WO2013012127A1 - 미세 세포 배양 장치 및 그 제조 방법, 및 미세 세포배양 장치를 이용한 세포 배양 방법 - Google Patents
미세 세포 배양 장치 및 그 제조 방법, 및 미세 세포배양 장치를 이용한 세포 배양 방법 Download PDFInfo
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M1/00—Apparatus for enzymology or microbiology
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
Definitions
- the present invention relates to a micro-device for culturing cells and a method for manufacturing the same, and more particularly to a low-cost, high-efficiency micro cell culture device, The method and the cell culturing method using this micro cell culture apparatus are related.
- the classical cell culture method was capable of cell culture on an open two-dimensional plane, but there is a disadvantage in that a large amount of expensive three-dimensional cultured polymeric material to serve as a scaffold is required for the three-dimensional culture.
- Korean Patent Laid-Open Publication No. 2011-0003526 Publication Date Jan. 12, 2011
- a device for culturing cells in the three-dimensional framework By culturing the cells in a microfluidic channel in a condition and composition close to the in vivo environment, a plurality of reagents and the like can be used to culture the cells with a very small amount of reagents, cultures, and cells as compared to the classical cell culture method described above.
- the stability of the three-dimensional skeleton is very high in the surface tension and injection pressure, temperature, size, shape, number and spacing of the microstructure. It is closely related and difficult to use universally and easily.
- the size, shape, number, and spacing of the microstructures should also seriously consider the surface characteristics of the material forming the microstructures.
- the process must additionally be provided.
- the amount of fluid movement through the three-dimensional skeleton, the concentration of the medium and chemicals supplied to the cells depending on the shape, size, number, and spacing of the posts.
- the gradient will be different. This makes it impossible to culture cells under uniform flow and concentration gradient conditions, and this uncertainty makes it difficult to precisely control the microenvironment using the microcellular culture system, thereby making it impossible to make accurate biological experiments and analysis.
- a microprocess is indispensable.
- the microstructure has a size of several tens of micrometers or less, photolithography and reactive ion etching are required. Efficient time and costly micro-etching process is indispensable.
- the minute error that may occur during this process also affects the size and shape of the post, which may render it impossible to form a three-dimensional skeleton, and thus, the successful production efficiency of the culture apparatus is not high, resulting in additional cost and time loss. There is a problem that can be.
- an object of the present invention is to provide a low-cost, high-efficiency fine cell culture apparatus and a method for producing the same.
- Micro-cell culture apparatus for achieving the above object, a plurality of microfluidic flow fluid is movable, and one or more injection ports for injecting the fluid into the microfluidic flow path,
- the microfluidic flow passages are characterized by having a different height from each other.
- the first microfluidic channel on the basis of the flat surface and the second microfluidic channel having a lower height than the first microfluidic channel corresponding to Forming a first microstructure having a height equal to the height of the second microfluidic channel in the region, and at a height of the first microfluidic channel in the region corresponding to the first microfluidic channel on the first microstructure; Forming a second microstructure having a height minus the height of the second microfluidic channel; applying and curing a liquid material forming the microfluidic channel on the first and second microstructures; And bonding the cured liquid material to a substrate to form the first and second microfluidic flow passages penetrating each other, and polymeric water in the second microfluidic flow passage. Injecting the vagina to form a three-dimensional skeleton, and injecting a fluid into the first microfluidic flow path.
- Cell culture method using a micro-cell culture apparatus having a plurality of microfluidic flow channels forming a three-dimensional skeleton in at least one microfluidic flow passage having a relatively low height of the plurality of microfluidic flow paths
- Injecting a polymeric material to make the polymer material curing the polymeric material to form a three-dimensional skeleton, and injecting a fluid for cell culture into a microfluidic channel in contact with the three-dimensional skeleton. It features.
- the present invention can be produced through a simple low-cost process, cell culture in a specific position inside the microchannel without sophisticated fluid control devices such as pumps, in particular cell culture in a specific region inside the microchannel
- micro-posts for the stability of the three-dimensional skeleton
- it can be applied to a variety of cells and skeletal forming materials, can be a low-cost process, all aspects of the three-dimensional skeleton It is open to the microfluidic flow path injected into the device, and precisely controls the flow of the interstitial fluid and the concentration gradient of the molecules in the three-dimensional skeleton, thereby enabling accurate cell migration and differentiation experiments.
- FIG. 1 is a perspective view showing a micro cell culture apparatus according to an embodiment of the present invention
- Figure 2 is a cross-sectional view of the micro cell culture apparatus of Figure 1
- FIG. 3 is a perspective view illustrating an example in which a three-dimensional skeleton and fluid are injected into a micro cell culture device of FIG. 1,
- FIG. 4 is a cross-sectional view of FIG. 3,
- FIG. 5 is a cross-sectional view showing a state of culturing cells in a three-dimensional skeleton using the present invention micro cell culture apparatus
- 6a and 6b are micrographs showing the cell culture of FIG. 5,
- Figure 7 is a cross-sectional view showing the appearance of inducing interstitial flow through the three-dimensional skeleton in the micro-cell culture device of the present invention
- FIG. 8A is a perspective view illustrating an embodiment in which a specific chemical is injected into one microfluidic channel to form a concentration gradient of a specific chemical inside a three-dimensional skeleton,
- FIG. 8B is a graph showing a concentration gradient of a specific chemical formed in a three-dimensional skeleton by injecting a specific chemical into one microfluidic channel
- FIG. 10 is a cross-sectional view showing an embodiment for co-culture of heterologous cells using the micro cell culture apparatus according to the present invention
- FIG. 11 is a photograph showing an example of co-culture of heterologous cells using the apparatus of FIG. 10,
- FIG. 12 is a cross-sectional view showing another embodiment for co-culture of heterologous cells using the micro cell culture apparatus according to the present invention.
- FIG. 13 is a photograph showing an example of co-culturing heterologous cells using the apparatus of FIG. 12.
- FIG. 14 is a cross-sectional view showing an example where a fluid is injected into a relatively high microfluidic flow path
- FIG. 15 is a process flowchart showing a method for producing a micro cell culture device according to the present invention.
- 16 is an exemplary view of a microstructure formed by cutting and then overlapping a tape.
- FIG. 1 is a perspective view showing a micro cell culture device according to the present invention as an embodiment to help understanding of the present invention
- Figure 2 is a cross-sectional view of the micro cell culture device according to the present invention.
- Micro-cell culture apparatus comprises a first microfluidic flow path 200, the fluid having a specific height is movable; A second microfluidic flow passage 201 penetrating with the first microfluidic flow passage 200 and having a height lower than that of the first microfluidic flow passage 200; A first injection hole 300 for injecting a fluid into the first microfluidic flow path 200; And a second injection hole 301 for injecting a fluid into the second microfluidic flow path 201.
- the microfluidic flow paths 200 and 201 may be formed by bonding the material 100 forming the first or second microfluidic flow paths 200 and 201 to the substrate 101 and having different heights.
- the plurality of microfluidic flow passages 200 and 201 may have one or more injection holes 300 and 301, respectively.
- the plurality of microfluidic flow passages 200 and 201 having different heights penetrate each other, and each of the microfluidic flow passages 200 and 201 is positioned on a plane parallel to the substrate 101 and has a specific height. Can be.
- the material 100 and the substrate 101 forming the microfluidic flow paths 200 and 201 are preferably optically transparent, and in the present invention, polydimethylsiloxane (PDMS) and polymethylmethacrylate formed of one or more of various plastics or glass such as polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins, polyimides, polyurethanes, and the like May be
- PDMS polydimethylsiloxane
- PMMA polymethylmethacrylate
- PMMA polyacrylates
- polycarbonates polycyclic olefins
- polyimides polyimides
- polyurethanes and the like
- the surface of the material 100 and the substrate 101 forming the microfluidic flow paths 200 and 201 is more hydrophilic, and when the contact angle of the fluid to be injected is 90 degrees or less, the fluid is not required without an additional peripheral device such as a pump. May be spontaneously injected into the microfluidic flow paths 200 and 201.
- a pump for injecting the fluid Additional peripherals may be required.
- the movement of fluid from the microfluidic flow path having a lower height to the relatively higher height microfluidic flow path is It is suppressed by the surface tension of the injected fluid, and vice versa, the fluid moves smoothly. Therefore, if the fluid is first injected into the lower microfluidic channel, the lower microfluidic channel is filled first.
- Figure 3 is a perspective view showing an example of injecting a three-dimensional skeleton and fluid for cell culture in the micro-cell culture apparatus according to the present invention
- Figure 4 is a three-dimensional skeleton and fluid for cell culture in the micro-cell culture apparatus of Figure 3 Is a cross-sectional view showing an example of injection.
- Micro-cell culture apparatus comprises a first microfluidic flow path 200, the fluid 400 is movable; A second microfluidic flow passage 201 in contact with the first microfluidic flow passage 200 and having a lower height than the first microfluidic flow passage 200; Three-dimensional skeleton 401; One or more inlets 300, 301 for fluid injection.
- the three-dimensional skeleton 401 may be injected into the second microfluidic channel 201 having a lower height than the first microfluidic channel 200. At this time, it is preferable to inject a material forming the three-dimensional skeleton 401 through the second injection hole 301 away from the substrate 101 by the height of the second microfluidic flow path 201, in which case the three-dimensional skeleton 401 Forming material is first filled only in the second microfluidic flow path (201).
- a three-dimensional skeleton 401 is formed.
- the sol-gel is changed to store for a predetermined time at a specific temperature at which a polymerization reaction can occur, or to irradiate a light having a specific wavelength or cause a polymerization reaction. You can also add additional substances that cause it.
- the specific temperature, time, wavelength of light, and material vary depending on the type of polymeric material forming the three-dimensional skeleton 401.
- the three-dimensional skeleton is intended to form a concentration gradient of the chemical through the diffusion of a specific chemical through the three-dimensional skeleton or cells in or on the surface thereof, to form the three-dimensional skeleton
- the polymeric material Matrigel, Puratrix, Collagen, Fibrin gel, PEGDA, or Alginate may be used.
- Alginate when the temperature rises above room temperature, sol-gel transition may occur within several ten minutes to form a skeleton, and in the case of PEGDA, when irradiated with light of UV ambient wavelength Polymerization can occur to form a backbone.
- Alginate Alginate
- PEGDA when irradiated with light of UV ambient wavelength Polymerization can occur to form a backbone.
- Alginate Alginate
- the material and the cells forming the three-dimensional skeleton can be mixed and injected into the second microfluidic flow path 201, and the three-dimensional skeleton 401 is formed. Thereafter, a medium for supplying nutrients to the cells may be injected into the first microfluidic flow path 200.
- the three-dimensional skeleton 401 If cell culture is to be performed on the surface of the three-dimensional skeleton 401, after forming the three-dimensional skeleton 401, it is preferable to inject the cells through the first microfluidic channel 200.
- the solution containing the specific chemical is injected through the first microfluidic flow path 200 Just do it.
- the injected chemical is moved through the three-dimensional skeleton 401 by diffusion, where the contact with the solution containing the chemical has the highest concentration, and the farther it is from the three-dimensional skeleton 401 The concentration of chemicals will be lowered.
- the concentration gradient can be formed, and the concentration gradient is influenced by the diffusion characteristics, the size, the injection time, the initial concentration difference, the type of the 3D skeleton, and the like of the specific chemical.
- the concentration gradient may vary depending on the flow rate of the injected solution.
- PDMS is used as the material for forming the microfluidic flow paths 200 and 201, and a polystyrene plate or a glass substrate is used as the substrate 101.
- the height of the second microfluidic channel 201 that is, the three-dimensional skeleton 401, which has a relatively low height is 100 ⁇ m
- the first microfluidic channel 200 that is, the medium is relatively high, is injected.
- the height of the flow path is 200 ⁇ m.
- the width of the three-dimensional skeleton 401 is 500 ⁇ m.
- Silane may be used to bond PDMS and polystyrene in this embodiment.
- the polystyrene is first immersed in a 1% silane solution, reacted at room temperature for 20 minutes, and then washed.
- Glass may also be used as a substrate.
- irreversible bonding is achieved immediately after the oxygen plasma treatment is applied to the glass and PDMS, respectively, without any special surface treatment.
- the surface properties of the material 100 and the substrate 101 forming the microfluidic flow paths 200 and 201 are advantageous as they are hydrophilic.
- the surfaces of the material 100 and the substrate 101 forming the microfluidic flow paths 200 and 201 can be made hydrophilic, and the contact angle of the fluid with the surface increases with time, and therefore, within 1 hour after the bonding process.
- the fluid includes a material, a medium, and the like forming the three-dimensional skeleton 401.
- a peripheral device such as a pump or the like may be used, or the surface treatment process such as the oxygen plasma may be performed once more.
- the properties of the surface can be kept hydrophilic for several days to several weeks.
- the surface hydrophilicity can last longer.
- the ratio (width / height) of the width and the height of the second microfluidic flow path 201 it is preferable to keep at 4 or more.
- the ratio of the width and height is smaller, only the second microfluidic flow path 201 may be filled with the fluid first, and if the fluid is injected with too much pressure, the first fineness beyond the surface tension of the fluid Fluid may leak into the fluid passage 200.
- the first microfluidic flow path (201) than the fluid of the second microfluidic flow path 201 Since the fluid of 200 is sucked first, the fluid can be easily filled only in the second microfluidic flow path 201.
- FIG. 5 is a cross-sectional view showing the culture of cells in a three-dimensional skeleton using the micro-cell culture apparatus according to the present invention
- Figures 6a and 6b is a three-dimensional skeleton using the micro-cell culture apparatus according to the present invention It is a micrograph showing the culture of cells inside.
- the used cell 500 is a type of breast cancer cell, and is MCF7 cell
- the used three-dimensional skeleton 401 uses 10 mg / ml Matrigel and 2 mg / ml collagen I (Collagen type I) 1: 1. Used to mix. In addition, a small amount of 5 ⁇ L or less in total was sufficient to form the framework.
- the 30% Matrigel-Collagen mixed solution of the above cells is first injected into a relatively low microfluidic channel, and then Matrigel- in a 37 ° C. incubator for 30 minutes.
- the collagen (Collagen) mixed solution was hardened to form a three-dimensional skeleton (401), and then a medium for culturing the cells was injected into a relatively high microfluidic channel (200, 201).
- a peripheral device such as a pump was not used at all, and when the fluid was dropped into the injection holes 300 and 301 using a pipette or the like, the fluid 400 was naturally filled in the microfluidic flow path 200 and 201. This is due to the capillary phenomenon due to the surface tension of the fluid, the more hydrophilic the surface properties of the microfluidic channel is filled faster.
- Figure 7 is a cross-sectional view showing the appearance of inducing interstitial flow through the three-dimensional skeleton in the micro-cell culture apparatus according to the present invention.
- the three-dimensional skeleton 401 is disposed in the second microfluidic channel 201 having a low height, and the first microfluidic channel 200 having two higher heights is disposed therebetween.
- the first microfluidic channel 200 on one side of the three-dimensional skeleton 401 is the first microfluidic channel 1-1, and the first microfluidic channel 200 on the other side is the 1-2 microfluidic channel. I will name it.
- the 1-1 microfluidic flow passage and the 1-2 microfluidic flow passage are separated by a three-dimensional skeleton 401 inside the second microfluidic flow passage 201.
- the three-dimensional skeleton 401 has a microscopic hole of a few um or less, the fluid can move in the direction of the arrow, this flow is called interstitial flow.
- the fluid pressures in the 1-1 microfluidic flow passage and the 1-2 microfluidic flow passage are equal to each other, so that only the diffusion of chemicals present in the fluid occurs through the three-dimensional skeleton 401.
- the fluid pressures in the 1-1 microfluidic flow passage and the 1-2 microfluidic flow passage are different from each other, the interstitial flow may occur.
- the heights of the fluids connected to the inlet of the 1-1 microfluidic flow passage and the inlet of the 1-2 microfluidic flow path are different. May occur.
- the pressure difference is expressed as the product of the density of the fluid, the acceleration of gravity and the height difference of the fluid. The greater the pressure difference, the higher the rate of interstitial flow.
- the speed of the interstitial flow increases, and vice versa, the speed of the interstitial flow decreases.
- the fluid conductivity of the three-dimensional skeleton 401 depends on the material forming the three-dimensional skeleton 401.
- collagen IV is present at 60%, laminin at 33%, heparin sulfate at 5.4%, and the radius of each fiber is 0.7 nm, 0.6 nm, 0.5 nm.
- the average distance from one fiber surface to another fiber surface is about 8 nm for 20 mg / ml matrigel and about 0.54 nm for 300 mg / ml matrigel.
- the higher the concentration of the Matrigel the smaller the size of the gap, and therefore, the fluid conductivity of the three-dimensional skeleton is also lowered, and the speed of the interstitial flow is also lowered.
- the rate of the interstitial flow and the concentration or gradient of chemicals moving through the three-dimensional skeleton through this flow or diffusion, or mechanical stimulation to the cell 500 cultured in the three-dimensional skeleton, Growth, differentiation and migration will be different.
- FIG. 8A illustrates a method of injecting a fluid containing a specific chemical into one microfluidic flow path and a fluid containing no specific chemical into the other microfluidic flow path to form a concentration gradient of a specific chemical inside the three-dimensional skeleton.
- FIG. 8B shows that when a specific chemical is injected into one microfluidic channel as shown in FIG. 8A, a concentration gradient of a specific chemical is formed in the three-dimensional skeleton.
- the concentration gradient of a specific chemical is formed by diffusion due to the difference in concentration of molecules, and is characterized by moving from a high concentration to a low position.
- continuously injecting fluids and specific chemicals into the microfluidic flow path not only maintains a constant concentration gradient, but can also freely control the shape of the concentration gradient by adjusting the flow rate. At this time, if the moving speed or the fluid pressure of the fluid in each microfluidic flow path is different, it may affect the interstitial flow or the concentration gradient of a specific chemical.
- 10 kDa dextran labeled with a fluorescein isothiocyanate (FITC) fluorescent material was used to optically identify the molecular concentration gradient inside the three-dimensional skeleton.
- FITC fluorescein isothiocyanate
- FIG. 9 illustrates the movement of cells in the three-dimensional skeleton according to the concentration gradient.
- two microfluidic channels completely isolated with a three-dimensional skeleton in which cells are cultured are positioned up and down.
- the chemotactic material that induces the migration of the cells into the lower microfluidic channel was mixed and injected into the medium for cell culture, and only the general medium was injected into the upper microfluidic channel.
- the concentration gradient of the chemotactic material is formed in the three-dimensional skeleton, it can be seen that the movement by the chemotaxis of the cell occurs.
- RAW 264.7 cells which are macrophages
- CCL2 a concentration gradient of a cell signal transduction material
- the cell culture device By using the cell culture device according to the invention it is also possible to culture different kinds of cells at the same time.
- our environment is composed of various cells located at regular intervals to interact with each other. Observing the growth, migration, and differentiation of cells in an environment similar to the body environment is very important for accurate testing in the fields of drug testing, cell biology and molecular biology research.
- FIG 10 shows another embodiment of co-culture of heterologous cells using the micro cell culture apparatus according to the present invention.
- the microfluidic flow passages 200, 201, and 202 having at least three different heights are positioned to penetrate each other.
- a three-dimensional skeleton 401 material including one kind of cell 500 composition is injected, cured, and then, into the microfluidic flow passage 202 having the lowest height.
- the three-dimensional framework 402 material including another kind of cell 501 composition is injected and then cured.
- a fluid 400 including a medium for cell culture or a chemical for stimulation may be injected through the microfluidic flow path 200 to perform co-culture of heterologous cells.
- Figure 11 is a photograph showing an experimental example of co-culture of heterologous cells.
- the macrophages stained with fluorescent material 500 were mixed with the hydrogel 401 and injected through the lowest microfluidic flow path 201 and cured in a 37 ° C. incubator for about 30 minutes. Thereafter, breast cancer cells 501 stained with another fluorescent material were mixed with the hydrogel 402 and injected into the microfluidic flow passage 202 penetrating and contacting with the lowest microfluidic flow passage to cure for about 30 minutes. Finally, a medium capable of simultaneously culturing the two kinds of cells was prepared and injected into the remaining microfluidic flow path 200. In order to maintain the three-dimensional shape of the cells in the curing step to reverse the micro-cell culture device to prevent the cells from sinking to the bottom by gravity.
- the two kinds of cells are effectively fixed in the three-dimensional skeleton formed in the microfluidic flow path, and since the microfluidic flow paths all penetrate each other, a medium for culturing and a signaling material between different cells can be well transmitted to each other.
- FIG 12 shows another embodiment of co-culture of heterologous cells using the micro cell culture device according to the present invention.
- each of the plurality of microfluidic flow paths 200, 201 having two different heights, two or more cells can be simultaneously cultured in five divided spaces.
- Each of the plurality of microfluidic flow paths is connected to a unique injection port, and thus, when a specific fluid is injected into each injection hole, each of the microfluidic flow paths can be selectively filled.
- the microfluidic channel 201 having a relatively low height is first injected with a three-dimensional skeleton 401 material including one kind of cell 500 composition, cured, and then a microfluidic channel having a relatively high height ( The 3D skeleton 402 material including another kind of cell 501 composition is injected into 203 and then cured. Finally, a fluid 400 including a medium for cell culture or a chemical for stimulation may be injected through the microfluidic flow path 200 to perform co-culture of heterologous cells.
- Figure 13 is a photograph showing an experimental example of co-culture of heterologous cells.
- MDA-MB-231 cells and fibroblasts one of breast cancer cells, were stained with different fluorescent materials.
- the three-dimensional framework material the Matrigel-collagen mixed hydrogel was used.
- the fibroblasts 500 stained with fluorescent material were mixed with the hydrogel 401 and injected through a relatively low microfluidic flow path 201 and cured in a 37 ° C. incubator for about 30 minutes. Thereafter, the breast cancer cells 501 stained with another fluorescent substance are mixed with the hydrogel 402 to a microfluidic flow passage 203 located between the microfluidic flow passage 201 having a relatively high height and having a relatively low height. Injection was also performed for about 30 minutes. Finally, a medium capable of simultaneously culturing the two kinds of cells was prepared and injected into the remaining microfluidic flow path 200. In order to maintain the three-dimensional shape of the cells in the curing step inverted the micro-cell culture apparatus to prevent the cells from sinking to the bottom by gravity.
- the two kinds of cells are effectively fixed in the three-dimensional skeleton formed in the microfluidic flow path, and since the microfluidic flow paths all penetrate each other, a medium for culturing and a signaling material between different cells can be well transmitted to each other.
- two different types of cells may be co-cultured to cross each other, or may be co-cultured by placing different types of cells in a plurality of microfluidic flow paths.
- a mesenchymal cell such as fibroblast (Fibroblast) may be cultured by mixing two or more types of cells in one compartment.
- a skeletal material including different combinations of cells into each different microfluidic channel, a multi-cell co-culture platform of a wide variety of forms can be constructed.
- the fluid is first injected into the relatively low height microfluidic flow path, and the fluid is first filled in the relatively low height microfluidic flow path by surface tension.
- FIG. 14 is a cross-sectional view illustrating an embodiment in which a fluid is injected into a microfluidic flow path having a relatively high height as described above.
- the micro cell culture apparatus of FIG. 14 includes two or more microfluidic flow paths 700 and 702 in which a fluid is movable; A microfluidic flow path 701 located between the two or more microfluidic flow paths 700 and 702 and having a lower height than the two or more microfluidic flow paths 700 and 702; It may include an injection hole (not shown) for injecting fluid into the microfluidic flow paths 700 and 702.
- the flow from the microfluidic flow path 701 to the microfluidic flow paths 700 and 702 is suppressed by the surface tension of the injected fluid, and the microfluidic flow paths 700 and 702 and the microfluidic flow path into which the fluid is injected Only up to 701 is fluid filled first.
- the micro-cell culture apparatus when using a three-dimensional skeleton, includes two or more microfluidic flow paths 700, 702 in which the fluid is movable; A three-dimensional skeleton 401 in contact with the microfluidic flow paths 700 and 702; Located between the microfluidic flow paths 700 and 702 and the three-dimensional skeleton 401, the fine fluid having a height lower than the height of the microfluidic flow paths 700, 702 and the three-dimensional skeleton 401 A fluid passage 701; It includes an injection port (not shown) for injecting fluid into the microfluidic flow paths 700 and 702.
- 15 is a process flowchart showing the method for producing a micro cell culture device according to the present invention.
- the method for manufacturing a micro cell culture device is a combination of a first microfluidic channel having a specific height on a flat plate 800 and a second microfluidic channel having a lower height.
- a microstructure 801 having the same height as that of the second microfluidic flow path is formed in the corresponding region (S100).
- a microstructure 803 having a height obtained by subtracting the height of the second microfluidic flow path from the height of the first microfluidic flow path is formed in a region corresponding to the first microfluidic flow path on the microstructure 801 (S200).
- liquid material 100 forming the microfluidic flow path is applied onto the overlapped microstructures 801 and 803, and then cured (S300).
- the material 100 forming the cured microfluidic flow path is bonded to the substrate 101 to form the microfluidic flow paths 200 and 201 (S400).
- a polymeric material for forming a three-dimensional skeleton is injected into the second microfluidic flow path (S500).
- microstructures 801 and 803 may be formed in steps S100 and S200.
- a photoresist in the case of using photolithography, a photoresist, a photoresist, may be coated, and a microstructure pattern may be formed by hardening or melting by irradiating UV light only to a specific area.
- the microstructures can be formed very cheaply and quickly by cutting and then attaching tapes as shown in FIGS. 15 and 16.
- the tape shown on the right in Figure 16 is a cut tape, and the one shown on the left is a tape to produce a microstructure.
- the material forming the microfluidic flow path was poured and solidified on the microstructure thus manufactured, and then bonded to the substrate to prepare the microfluidic flow path.
- PDMS was used as a material for forming the microfluidic flow path, which was mixed with a curing agent and 10: 1, and cured at 65 ° C. for 2 hours.
- the PDMS was removed and bonded to the glass substrate through an oxygen plasma treatment, followed by sequentially injecting a fluid including a three-dimensional skeleton, thereby manufacturing a micro cell culture device according to the present invention.
Abstract
Description
Claims (21)
- 미세 세포 배양장치에 있어서,유체가 이동 가능한 복수개의 미세유체유로;상기 미세유체유로에 상기 유체를 주입하기 위한 하나 이상의 주입구를 포함하되, 상기 미세유체유로는 서로 관통하며 서로 다른 높이를 가지는 것을 특징으로 하는 미세 세포 배양 장치.
- 제 1 항에 있어서,상기 주입구를 통해 유체를 주입할 경우, 상기 복수개의 미세유체유로 중 상대적으로 낮은 높이를 갖는 미세유체유로에 유체가 우선적으로 채워지는 것을 특징으로 하는 미세 세포 배양 장치.
- 제 1항에 있어서,어느 하나의 상기 미세유체유로는 세포 배양을 위해 고분자성 물질로 형성된 3차원 골격을 포함하는 것을 특징으로 하는 미세 세포 배양 장치.
- 제3 항에 있어서,상기 고분자성 물질의 경화를 유발하기 위해 특정 온도, 시간, 빛의 파장, 및 물질이 추가적으로 구비되는 것을 특징으로 하는 미세 세포 배양 장치.
- 제 3 항에 있어서,상기 고분자성 물질은 매트리젤(Matrigel), 푸라마트릭스(Puramatrix), 콜라겐(Collagen), 피브린 겔(Fibrin gel), PEGDA, 또는 알지네이트(Alginate), 또는 이들의 혼합물을 포함하는 미세 세포 배양 장치.
- 제 3 항에 있어서,상기 어느 하나의 미세유체유로에서 형성된 상기 3차원 골격 내에서 세포 배양을 수행하기 위하여, 상기 어느 하나의 미세유체유로와 다른 적어도 하나의 미세유체유로에는 세포에 영양분을 공급하기 위한 배지가 주입되며, 상기 3차원 골격은 상기 3차원 골격을 형성하는 물질과 세포와의 혼합물에 의해 형성되는 미세 세포 배양 장치.
- 제 3 항에 있어서,상기 어느 하나의 미세유체유로에서 형성된 상기 3차원 골격의 표면에서 세포 배양을 수행하기 위하여, 상기 어느 하나의 미세유체유로에 상기 배양을 위한 세포를 주입하는 미세 세포 배양 장치.
- 제 3 항에 있어서,상기 어느 하나의 미세유체유로에서 형성된 상기 3차원 골격에 상기 어느 하나의 미세유체유로와 다른 적어도 하나의 미세유체유로를 통해 화학물질을 포함한 용액을 주입하여 상기 3차원 골격 내에 상기 화학물질의 농도 구배를 형성시키며,상기 농도 구배는 상기 화학물질의 확산 특성, 크기, 주입 시간, 초기 농도 차이, 상기 3차원 골격의 종류 및 상기 주입된 용액의 유속에 의해서 달라지는 미세 세포 배양 장치.
- 제 3 항에 있어서,이종세포 동시 배양을 위하여 상기 3차원 골격은 상기 서로 높이가 다른 미세유체유로 내에 형성되어 있으며, 상기 서로 다른 높이의 미세유체유로마다 서로 다른 종류 또는 조합의 세포 및 3차원 골격이 채워져 있는 것을 특징으로 하는 미세 세포 배양 장치.
- 제 1 항에 있어서,상기 미세유체유로는 폴리디메틸실록산 (poly(dimethylsiloxane), PDMS), 폴리메틸메타클릴레이트 (polymethylmethacrylate, PMMA), 폴리아크리레이트(polyacrylates), 폴리카보네이트(polycarbonates), 폴리실릭 올레핀(polycyclic olefins), 폴리이미드(polyimides), 폴리우레탄(polyurethanes), 폴리스티렌 (polystyrene), 및 유리 중 하나 이상의 물질로 형성되는 것을 특징으로 하는 미세 세포 배양 장치.
- 세포를 배양하기 위한 장치를 제조하는 방법에 있어서,평탄한 면을 기준으로 제 1 미세유체유로와 상기 제1 미세유체유로보다 더 낮은 높이를 갖는 제2 미세유체유로의 합집합에 해당하는 영역에 상기 제 2 미세유체유로의 높이와 같은 높이를 지닌 제1 미세구조물을 형성시키는 단계;상기 제1 미세구조물 상에서 제 1 미세유체유로에 해당하는 영역에 상기 제 1 미세유체유로의 높이에서 상기 제 2 미세유체유로의 높이를 뺀 높이를 지닌 제2 미세구조물을 형성시키는 단계;상기 제1 및 제2 미세구조물 상에 상기 미세유체유로를 형성하는 액상의 물질을 도포하고 경화시키는 단계;상기 경화된 액상의 물질을 기판과 접합하여 서로 관통하는 상기 제1 및 제2 미세유체유로를 형성시키는 단계;상기 제 2 미세유체유로에 고분자성 물질을 주입하여 3차원 골격을 형성시키는 단계;상기 제 1 미세유체유로에 유체를 주입하는 단계를 포함하는 것을 특징으로 하는 미세 세포 배양 장치의 제조 방법.
- 제 11 항에 있어서,상기 미세유체유로를 형성하는 액상의 물질은 폴리디메틸실록산 (poly(dimethylsiloxane), PDMS), 폴리메틸메타클릴레이트(polymethylmethacrylate, PMMA), 폴리아크리레이트(polyacrylates), 폴리카보네이트(polycarbonates), 폴리실릭 올레핀(polycyclic olefins), 폴리이미드(polyimides), 폴리우레탄(polyurethanes), 폴리스티렌(polystyrene), 및 유리 중 하나 이상의 물질로 형성되는 것을 특징으로 하는 미세 세포 배양 장치의 제조 방법.
- 제 11 항에 있어서,상기 미세유체유로의 표면이 친수성이고, 주입시키고자 하는 유체의 접촉각이 90도 이하에서 상기 유체가 상기 제1 미세유체유로에 주입되는 미세 세포 배양 장치의 제조 방법.
- 제 11 항에 있어서,상기 고분자성 물질은 매트리젤(Matrigel), 푸라마트릭스(Puramatrix), 콜라겐(Collagen), 피브린 겔(Fibrin gel), PEGDA, 또는 알지네이트(Alginate), 또는 이들의 혼합물을 포함하는 미세 세포 배양 장치의 제조 방법.
- 제 11 항에 있어서,상기 고분자성 물질의 경화 반응을 유발하기 위해 특정 온도, 시간, 빛의 파장, 및 물질이 선택적으로 추가되는 미세 세포 배양 장치의 제조 방법.
- 제 11 항에 있어서,상기 제1 및 제2 미세구조물은 테이프를 오린 다음 겹쳐 붙여서 형성되거나, 광식각공정, 임프린팅 공정, 핫 엠보싱 공정 중 하나를 통해 형성되는 미세 세포 배양 장치의 제조 방법.
- 제 11 항에 있어서,이종 세포 동시배양을 위해 상기 제 2 미세유체유로는 두 개 이상의 서로 다른 높이의 서로 관통하는 미세유체유로로 구성되어 있으며, 높이가 상대적으로 낮은 미세유체유로부터 순차적으로 3차원 골격을 형성시키는 미세 세포 배양 장치의 제조 방법.
- 복수개의 미세유체유로를 갖는 세포를 배양하기 위한 장치를 이용한 세포 배양 방법에 있어서,복수개의 미세유체유로 중 상대적으로 낮은 높이를 갖는 하나 이상의 미세유체유로에 3차원 골격을 형성시키기 위한 고분자성 물질을 주입하는 단계;상기 고분자성 물질을 경화시켜 3차원 골격을 형성하는 단계;상기 3차원 골격과 접하는 미세유체유로에 세포 배양을 위한 유체를 주입하는 단계를 포함하는 것을 특징으로 하는 미세 세포 배양 장치를 이용한 세포 배양 방법.
- 제 18 항에 있어서,상기 어느 하나의 미세유체유로에서 형성된 상기 3차원 골격 내에서 세포 배양을 수행하기 위하여, 상기 어느 하나의 미세유체유로와 다른 적어도 하나의 미세유체유로에는 세포에 영양분을 공급하기 위한 배지가 주입되며, 상기 3차원 골격은 상기 3차원 골격을 형성하는 물질과 세포와의 혼합물에 의해 형성되는 미세 세포 배양 장치를 이용한 세포 배양 방법.
- 제 18 항에 있어서,상기 어느 하나의 미세유체유로에서 형성된 상기 3차원 골격의 표면에서 세포 배양을 수행하기 위하여, 상기 어느 하나의 미세유체유로에 상기 배양을 위한 세포를 주입하는 미세 세포 배양 장치를 이용한 세포 배양 방법.
- 제 18 항에 있어서,상기 어느 하나의 미세유체유로에서 형성된 상기 3차원 골격에 상기 어느 하나의 미세유체유로와 다른 적어도 하나의 미세유체유로를 통해 화학물질을 포함한 용액을 주입하여 상기 3차원 골격 내에 상기 화학물질의 농도 구배를 형성시키며,상기 농도 구배는 상기 화학물질의 확산 특성, 크기, 주입 시간, 초기 농도 차이, 상기 3차원 골격의 종류 및 상기 주입된 용액의 유속에 의해서 달라지는 미세 세포 배양 장치를 이용한 세포 배양 방법.
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WO2024033549A1 (es) * | 2022-08-11 | 2024-02-15 | Readycell, S.L. | Dispositivo de órgano en chip |
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Also Published As
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US20140273223A1 (en) | 2014-09-18 |
KR101307196B1 (ko) | 2013-09-12 |
KR20130009260A (ko) | 2013-01-23 |
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