing the three characteristics of microfluidic device, it is possible to use drugs for cell differentiation induction and functional analysis to continuously screen drug.

Fig. 11 is a photograph showing the result of measuring an experiment for confirming the activity of human hepatocytes by maintaining the cell culture for 3 days and staining them. As can be seen from FIG. 11A, when human primary hepatocytes, which are not in good condition, are cultured in Comparative Example 2 in which there is no fluid flow, more than half of the cells are dead, as seen from the Live / Dead assay, The result is that the dye itself is not good. On the other hand, in Example 2 (FIG. 11B) in which the flow of the fluid is observed, it can be seen that the cell viability is encouragingly different from the result in Comparative Example 2 in which there is no flow. Considering that the viability of the first human liver cell was about 40-60%, confirming that viability improves with time in Example 2 with flow, the living cells remain more firmly, Can be confirmed. This demonstrated that when poorly conditioned cells were cultured in Example 2 with flow, the condition was improved and this showed the possibility of making human primary cells, which are the poorest but most accessible cell source, available for experimentation .

On the other hand, FIG. 12 shows a comparison between 2D (FIGS. 12A and 12B) and Comparative Example 2 (FIGS. 12C and 12D) (Fig. 12B, D), Live / Dead (Fig. 12E) and SEM (Fig. 12F).

Experimental Example  3: Plural  Three-dimensional co-culture with direct binding of human cells ( Co - culture )

When human liver cells and hADSC were mixed and incubated in a microsphere array plate according to Example 1, experiments were carried out to confirm that these cells could be directly coupled and three-dimensional co-culture was possible. The results are shown in FIG. FIG. 13A shows that two kinds of cells are united together. Also, in FIG. 13B, the vitality expressed in green is also very high, suggesting that one sphere composed of two cells is cultured in the hemisphere in a very healthy state. In Fig. 13C, SEM photograph taken on the third day after culturing showed that the two cells were completely united with each other without any boundary or discrimination. In Fig. 13D, the photograph of the ninth day after the incubation showed that they were more tightly packed together than the third day, and that the apparent boundary completely disappeared. This shows that the two cells are directly coupled to each other to form a complete unit, and it can be said that it is a new three-dimensional co-cultivation model by direct coupling.

Fig. 14 shows the result of performing the functional test with the three-dimensional co-culture model by direct coupling. In FIGS. 14A and 14B, secretion of activated albumin (FIG. 14A) and Urea (FIG. 14B) was secreted similarly to that of normal hepatocytes alone, have. Also, the level was also high in the Cytochrome P450 reductase staining indicated by red in C and D of FIG. 14, and the graph showing quantitative CYP3A4 activity in E of FIG. 14 showed a high level continuously, metabolism-related functions. By demonstrating that the cells of this new unitary mass can function in a functional way, we have realized a similar state to that of various intercellular junctions in actual organs and have shown that this unit can be used in vitro and in vivo Giving.

As can be seen from FIG. 15, when the inside of the cell co-cultured by TEM is taken, it can be seen that various activated cell characteristics are shown as shown in FIG. Many mitochondria, healthy vasculature, tight junctions specific to hepatocytes and even bile canaliculi are observed, and glycogen and collagen, ECM, are also observed. Peroxisome and rough ER were also confirmed and endocytosis was observed, confirming that the cells were in a very healthy state, morphologically and functionally.

FIG. 16 is a photograph showing the viability of a cell by performing a Live / Dead assay by taking out a three-dimensionally co-cultured cell population (FIG. 16B) by direct binding with a poorly sorted hepatocyte (FIG. 16A). As can be seen in FIG. 16, the number of living and dead cells in most of the cells is very small. Thus, it can be seen that the process of taking out the cells from the microsphere array plate according to Example 1 does not damage the cells , Which suggests that cells can be taken out of the cell only in three dimensions in a hemisphere, taken out and used in different ways elsewhere.

Also shown in FIG. 17 are human primary hepatocytes (FIG. 17A) and Hadsc (FIG. 17B), and those cells were co-cultured in 2D (FIG. Although there is direct binding, it can not be seen that the two cells become a single unit, and it can be seen that they are co-cultured only to the extent that two cells are attached to one space. When the two-dimensional co-cultured model was used to stain albumin-secreted regions, the activity of the cells was confirmed (FIG. 18), indicating that the cells exhibited almost no activity in red.

On the contrary, when co-cultured in the microsphere array plate prepared according to Example 1 (FIG. 19), albumin expressed in green is very active and the activity of the cells is much better. FIG. 20 is a photograph showing the 3D case (FIGS. 20C, 20D, 20F, and 20F) in a 2D environment (FIGS. 20A and 20B), which is the same as the embodiment of the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is natural.

1. Sample injection section
2. Sample mixing section
3. Cell aggregation forming part
4. Channel
5. Flow channel

Claims (16)

1) attaching a photosensitive photoresist on a silicon substrate;
2) adjusting the height of the photosensitive photoresist through spin coating;
3) hemispherically etching the photoresist through overcuring;
4) depositing a primary metal layer on the etched surface after step 3);
5) depositing a secondary metal layer on the primary metal layer after deposition;
6) forming a mold core layer on the secondary metal layer;
7) planarizing the upper surface of the mold core layer after the step 6);
8) separating the mold core layer after step 7);
9) injection molding the microsphere array plate using the separated mold core layer as a mold; And
10) imparting hydrophilicity or hydrophobicity to the surface of the microsphere array plate;
&Lt; / RTI &gt;
The method according to claim 1,
Wherein the photosensitive photoresist has a length of 100-1,000 占 퐉.
The method according to claim 1,
Wherein the material of the primary metal layer is at least one selected from the group consisting of Cr, Ti, Au, Ni, Cu, Al and Fe.
The method according to claim 1,
Wherein the material of the secondary metal layer is at least one selected from the group consisting of Au, Ag, Pt, Ni, and Cu.
The method according to claim 1,
Wherein the material of the mold core layer is at least one selected from the group consisting of nickel, titanium, and aluminum.
The method according to claim 1,
Wherein the injection molding of the microsphere array plate is performed using at least one selected from the group consisting of polycarbonate (PC), polymethylmethacrylate (PMMA), polystyrene (PS), and cyclic olefin copolymer (COC) A method of manufacturing a sphere array plate.
The method according to claim 1,
Wherein the hemispheres of the microsphere array fabricated by the above manufacturing method have a diameter of 100-1000 μm.
delete A method for culturing a cell aggregate in which a cell aggregate is cultured from a microsphere sphere array plate produced by the production method according to any one of claims 1 to 7.
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