WO2018018614A1 - 微流体装置及其用途与使用方法 - Google Patents

微流体装置及其用途与使用方法 Download PDF

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Publication number
WO2018018614A1
WO2018018614A1 PCT/CN2016/092343 CN2016092343W WO2018018614A1 WO 2018018614 A1 WO2018018614 A1 WO 2018018614A1 CN 2016092343 W CN2016092343 W CN 2016092343W WO 2018018614 A1 WO2018018614 A1 WO 2018018614A1
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Prior art keywords
layer
microfluidic device
culture
fluid
cell
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PCT/CN2016/092343
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English (en)
French (fr)
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苏文弘
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苏文弘
庄佩锦
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Priority to CN201680046542.XA priority Critical patent/CN107922910B/zh
Priority to PCT/CN2016/092343 priority patent/WO2018018614A1/zh
Publication of WO2018018614A1 publication Critical patent/WO2018018614A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a microfluidic device, and more particularly to a microfluidic device that mimics cellular activity for a long period of time.
  • the invention further relates to the use of a microfluidic device as described above, and in particular to a use which provides both cell culture, cell observation, cell transfer, cell invasion or cell attachment.
  • the invention also relates to a method of using the aforementioned microfluidic device.
  • transwell migration assay transwell invasion assay
  • cell adhesion assay cell adhesion assay
  • flow chamber rolling-adhesion assay or anoikis-analysis Assays which are distinguished by the five stages of the aforementioned distant metastasis, ⁇ time metastasis analysis, wound healing analysis, matrix metalloproteinase activity assay, cell transfer assay, and cell invasion assay can only assess local invasion of cancer cells (local invasion)
  • the ability, anoikis analysis can only assess the ability of cancer cells to survive in the circulation; fluid chamber rolling attachment analysis and cell attachment analysis can only assess the distal arrest ability of cancer cell parts. In the prior art, it is more commonly used to assess trans-endothelial migration and extravasation.
  • the fluid chamber system is also used to observe the translocation of leukocytes across the endothelium, but the rate of movement of the cancer cells is too slow.
  • the prior art uses the fluid chamber system to evaluate that cancer cells transend the endothelium is not smooth.
  • the fluid chambers in the prior art include the following two types: 1.
  • the cells and the culture solution are only left in the fluid chamber once, and the remaining cells and the culture solution enter the waste liquid tank. Such devices not only do not conform to the actual cells in the flowing blood. State, if you need to operate for a long time, you need to use a lot of culture fluid. 2.
  • the culture solution can be recycled, but due to the design of the aeration device or liquid circulation, the cells will accumulate somewhere in the system. For example, the ibidi pump system fluid system of the German ibidi company will accumulate cells.
  • the bottom of the syringe on one side may cause cells to adhere to it or stimulate each other, and may even cause cell agglutination. In addition to increasing the amount of cells, it may interfere with the experimental results.
  • the concentration of the prolonged catalyzed substance is maintained by continuously changing the two sides of the solution, the experimental results may be affected, for example, the unattached cells may It is washed away or the pressure difference between the two sides causes experimental errors, etc., but most importantly, such devices use only a single type of flooding substance to attract cells to travel between endothelial cells and extracellular matrices, and have a general physiological condition. Big difference. Under normal physiological conditions, tissue cells may release a variety of substances, in addition to partially attracting cells to pass through, and another part can directly act on vascular endothelial cells to change the nature of endothelial cells and increase the entry of specific cells into tissues.
  • an in vitro system for the complete and continuous observation of the ability of cancer cells to be transferred to various organs via the distal end of the blood vessel is provided, and it is an object of the present invention to provide a microfluidic device by means of the microfluidic device The purpose of the active process of cell transfer, invasion or attachment is observed continuously, for a long time and completely.
  • the present invention provides a microfluidic device comprising: at least one fluid chamber, an aeration tank, At least one connecting tube and a peristaltic pump, wherein the fluid chamber comprises a cap layer, a fluid layer and a culture layer interposed between the cap layer and the fluid layer, the cap layer being provided with a hole, the culture layer comprising a base layer and a stack of interlayers disposed on the substrate layer, and the interlayer is further provided with at least one through hole penetrating the interlayer such that at least one groove is formed in the culture layer; the fluid layer comprises a hard layer, a hard layer, and a hard layer a soft layer and a light transmissive layer disposed between the hard layer and the soft layer, wherein the hard layer is provided with two channels and a uniform hole, the through hole penetrating through the hard layer and disposed between the two channels, wherein one side of the soft layer Providing at least one through hole, the other side of the soft layer is provided with a groove, the groove is
  • the grooves are respectively connected to the respective channels of the hard layer, and the positions of the at least one through holes correspond to the positions of the respective grooves of the culture layer.
  • One end of at least one of the communication tubes is respectively inserted into two channels of the fluid chamber, and the other end of the at least one communication tube is connected to the aeration tank and the peristaltic pump.
  • each channel of the hard layer is formed by a through hole and a through hole, wherein the two through holes are respectively formed on the surface of the hard layer near the two end edges, wherein the two through holes are respectively formed on opposite sides of the hard layer. wall.
  • the outer edge shape of the light transmissive layer of the fluid layer is larger than the outer edge shape of the through hole of the soft layer, and the outer edge shape of the light transmissive layer is equal to the outer edge shape of the groove of the soft layer to make the light transmissive layer Can be placed in the groove.
  • the cover layer further comprises a plurality of magnets which are equally spaced around the hole and are disposed in the cover layer.
  • the fluid layer further comprises a plurality of magnets, and the plurality of magnets are equally spaced apart in the hard layer and surround the through holes and the grooves of the soft layer.
  • the majority of the magnets are made of neodymium iron boron magnets.
  • the material of the cap layer and the hard layer of the fluid layer are made of a hard material, including but not limited to hard plastic, acrylic, epoxy, stainless steel or aluminum.
  • the material of the interlayer of the culture layer and the soft layer of the fluid layer are plastic and elastic, including, but not limited to, silicone, latex, rubber, fluorosilicone (FVMQ) or butadiene rubber. (butadiene rubber, BR).
  • plastic and elastic including, but not limited to, silicone, latex, rubber, fluorosilicone (FVMQ) or butadiene rubber. (butadiene rubber, BR).
  • the inner wall of each channel of the soft layer may be coated with anti-cell adhesion materials including, but not limited to, poly(2-hydroxyethyl methacrylate), poly(HEMA), organic Silicon, polytetrafluoroethene (PTFE) or silicone oil.
  • anti-cell adhesion materials including, but not limited to, poly(2-hydroxyethyl methacrylate), poly(HEMA), organic Silicon, polytetrafluoroethene (PTFE) or silicone oil.
  • the aeration tank and the inner wall of each connecting tube may be coated with anti-cell adhesion materials including, but not limited to, poly(hydroxyethyl methacrylate), silicone, polytetrafluoroethylene polymer or silicone oil.
  • anti-cell adhesion materials including, but not limited to, poly(hydroxyethyl methacrylate), silicone, polytetrafluoroethylene polymer or silicone oil.
  • the material of the base layer of the culture layer and the light transmissive layer of the fluid layer are permeable materials, including but not limited to transparent slides.
  • the at least one groove can be filled with a biocompatible gel, including collagen, gelatin, hyaluronic acid, polylactic acid-glycolic acid. Copolymer [poly(lactic-co-glycolic acid), PLGA], matrigelTM or tissue decellularized extracellular matrix.
  • the number of at least one through hole of the fluid layer is one, and the number of at least one groove of the culture layer is one.
  • the number of at least one through hole of the fluid layer is one, and the number of at least one groove of the culture layer is two or more.
  • the cap layer, the culture layer and the fluid layer are closely connected between the cap layer, the culture layer and the fluid layer by a plurality of magnets, screw nuts, clamps, vacuum suction, adhesive or other combination.
  • the cap layer, the culture layer, and the fluid layer are closely connected by attracting a plurality of magnets respectively disposed between the cap layer and the fluid layer.
  • the cap layer, the culture layer, and the fluid layer may be sandwiched by a clamp, or may be coated between the cap layer and the culture layer and between the culture layer and the fluid layer by an adhesive.
  • the cover layer, the culture layer and the fluid layer can connect the cover layer, the culture layer and the fluid layer of the present invention in various ways according to requirements, and the combination of the cover layer, the culture layer and the fluid layer should not be unduly restricted to Specific implementation aspects.
  • the number of the at least one fluid chamber is two or more.
  • the invention further provides the use of a microfluidic device as described above, which can simultaneously provide for cell culture, cell observation, cell transfer, cell invasion or cell attachment.
  • the present invention further provides a method of using the microfluidic device as described above, the method comprising: preparing (preparing) a cell; culturing the cell in at least one groove, and perforating the pore of the cap layer through the microscope and The cells in the at least one groove are observed in the base layer of the culture layer, or the cells in the at least one groove are observed through the through holes of the fluid layer and the light transmissive layer.
  • the step of preparing a cell further comprises mixing the cell with the biocompatible gel and then culturing in at least one groove.
  • the method further comprises adding a drug or a cell strain having a mobile characteristic.
  • the present invention can perform tissue 3D culture in an environment suitable for cell culture (in vitro system) and pass through light or fluorescence continuously, for a long time (may exceed 24 hours) and Completely observe the active processes such as cell transfer, invasion or attachment; in addition, by means of at least one groove of the fluid chamber of the present invention, multiple items can be simultaneously tested, saving time, labor and consumables costs, for a small number of cells [For example, circulating tumor cells (CTCs) isolated from the human body can also reduce the amount of cells used.
  • CTCs circulating tumor cells isolated from the human body can also reduce the amount of cells used.
  • the cells can be repeatedly circulated in the microfluidic device of the present invention to achieve a process that mimics the distant metastasis of cancer cells in the blood in a physiological state of the human body.
  • the cap layer, the culture layer and the fluid layer of the fluid chamber of the present invention are separably phased The connection is convenient for tissue culture, drug addition regulation, cell immunostaining and the like in the fluid experiment process, and even the tissue or the cells can be separated from at least one groove in a sterile environment and then the cell culture is continued.
  • Figure 1A is a schematic side elevational view of the microfluidic device of the present invention.
  • Figure 1B is a top plan view of the microfluidic device of the present invention.
  • FIG. 2 is a side cross-sectional view showing the fluid chamber and the connecting tube of the microfluidic device of the present invention.
  • 3A is a top plan view of a cap layer of the microfluidic device of the present invention.
  • Figure 3B is a side cross-sectional view of the cap layer of the microfluidic device of the present invention.
  • FIG. 4A is a side cross-sectional view of a culture layer of the microfluidic device of the present invention.
  • 4B is a top plan view of a culture layer of the microfluidic device of the present invention.
  • Figure 5A is a side cross-sectional view of a fluid layer of a microfluidic device of the present invention.
  • Figure 5B is a schematic perspective view of the soft layer of the fluid layer of the microfluidic device of the present invention.
  • Figure 5C is a top plan view of a soft layer of a fluid layer of the microfluidic device of the present invention.
  • Figure 5D is a top plan view of the soft lamination of the fluid layer of the microfluidic device of the present invention in a culture layer.
  • Figure 6 is a perspective exploded view of the cap layer, the culture layer and the fluid layer of the microfluidic device of the present invention.
  • Figure 7 is a side elevational view of the aeration tank of the microfluidic device of the present invention.
  • Figure 8 is a top plan view showing the culture layer of the microfluidic device of the present invention having a single groove and a fluid layer provided with a single through hole.
  • Figure 9 is a top plan view showing the culture layer of the microfluidic device of the present invention having two grooves and a fluid layer provided with a single through hole.
  • Fig. 10 is a top plan view showing the culture layer of the microfluidic device of the present invention having five grooves and a fluid layer provided with five through holes.
  • Figure 11A is a first preferred embodiment of the microfluidic device of the present invention, wherein the culture layer is provided with two grooves, the fluid layer is provided with a single through hole, and the bone marrow cells are cultured in one of the grooves.
  • Figure 11B is a first preferred embodiment of the microfluidic device of the present invention, wherein the culture layer is provided with two grooves, the fluid layer is provided with a single through hole, and the bone marrow cells 3D are cultured in another concave containing collagen gel. The cell is magnified 200 times the cell image.
  • Figure 12A is a magnified 50-fold cell image of a control group of a first preferred embodiment of the microfluidic device of the present invention, wherein red fluorescence shows human umbilical vein endothelial cells (HUVEC).
  • HUVEC human umbilical vein endothelial cells
  • Figure 12B is a magnified 50-fold cell image of a control group of the first preferred embodiment of the microfluidic device of the present invention, wherein the green fluorescence shows prostate cancer cell line PC3.
  • Figure 12C is a magnified 50-fold cell image of the control group of the first preferred embodiment of the microfluidic device of the present invention, which is merged with Figure 12A and Figure 12B, wherein red fluorescence shows human umbilical vein endothelial cells, green fluorescent display Prostate cancer cell line PC3.
  • Figure 13A is a magnified 50-fold cell image of a 3D culture set of a first preferred embodiment of the microfluidic device of the present invention, wherein red fluorescence shows human umbilical vein endothelial cells.
  • Figure 13B is a magnified 50-fold cell image of a 3D culture set of the first preferred embodiment of the microfluidic device of the present invention, wherein the green fluorescence shows prostate cancer cell line PC3.
  • Figure 13C is a magnified 50-fold cell image of the 3D culture group of the first preferred embodiment of the microfluidic device of the present invention, which is a combination of Figure 13A and Figure 13B, wherein red fluorescence shows human umbilical vein endothelial cells and green fluorescence shows prostate cancer.
  • FIG. 14A to 14D are fluorescence cell images of a magnified 600-fold conjugated focus microscope of a first preferred embodiment of the microfluidic device of the present invention, wherein blue fluorescence is a position where a DAPI stain exhibits a nucleus, and red fluorescence indicates a human.
  • the location of umbilical vein endothelial cells, as well as green fluorescence, showed the location of prostate cancer cell line PC3.
  • the depth position of FIG. 14A as a base point (0 ⁇ m)
  • the depth of FIG. 14B is 3 ⁇ m (ie, ⁇ 3 ⁇ m) below FIG. 14A
  • the depth of FIG. 14C is 5 ⁇ m (ie, ⁇ 5 ⁇ m) below FIG. 14A
  • the depth of FIG. 14D is FIG. 14A.
  • Below 8 ⁇ m (ie -8 ⁇ m) the group of Figures 14A to 14D shows that the location of the prostate cancer cell line PC3 is below the endothelial
  • Figure 15A is a magnified 50-fold cell image of a control group of a first preferred embodiment of the microfluidic device of the present invention, wherein red fluorescence shows the location of human umbilical vein endothelial cells and green fluorescence indicates the location of prostate cancer cell line LNCaP .
  • Figure 15B is a magnified 50-fold cell image of a control group of the first preferred embodiment of the microfluidic device of the present invention, wherein red fluorescence shows the position of human umbilical vein endothelial cells, and green fluorescence indicates the location of prostate cancer cell line PC3. .
  • Figure 15C is a magnified 50-fold cell image of the 3D culture group of the first preferred embodiment of the microfluidic device of the present invention, wherein red fluorescence shows the position of human umbilical vein endothelial cells, and green fluorescence shows the prostate cancer cell line LNCaP. position.
  • 15D is a magnified 50-fold cell image of a 3D culture group of the first preferred embodiment of the microfluidic device of the present invention, wherein red fluorescence shows the position of human umbilical vein endothelial cells, and green fluorescence shows prostate cancer cell line PC3. position.
  • Figure 16 is a side elevational view showing the appearance of a microfluidic device of a third embodiment of the present invention.
  • the microfluidic device of the present invention comprises a fluid chamber 10, an aeration tank 20, at least one communication tube 30, and a peristaltic pump 40.
  • the fluid chamber 10 includes a cap layer 11, a culture layer 12 and a fluid layer 13, and the cap layer 11 is detachably connected to the culture layer 12, and the culture layer 12 is opposite to the cap layer 11 The joined side is then detachably connected to the fluid layer 13 such that the culture layer 12 is interposed between the cap layer 11 and the fluid layer 13.
  • a hole 111 is formed in the middle of the cap layer 11, and the hole 111 penetrates the cap layer 11 and can be observed by a microscope.
  • the cover layer 11 is a hard material, and the type thereof includes hard plastic, acrylic, epoxy, stainless steel or aluminum.
  • the cap layer 11 further includes a plurality of magnets 112. The magnets 112 are equally spaced around the holes 111 and disposed in the cap layer 11, and the majority of the magnets 112 are made of neodymium iron boron magnets.
  • the culture layer 12 includes a base layer 121 and an interlayer 122, wherein the interlayer 122 is superposed on the base layer 121, and the material of the interlayer 122 is a moldable material such as silica gel and a base layer.
  • the material of 121 is a light transmissive material, including a transparent glass slide; and the interlayer 122 is provided with at least one through hole penetrating through the interlayer 122 such that at least one groove 123 is formed on the culture layer 12.
  • the number of at least one groove 123 may be one, two (as shown in FIG. 4B) or five, and at least one groove 123 may be filled with a biocompatible gel.
  • Capacitive gels for cells to grow in or on the surface include collagen, gelatin, hyaluronic acid, poly(lactic-co-glycolic acid). PLGA], matrigelTM, tissue decellularized extracellular matrix or other gels for mock tissue 3D culture.
  • different cancer cells, cancer tissues, cell lines, single types of tissue cells, tissue cell populations, small group tissues, tissue sheets, drug sustained release granules, and the like may be added to the biocompatible gel.
  • Micro-nanoparticles, sustained-release gels, and the like, various types of cells, drugs, or sustained-release substances; or when biocompatible gels are filled into the grooves 123 and solidified, tissue cells can be cultured on the surface of the biocompatible gel. , stem cells or cell lines.
  • the fluid layer 13 includes a hard layer 131 , a soft layer 132 , and a light transmissive layer 133 .
  • the surface of the hard layer 131 is respectively formed with a through hole 1311 near the two end edges, and the hard layer 131 is formed.
  • a through hole 1312 is formed in each of the two side walls, and each of the through holes 1311 communicates with each of the through holes 1312 to form two channels 1313, and a uniform hole 1314 is formed in the middle of the hard layer 131.
  • the through hole 1314 is formed through the hard layer 131. Between the two channels 1313.
  • the soft layer 132 is stacked on the hard layer 131, and one side of the soft layer 132 is provided with at least one through hole 1321, and at least one through hole 1321.
  • the opposite sides extend away from each other to form grooves 1322, respectively, which communicate with the respective channels 1313 of the hard layer 131.
  • the position of at least one of the through holes 1321 corresponds to the position of each of the grooves 123 of the culture layer 12.
  • the other surface of the soft layer 132 is further provided with a recess 1323 which communicates with at least one through hole 1321, and the outer edge shape of the transparent layer 133 is larger than the outer edge shape of the through hole 1321, and the transparent layer 13
  • the outer edge shape is equal to the outer edge shape of the recess 1323 so that the light transmissive layer 133 can be placed in the recess 1323.
  • the material of the hard layer 131 comprises hard plastic, acrylic, epoxy, stainless steel or aluminum; the soft layer 132 is made of silica gel; and the transparent layer 133 is made of light transmissive material, including Transparent glass; the inner wall of each channel 1313 can be coated with anti-cell adhesion substances including poly(2-hydroxyethyl methacrylate), poly(HEMA) to prevent cell adhesion or activation.
  • the fluid layer 13 further includes a plurality of magnets 134 disposed at equal intervals in the hard layer 131 and surrounding the through holes 1321 and the recesses 1323 of the soft layer 132, and the plurality of magnets 112
  • the material is a neodymium iron boron magnet.
  • the top of the aeration tank 20 is provided with a gas permeable anti-bacterial cap 21, and the bottom of the aeration tank 2 is a conical tip to prevent cell accumulation, and the anti-cell is coated on the inner wall of the aeration tank 20.
  • Adhesive substances include poly(2-hydroxyethyl methacrylate), poly(HEMA), which prevents cell adhesion.
  • At least one connecting tube 30 is one in a preferred embodiment, wherein both ends of the connecting tube 30 are respectively inserted into the passage 1313 of the hard layer 131 of the fluid layer 13 of the fluid chamber 10,
  • the trenches 1322 of the soft layer 132 are in communication with the respective channels of the hard layer 131, that is, the respective connecting tubes 30 are in communication with the respective channels 1313 of the hard layer 131 and the trenches 1322 of the soft layer 132.
  • the inner wall of each connecting tube 30 may be coated with an anti-cell adhesion substance including poly(2-hydroxyethyl methacrylate), poly(HEMA) to prevent cell adhesion or activation.
  • the two ends of the connecting pipe 30 are connected to the aeration tank 20 and the peristaltic pump 40.
  • one end of the fluid chamber 10 is connected to the aeration tank 20 by one of the communication tubes 30, and the aeration tank 20 is further connected to the peristaltic pump 40.
  • the other end of the fluid chamber 10 is connected to the peristaltic pump 40 by another communication tube 30.
  • the fluid chamber 10 is sequentially stacked from bottom to top, the base layer 121 of the culture layer 12, the interlayer 122 of the culture layer 12, the soft layer 132 of the fluid layer 13, and the hard layer 131 of the fluid layer 13, wherein the hard layer 131
  • Each of the channels 1313 communicates with the grooves 1322 of the soft layer 132, and the cap layer 11, the culture layer 12, and the fluid layer 13 are tightly attracted by the plurality of magnets 112 and 134 respectively provided on the cap layer 11 and the fluid layer 13.
  • the microfluidic device of the present invention can closely cover the cap layer 11, the culture layer 12, and the fluid layer 13 by means of a screw nut, a jig, a vacuum suction, a glue, or the like. connection.
  • the two connecting tubes 30 are respectively inserted into the communicating passages 1313 by the through holes 1312 of the hard layer 131, that is, the two connecting tubes 135 are respectively connected to the respective channels 1313 and the grooves 1322 of the soft layer 132.
  • the opposite end of one of the connecting tubes 30 connected to the fluid chamber 10 is connected to the peristaltic pump 40 by the aeration tank 20, and the other connecting tube 30 is connected to the fluid chamber 10. Since one end is connected to the peristaltic pump 40, the culture solution can be passed through the aeration tank 20 by the peristaltic pump 40, and then through the channels 1313 of the hard layer 131 of the fluid layer 13 of the fluid chamber 10 and the groove of the soft layer 132.
  • the tank 1322 flows into the fluid chamber 10, and the culture fluid flowing into the fluid chamber 10 flows out of the fluid chamber 10 from the other groove 1322 and the passage 1313 of the fluid chamber 10 and re-enters the peristaltic pump 40 to repeat the circulation.
  • the microfluidic device of the present invention can also observe at least one groove 123 of the culture layer 12 from the bottom through the hole 111 of the cap layer 11 and the base layer 121 of the culture layer 12 by a microscope, a fluorescence microscope or a conjugate microscope.
  • the cells, or the cells in at least one of the grooves 123 of the culture layer 12 are observed through the through holes 1314 and the light transmissive layer 133 of the fluid layer 13 from the top to the bottom.
  • the number of at least one through hole 1321 is one, and the number of at least one groove 123 is one.
  • the number of at least one through hole 1321 is one, and the number of at least one groove 123 is two.
  • the number of at least one through hole 1321 is five, and the number of at least one groove 123 is five, and faces away from each other on opposite sides of each through hole 1321.
  • the direction extensions respectively form two grooves 1322 which are merged and communicate with the respective channels 1313 of the hard layer 131.
  • FIG. 1A to FIG. 2 Please refer to FIG. 1A to FIG. 2 for the following embodiments.
  • First Embodiment Simulating the organ microenvironment to detect the ability of cancer cells to enter the tissue from blood vessels
  • two grooves 123 are provided in the culture layer 13, and the soft layer 132 is provided with a through hole 1321.
  • mice Four-week-old Balb/c mice were sacrificed in an aseptic workstation. The femur bones were removed and immersed in a sterile 4°C medium for storage. The aseptic technique was used to remove the mouse thigh bones. Mice bone marrow cells (MBM cells) were cultured (as shown in Fig. 11A). Next, the bone marrow cells are mixed into the collagen solution, and the collagen solution containing the bone marrow cells is filled with one of the grooves 123 of the culture layer 12 to form a gel and serve as a 3D culture group; another groove of the culture layer 12 123 was filled with a collagen solution containing no bone marrow cells to form a gel and served as a control group.
  • MBM cells mouse bone marrow cells
  • the human umbilical vein endothelial cells (HUVEC) pre-stained with red fluorescent dye were stained in the 3D culture group and the control group, respectively, and the cell-containing slides were cultured in a cell culture solution, as shown in Fig. 11B. .
  • the tube of the microfluidic device is filled with the cell culture solution at the aseptic workstation, and then the cap layer 11, the cell-containing culture layer 12 and the fluid layer 13 are sequentially assembled into the fluid chamber 10, and then the assembled fluid chamber 10 is respectively
  • the aeration tank 40 and the aeration tank 20 which does not leave cells are connected by a connecting pipe 30 to form a pipe in which the cell culture liquid can circulate in a sterile environment, and the entire microfluidic device is placed in a cell incubator for cultivation. It was confirmed by red fluorescence that the endothelial cells remained intact.
  • Prostate cancer cell line PC3 or LNCaP Fluorescence or conjugated-focus microscopy can be used to observe the process of cancer cells rolling, adhering, moving and passing through endothelial cells, phase difference, red fluorescence (endothelial cells) and green fluorescence (prostate cancer cells) on endothelial cells.
  • the microfluidic device of the present invention was tested for assessing the ability of distant metastasis of cancer cells.
  • the PC3 cell line is obtained from human prostate cancer bone metastasis cancer cells, and is generally considered to be a relatively aggressive metastatic prostate cancer cell line.
  • LNCaP is obtained from human prostate cancer lymph node metastatic cancer cells. It is generally considered to be a relatively mild (indolent) metastatic prostate cancer cell line. Injecting PC3 cells into the circulatory system of mice can successfully form bone metastases, whereas the original LNCaP cells cannot form distant metastases.
  • Fig. 12A and Fig. 13A after 24 hours of prostate cancer cell addition, red fluorescent endothelial cell images were observed to confirm that endothelial cells remained intact; as shown in Fig. 12B and Fig. 13B, green fluorescent prostate cancer cells were observed.
  • the images can compare the number of cancer cell adhesions and the changes in cancer cell appearance in the control and experimental groups, respectively.
  • the cells were sequentially filled with Phosphate buffered saline (PBS) and 5% paraformaldehyde for 3 minutes. After the cells and collagen gel were fixed, the 3D culture group and The basal layer 121 (i.e., the slide) of the control group was taken out from the fluid chamber 10 for immunofluorescence staining, and after the staining was completed, the endothelial cells and the cancer cells were observed using a conjugated focus microscope to determine whether the cancer cells passed through the endothelial cell layer.
  • PBS Phosphate buffered saline
  • the bone marrow tissue cells were cultured in 3D and covered with a layer of vascular endothelial cells to form a microenvironment similar to the microvessels between the bone marrow tissues (ie, the 3D culture group), and the control group with only endothelial cells alone (Fig. 12B)
  • the 3D culture group successfully attracted more metastatic prostate cancer cells PC3 attachment (as shown in Fig. 13B).
  • the appearance of prostate cancer cell line PC3 in the control group was round granular, while the appearance of prostate cancer cell PC3 in the 3D culture group showed an irregular amoeba-like appearance, so the cancer cells in the 3D culture group were significantly activated.
  • FIG. 14A to 14D are the activated prostate cancer cells PC3 observed by a conjugate focal microscope, wherein the depth position of FIG. 14A is the base point [0 micrometer ( ⁇ m)].
  • the depth of FIG. 14B is 3 ⁇ m below the FIG. 14A (ie, - 3 ⁇ m)
  • the depth of FIG. 14C is 5 ⁇ m (ie, -5 ⁇ m) below FIG. 14A
  • the depth of FIG. 14D is 8 ⁇ m (ie, -8 ⁇ m) below FIG. 14A
  • the group of FIG. 14A to FIG. 14D shows prostate cancer cell PC3. Positioned below the endothelial cells, it was found that most of the activated cancer cells have passed through the endothelial cell layer. As shown in Fig.
  • LNCaP cells in the 3D culture group were attached in the same experiment with LNCaP cells having weak metastatic ability. And activation is significantly less than PC3 cells.
  • the fluid chamber 10 of the microfluidic device of the present invention has the following effects:
  • the present invention can be used to simulate 3D tissue culture of an organ microenvironment, and can perform 3D tissue culture in one of the grooves without affecting cells of the control group.
  • the microfluidic device of the present invention can observe a clear cell fluorescence image under a fluorescence microscope without the disassembly of the cells while the cells are still alive and the culture solution continues to circulate continuously.
  • the present invention simulates the interaction between tissue and intravascular cells.
  • the prostate cancer cells PC3 in the 3D culture group and the control group were significantly different in appearance and adhesion and the number of layers crossing the endothelial cells.
  • the adherent and activated cancer cells of the 3D culture group were significantly less than the PC3 cells compared with the control group.
  • the base layer 121 (slide) of the culture layer 12 of the fluid chamber 10 can be easily disassembled after the experiment, and can be subjected to immunostaining and conjugated focus microscopic observation again.
  • Second Embodiment Simulating the organ microenvironment to detect the ability of cancer cells to enter the blood vessels from tissues
  • the two cancer cells to be tested are stained with red or green fluorescence respectively on the aseptic console, and the two cancer cells stained are respectively mixed into the collagen solution, and the collagen solution is respectively filled with the culture layer 12
  • the two grooves 123 are formed into a gel, and finally the human umbilical vein endothelial cells are covered on the 3D culture slides and the gel, and the cell-containing 3D culture slides are cultured in a cell culture medium for use.
  • the tube of the microfluidic device is filled with the cell culture solution at the aseptic workstation, and then the cap layer 11, the cell-containing culture layer 12 and the fluid layer 13 are sequentially assembled into the fluid chamber 10, and then the assembled fluid chamber 10 is respectively
  • the aeration pump 20 and the aeration tank 20 that does not leave cells are connected by a connecting tube 30 to form a pipe in which the cell culture liquid can circulate in a sterile environment, and then the entire microfluidic device is placed in a cell culture incubator. 24 hours.
  • the culture solution in the microfluidic device was collected, the culture solution was centrifuged, and the collected cells were counted under a fluorescence microscope, and the number of red and green fluorescent cells and the ratio of the two cells were calculated, respectively, in the solution.
  • the higher proportion of cancer cells measured indicates that the ability of the cells to enter the blood vessels from tissues is also higher, and the results indicate the distant metastatic ability of different cancer cells.
  • two fluid chambers 10 are used, which are respectively a first fluid chamber 10A and a second fluid chamber 10B, and each of the culture layers 13A, 13B is provided with two concave grooves 123A, 123B and soft layers 132A and 132B, respectively.
  • a through hole 1321A, 1321B is provided.
  • the two cancer cells to be tested were stained with red and green fluorescence, respectively, and the two cancer cells were separately mixed into the collagen solution, and the solution was respectively filled with the two grooves 123A of the culture layer 12A.
  • the cells were gelled, and the human umbilical vein endothelial cells were covered on the 3D culture slides and gels, and the cell-containing 3D culture slides were cultured in a cell culture medium for use.
  • the cells are separated from the specific organ tissues of the animal, and the cells are mixed into another collagen solution, and the solution is filled with one of the grooves 123B of the culture layer 12B to form a gel; the other groove 123B is filled.
  • the cell-containing collagen solution is also gelled; and the 3D culture slides and gels are covered with human umbilical vein endothelial fine
  • the cell-containing 3D culture slide was cultured in a cell culture medium for use.
  • the cap layer 11A, the cell-containing culture layer 12A, and the fluid layer 13A are sequentially assembled into the first fluid chamber 10A in the aseptic processing table, and the cap layer 11B, the cell-containing culture layer 12B, and the fluid layer 13B are assembled into the second fluid.
  • the chamber 10B, then the assembled first fluid chamber 10A, second fluid chamber 10B, peristaltic pump 40 and aeration tank 20 without residual cells are connected by a connecting tube 30 (as shown in FIG. 16) to form
  • the cell culture solution can be circulated in a bacterial environment, and the entire microfluidic device is placed in a cell culture incubator for 24 hours.
  • the cancer cells originally located in the first fluid chamber 10A are moved from the gel of the culture layer 12A into the circulating culture solution, and then adhered to the endothelial cells of the culture layer 12B of the second fluid chamber 10B along the connection tube 30, and finally cancer The cells are transferred to the cell-containing collagen gel of the second fluid chamber 10B (i.e., mimicked into the animal tissue), so that each step of simulating the distal rotation of the cancer cells in vitro is observed and observed.
  • the process of the passage of the second culture layer 12B cells through the endothelial cells can be observed by a fluorescence microscope or a conjugated focus microscope at any time according to the needs of the experiment.
  • the second fluid chamber 10B was placed under a fluorescence microscope to observe the phase difference, red fluorescence and green fluorescence images of the control layer and the 3D culture side without tissue cells, and compare the number of cancer cells adhered to the cancer cells. Type change.
  • the culture layer containing the tissue cells was poured into the phosphate buffer solution and 5% paraformaldehyde for 3 minutes, and after the cells and the collagen gel were fixed, the culture layers 12A and 12B were taken out from the fluid chambers 10A and 10B, respectively.
  • Immunofluorescence staining after the staining is completed, the endothelial cells and cancer cells were observed using a conjugated focus microscope to determine whether the cancer cells passed through the endothelial cell layer.

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Abstract

一种微流体装置及其用途与使用方法，所述微流体装置包括至少一流体室(10)、一曝气槽(20)、至少一连通管(30)以及一蠕动泵(40)，其中至少一流体室包含一盖层(11)、一流体层(13)以及一培养层(12)，该培养层(12)包括一基底层(121)以及一夹层(122)，且夹层(122)还设有至少一透孔，使得于该培养层(12)形成至少一凹沟(123)；该流体层(13)包括一硬层(131)、一软层(132)以及一透光层(133)，其中该硬层(131)设有两通道(1313)，其中该软层(132)设有至少一透孔(1321)，至少一透孔(1321)的位置对应于培养层(12)的至少一凹沟(123)的位置。其中至少一连通管(30)与至少一流体室(10)的两通道相连接，连通管(30)再与曝气槽(20)及蠕动泵(40)相连接。该装置可连续、长时间且完整地观察细胞转移、入侵或附着等活动过程。

Description

微流体装置及其用途与使用方法 技术领域
本发明关于一种微流体装置，尤指一种可长时间模拟细胞活动的微流体装置。本发明还关于一种前述的微流体装置的用途，尤指一种可同时提供细胞培养、细胞观察、细胞转移、细胞入侵或细胞附着的用途。本发明还关于一种前述的微流体装置的使用方法。
背景技术
远端转移对癌症致病的机转是一个很复杂并且很重要的过程，但是很不幸的，在体外系统用来评估癌细胞远端转移能力的方法及工具却相对较少，而且评估的方法也并不全面。现有技术中，癌症研究中常见评估远端转移能力的方法有旷时转移分析(time lapse migration assay)、伤口愈合分析(wound healing assay)、基质金属蛋白酶活性分析(matrix metalloproteinases,MMPs activity assay)、细胞转移分析(transwell migration assay)、细胞侵入分析(transwell invasion assay)、细胞附着分析(cell adhesion assay)、流体室滚动附着分析(flow chamber rolling-adhesion assay)或失巢凋亡分析(anoikis-assay)，以前述远端转移的5个分期来区分，旷时转移分析、伤口愈合分析、基质金属蛋白酶活性分析、细胞转移分析以及细胞侵入分析皆只能评估癌细胞部分的局部入侵(local invasion)能力，失巢凋亡分析仅能评估癌细胞的于循环中的存活率(survival)能力；流体室滚动附着分析与细胞附着分析仅能评估癌细胞部分的远端停滞(arrest)能力。现有技术中，用于评估跨内皮迁移及外渗(trans-endothelial migration and extravasation)较常用的是
Figure PCTCN2016092343-appb-000001
[可用于评估癌细胞转移时，癌细胞跨越内皮障碍(endothelial barrier)]。另外，现有技术中也有利用流体室系统观察白血球的跨越内皮的转移，但癌细胞移动速率太慢，现有技术利用流体室系统评估癌细胞跨越内皮转移并不顺利。
前述的现有技术都不是很好的远端转移评估系统，大部分只能评估单一个远端转移能力，例如转移(migration)、蛋白酶(protease)活性、失巢(anoikis)等等，
Figure PCTCN2016092343-appb-000002
虽然可以评估多种远端转移能力，但是
Figure PCTCN2016092343-appb-000003
中间的多孔膜会严重干扰光线穿透，因此很难即时观测活的癌细胞在内皮细胞及细胞外基质穿行的状况，此外
Figure PCTCN2016092343-appb-000004
仍有无法模拟血液流动及实验进行时间如果过长会有驱化物质(chemoattractant)浓度差降低等缺点。在现有技术的装置中，只有流体室及类似的微流体装置具有模拟血液流动的功能，并且具有良好的光学品质，但此类装置的缺点是无法长期稳定维持驱化物质的浓度梯度，因此多只运用在滚动附着分析上。
利用以上的技术评估癌细胞远端转移能力，除了需要使用多种技术交互验证之外，更无法持续观察远端转移的过程，例如利用伤口愈合分析或
Figure PCTCN2016092343-appb-000005
评估癌细胞转移能力时，很难排除细胞增生或存活率的干扰。更麻烦的是，不同器官的微环境，也会影响到癌细胞远端转移到不同器官的比率。以前列腺癌为例，几乎大部分的病患的癌细胞都是转移到骨组织，其他不同的癌症也有偏好转移的器官，至今仍然没有一套体外系统可以评估此一现象。
现有技术中的流体室包括以下两类：1.细胞与培养液只留经流体室一次，剩余的细胞与培养液就进入废液桶，此类装置不但不符合细胞在流动血液中的实际状态，若需长时间运作需要耗费大量培养液。2.培养液可以循环使用，但是由于曝气装置或液体循环的设计，会使细胞堆积在系统某处，以德国ibidi公司的气动式泵(ibidi pump system)流体系统为例，细胞会堆积在一侧的针筒底部，可能造成细胞黏着在此处或是互相刺激活化，甚至可能造成细胞凝集，除了会增加细胞的用量，更会干扰实验结果。
此外，为了吸引细胞在内皮细胞及细胞外基质间穿行，现有技术的装置中大多使用以多孔膜或凝胶分隔不同浓度的单一种类驱化物质，以形成浓度差梯度，此类装置在经过一段时间后两边驱化物质的浓度会趋于平衡而使浓度差梯度减弱甚至消失，如果要维持长时间驱化物质浓度差，只能靠缩小膜的面积或是增加膜两侧液体的体积，这两种方式都有其极限，并且可能干扰实验的操作及结果。再者，多孔膜会增加观测的难度，而凝胶本身结构很脆弱，若藉由持续更换两侧溶液以维持长时间驱化物质浓度差也会影响到实验结果，例如未贴附的细胞可能被冲走或两侧压力差造成实验误差等，但最重要的是，此类装置多只用单一种驱化物质去吸引细胞在内皮细胞及细胞外基质间穿行，而与一般生理状况有极大的差异。在一般生理状况下，组织细胞可能释放多种物质，这些物质除了部分是吸引细胞穿行之外，另一部分可以直接作用到血管内皮细胞上，以改变内皮细胞的性质，增加特定细胞进入组织间。
因此不管是学术界或是医疗界，都亟需一套完整、可持续观测而且可以客观评估癌细胞经由血管远端转移到各器官能力的体外系统，以进行癌转移相关研究及癌症病患预后的评估。
发明内容
有鉴于现有技术的缺乏可完整并持续观测评估癌细胞经由血管远端转移到各器官能力的体外系统，故本发明的目的在于提供一种微流体装置，藉由该微流体装置，而达到连续、长时间且完整地观察细胞转移、入侵或附着等活动过程的目的。
为达上述目的，本发明提供一种微流体装置，其包括：至少一流体室、一曝气槽、 至少一连通管以及一蠕动泵，其中流体室包含一盖层、一流体层以及一介于盖层与流体层之间的培养层，该盖层其设有一孔洞，该培养层包括一基底层以及一叠置于基底层上的夹层，且夹层还设有至少一穿透于夹层的透孔，使得于该培养层形成至少一凹沟；该流体层包括一硬层、一与硬层叠置的软层以及一置于硬层与软层之间的透光层，其中该硬层设有两通道及一贯孔，该贯孔贯穿硬层并设于两通道之间，其中该软层的一面设有至少一透孔，软层的另一面设有一凹槽，该凹槽与至少一透孔相连通，且至少一透孔的相对两侧朝向远离彼此方向延伸分别形成一沟槽，所述沟槽分别与硬层的各通道相连通，至少一透孔的位置对应于培养层的各凹沟的位置。其中至少一连通管的一端分别插入流体室的两通道，至少一连通管的另一端与曝气槽以及蠕动泵相连接。
优选的，所述的硬层的各通道是有一穿孔及一通孔相连通所形成，其中两穿孔分别形成于硬层的表面靠近两端缘处，其中两通孔分别形成于硬层的相对两侧壁。
优选的，所述的流体层的透光层的外缘形状大于软层的透孔的外缘形状，且透光层的外缘形状等于软层的凹槽的外缘形状而使透光层可置于凹槽内。
优选的，所述的盖层还包含有多数磁铁，所述磁铁等间距并围绕孔洞且设于盖层中。
优选的，所述的流体层还包含多数磁铁，所述多数磁铁等间距设于硬层中且围绕软层的透孔及凹槽。
更优选的，所述的多数磁铁的材质是钕铁硼磁铁。
优选的，所述的盖层的材质与流体层的硬层的材质是硬性材质，包括，但不限于硬塑胶、压克力、环氧树脂、不锈钢或铝。
优选的，所述的培养层的夹层的材质与流体层的软层的材质是可塑形且具有弹性，包括，但不限于硅胶、乳胶、橡胶、氟硅橡胶(fluorosilicone，FVMQ)或顺丁橡胶(butadiene rubber，BR)。
优选的，所述的软层的各通道的内壁可涂覆抗细胞黏附物质包括，但不限于聚(甲基丙烯酸羟乙酯)[poly(2-hydroxyethyl methacrylate)，poly(HEMA)]、有机硅、聚四氟乙烯聚合物(polytetrafluoroethene，PTFE)或硅油。
优选的，所述的曝气槽与各连接管的内壁可涂覆抗细胞黏附物质包括，但不限于聚(甲基丙烯酸羟乙酯)、有机硅、聚四氟乙烯聚合物或硅油。
优选的，所述的培养层的基底层以及流体层的透光层的材料为可透光的材质，包括，但不限于透明玻片。
优选的，所述的至少一凹沟可填入生物相容性凝胶，该生物相容性凝胶包括胶原蛋白(collagen)、明胶(gelatin)、玻尿酸(hyaluronic acid)、聚乳酸-羟基乙酸共聚物 [poly(lactic-co-glycolic acid)，PLGA]、基质胶(matrigelTM)或组织脱细胞细胞外间质(tissue decellularized extracellular matrix)。
优选的，所述的流体层的至少一透孔的数量为一个，培养层的至少一凹沟的数量为一个。
优选的，所述的流体层的至少一透孔的数量为一个，培养层的至少一凹沟的数量为两个以上。
优选的，所述的盖层、培养层以及流体层之间可藉由多数磁铁、螺丝螺帽、夹具、真空吸引、黏胶或其他结合方式将盖层、培养层以及流体层紧密相连接。
依据本发明较佳实施例中，是藉由分别设于盖层与流体层的多数磁铁相互吸引而使盖层、培养层以及流体层紧密相连接。在其他的具体实施例中，可藉由夹具将盖层、培养层以及流体层相夹固定，亦或可藉由黏胶涂布在盖层与培养层之间以及培养层与流体层之间，以达成盖层、培养层以及流体层紧密连接的效果。熟悉该项技术的人员，可根据需求利用各种方式将本创作的盖层、培养层以及流体层相连接，且盖层、培养层以及流体层之间的结合方式不应被不当地限制于特定具体实施态样。
优选的，所述的至少一流体室的数量为两个以上。
本发明另提供一种如前述的微流体装置的用途，该微流体装置可同时提供细胞培养、细胞观察、细胞转移、细胞入侵或细胞附着的用途。
本发明再提供一种如前述的微流体装置的使用方法，其步骤包括：齐备(备齐)一细胞；将该细胞培养于至少一凹沟内，以及藉由显微镜透过盖层的孔洞及培养层的基层观察至少一凹沟内的细胞，或透过流体层的贯孔及透光层观察至少一凹沟内的细胞。
优选的，所述的齐备一细胞的步骤还包括将细胞与生物相容性凝胶混合后再培养于至少一凹沟内。
优选的，所述的将该细胞培养于至少一凹沟内的步骤后，还包括添加药物或具有移动特性的细胞株。
藉由本发明的微流体装置的流体室的组合，本发明可在适于细胞培养的环境下(体外系统)进行组织3D培养并以穿透光或荧光连续、长时间(可超过24小时)且完整地观察细胞转移、入侵或附着等活动过程；此外，藉由本发明的流体室的至少一凹沟，可于同时检验多个项目，不仅可节省时间、人力及耗材成本，对于数量稀少的细胞[诸如从人体分离出的循环肿瘤细胞(circulating tumor cells，CTCs)]而言，还能减少细胞用量。此外，细胞可在本发明的微流体装置中重复循环，进以达成模拟在人体生理状态下癌细胞在血液中远端转移的过程。再者，本发明的流体室的盖层、培养层及流体层可分离地相 连接，以于流体实验过程中方便组织细胞培养、药物添加调控、细胞免疫染色等各式检测，甚至可在无菌的环境由至少一凹沟分离出组织或细胞再继续进行细胞培养。
附图说明
图1A是本发明的微流体装置的外观侧视示意图。
图1B是本发明的微流体装置的俯视示意图。
图2是本发明的微流体装置的流体室与连接管的侧视剖面示意图。
图3A是本发明的微流体装置的盖层的俯视示意图。
图3B是本发明的微流体装置的盖层的侧视剖面示意图。
图4A是本发明的微流体装置的培养层的侧视剖面示意图。
图4B是本发明的微流体装置的培养层的俯视示意图。
图5A是本发明的微流体装置的流体层的侧视剖面示意图。
图5B是本发明的微流体装置的流体层的软层的立体外观示意图。
图5C是本发明的微流体装置的流体层的软层的俯视示意图。
图5D是本发明的微流体装置的流体层的软层叠合于培养层的俯视示意图。
图6是本发明的微流体装置的盖层、培养层与流体层的立体分解示意图。
图7是本发明的微流体装置的曝气槽的侧视示意图。
图8是本发明的微流体装置的培养层设有单一凹槽、流体层设有单一透孔的俯视示意图。
图9是本发明的微流体装置的培养层设有两凹槽、流体层设有单一透孔的俯视示意图。
图10是本发明的微流体装置的培养层设有五凹槽、流体层设有五透孔的俯视示意图。
图11A是本发明的微流体装置的第一较佳实施例，其中培养层设有两凹槽、流体层设有单一透孔，并将骨髓细胞培养于其中一凹槽的放大200倍细胞影像图。
图11B是本发明的微流体装置的第一较佳实施例，其中培养层设有两凹槽、流体层设有单一透孔，并将骨髓细胞3D培养于另一含有胶原蛋白凝胶的凹槽的放大200倍细胞影像图。
图12A是本发明的微流体装置的第一较佳实施例的对照组的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞(human umbilical vein endothelial cells，HUVEC)。
图12B是本发明的微流体装置的第一较佳实施例的对照组的放大50倍细胞影像图，其中绿色荧光显示前列腺癌细胞株PC3。
图12C是本发明的微流体装置的第一较佳实施例的对照组将图12A与图12B合并(merge)的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞、绿色荧光显示前列腺癌细胞株PC3。
图13A是本发明的微流体装置的第一较佳实施例的3D培养组的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞。
图13B是本发明的微流体装置的第一较佳实施例的3D培养组的放大50倍细胞影像图，其中绿色荧光显示前列腺癌细胞株PC3。
图13C是本发明的微流体装置的第一较佳实施例的3D培养组将图13A与图13B合并的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞、绿色荧光显示前列腺癌细胞株PC3。
图14A至图14D是本发明的微流体装置的第一较佳实施例的放大600倍共轭焦显微镜荧光细胞影像图，其中蓝色荧光是以DAPI染剂呈现细胞核的位置、红色荧光显示人类脐静脉内皮细胞的位置，以及绿色荧光显示前列腺癌细胞株PC3的位置。以图14A的深度位置为基点(0μm)图14B的深度是图14A下方3μm(即为-3μm)，图14C的深度是图14A下方5μm(即为-5μm)，图14D的深度是图14A下方8μm(即为-8μm)，图14A至图14D图组显示前列腺癌细胞株PC3的位置在内皮细胞之下。
图15A是本发明的微流体装置的第一较佳实施例的对照组的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞的位置、以及绿色荧光显示前列腺癌细胞株LNCaP的位置。
图15B是本发明的微流体装置的第一较佳实施例的对照组的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞的位置、以及绿色荧光显示前列腺癌细胞株PC3的位置。
图15C是本发明的微流体装置的第一较佳实施例的3D培养组的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞的位置、以及绿色荧光显示前列腺癌细胞株LNCaP的位置。
图15D是本发明的微流体装置的第一较佳实施例的3D培养组的放大50倍细胞影像图，其中红色荧光显示人类脐静脉内皮细胞的位置、以及绿色荧光显示前列腺癌细胞株PC3的位置。
图16是本发明的第三实施例的微流体装置的外观侧视示意图。
具体实施方式
以下配合附图及本发明的较佳实施例，进一步阐述本发明为达成预定发明目的所采取的技术手段。
如图1A与图1B所示，本发明的微流体装置包含一流体室10、一曝气槽20、至少一连通管30以及一蠕动泵40。
如图2所示，该流体室10包含一盖层11、一培养层12及一流体层13，该盖层11与培养层12可分离地相连接，该培养层12相对于与盖层11相连接的一面再与流体层13可分离地相连接，使得培养层12介于盖层11与流体层13之间。
如图3A及图3B所示，该盖层11中间设有一孔洞111，该孔洞111穿透盖层11并可供显微镜观测。于具体的实施例中，盖层11是硬性材质，其种类包括硬塑胶、压克力、环氧树脂、不锈钢或铝。在另一具体实施例中，该盖层11还包含有多数磁铁112，所述磁铁112等间距并围绕孔洞111且设于盖层11中，且多数磁铁112的材质是钕铁硼磁铁。
如图4A及图4B所示，培养层12包含一基底层121以及一夹层122，其中该夹层122叠置于基底层121上，且该夹层122的材料为可塑形的材质例如硅胶，基底层121的材料为可透光的材质，包括透明玻片；夹层122设有至少一穿透于夹层122的透孔，使得于培养层12上形成至少一凹沟123。于具体的实施例中，至少一凹沟123的数量可为一个、两个(如图4B所示)或五个，且至少一凹沟123可填入生物相容性凝胶，该生物相容性凝胶可供细胞生长于其中或表面的物质包括胶原蛋白(collagen)、明胶(gelatin)、玻尿酸(hyaluronic acid)、聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid)，PLGA]、基质胶(matrigelTM)、组织脱细胞细胞外间质(tissue decellularized extracellular matrix)或其他凝胶等，以供模拟组织3D培养。在其他具体实施例中，生物相容性凝胶中可加入不同的癌细胞、癌组织、细胞株、单一种类的组织细胞、组织细胞群、小团的组织、组织薄片、药物缓释颗粒、微纳米颗粒、缓释凝胶等等各类细胞、药物或缓释物质；或当生物相容性凝胶填入各凹沟123并凝固后，可于生物相容性凝胶表面培养组织细胞、干细胞或细胞株。
如图5A至图6所示，流体层13包含一硬层131、一软层132、一透光层133，其中硬层131的表面靠近两端缘处分别形成穿孔1311，且硬层131的相对两侧壁亦分别形成通孔1312，各穿孔1311与各通孔1312相连通分别形成两通道1313，且硬层131的中间还设有一贯孔1314，该贯孔1314贯穿硬层131并设于两通道1313之间。该软层132叠置于硬层131上，且该软层132的一面设有至少一透孔1321，且至少一透孔1321 的相对两侧朝向远离彼此方向延伸分别形成沟槽1322，所述沟槽1322分别与硬层131的各通道1313相连通。至少一透孔1321的位置对应于培养层12的各凹沟123的位置。软层132的另一面还设有一凹槽1323，该凹槽1323与至少一透孔1321相连通，且该透光层133的外缘形状大于透孔1321的外缘形状，透光层13的外缘形状等于凹槽1323的外缘形状而使透光层133可置于凹槽1323内。于具体的实施例中，硬层131的材质包括硬塑胶、压克力、环氧树脂、不锈钢或铝；软层132的材质是硅胶；透光层133的材料为可透光的材质，包括透明玻片；各通道1313的内壁可涂覆抗细胞黏附物质包括聚(甲基丙烯酸羟乙酯)[poly(2-hydroxyethyl methacrylate)，poly(HEMA)]，进以防止细胞黏着或活化。再另一具体的实施例中，流体层13还包含多数磁铁134，所述多数磁铁134等间距设于硬层131中且围绕软层132的透孔1321及凹槽1323，且多数磁铁112的材质是钕铁硼磁铁。
如图7所示，曝气槽20的顶部设有一可透气的防菌盖21，且曝气槽2的底部为圆锥尖底，以防止细胞堆积，并于曝气槽20内壁涂覆抗细胞黏附物质包括聚(甲基丙烯酸羟乙酯)[poly(2-hydroxyethyl methacrylate)，poly(HEMA)]，进以防止细胞黏着。
如图1A至图2所示，至少一连接管30于较佳实施例中的数量为一个，其中连接管30的两端分别插入流体室10的流体层13的硬层131的通道1313，又因软层132的沟槽1322与硬层131的各通道相连通，即各连接管30分别与硬层131的各通道1313以及软层132的沟槽1322相连通。各连接管30的内壁可涂覆抗细胞黏附物质包括聚(甲基丙烯酸羟乙酯)[poly(2-hydroxyethyl methacrylate)，poly(HEMA)]，进以防止细胞黏着或活化。其中连接管30两端之间则与曝气槽20及蠕动泵40相连接。
本发明的微流体装置于使用时，如图1A至图2所示，该流体室10的一端藉由其中一连通管30与曝气槽20相连接，曝气槽20再与蠕动泵40相连接；流体室10的另一端藉由另一连通管30与蠕动泵40相连接。其中流体室10由下至上依序叠置盖层11、培养层12的基底层121、培养层12的夹层122、流体层13的软层132及流体层13的硬层131，其中硬层131的各通道1313与软层132的沟槽1322相连通，并藉由分别设于盖层11与流体层13的多数磁铁112与134相互吸引而使盖层11、培养层12以及流体层13紧密相连接；在另一较佳的实施中，本发明的微流体装置可藉由螺丝螺帽、夹具、真空吸引、黏胶或其他结合方式将盖层11、培养层12以及流体层13紧密相连接。两连接管30分别由硬层131的各通孔1312插入相连通的通道1313中，即两连接管135分别与各通道1313以及软层132的沟槽1322相连通。其中一连接管30与流体室10连接的相对一端藉由曝气槽20与蠕动泵40相连接，另一连接管30与流体室10连接的相 对一端与蠕动泵40相连接，故可藉由蠕动泵40将培养液流经曝气槽20后，再经由流体室10的流体层13的硬层131的各通道1313以及软层132的沟槽1322流入流体室10，并使流入流体室10的培养液从流体室10的另一沟槽1322及通道1313流出流体室10并再进入蠕动泵40重复循环。本发明的微流体装置还可藉由显微镜、荧光显微镜或共轭交显微镜由下往上透过盖层11的孔洞111及培养层12的基层121观察培养层12的至少一凹沟123内的细胞，或是由上往下透过流体层13的贯孔1314及透光层133观察培养层12的至少一凹沟123内的细胞。
在第一较佳的实施例中，如图8所示，至少一透孔1321的数量为一个，且至少一凹沟123的数量为一个。在第二较佳的实施例中，如图9所示，至少一透孔1321的数量为一个，且至少一凹沟123的数量为二个。在第三较佳的实施例中，如图10所示，至少一透孔1321数量为五个、至少一凹沟123的数量为五个，并于各透孔1321的相对两侧朝向远离彼此方向延伸分别形成两沟槽1322，所述沟槽1322汇整再与硬层131的各通道1313相连通。
以下实施例请参考图1A至图2所示。
第一实施例：模拟器官微环境进以检测癌细胞由血管进入组织的能力
本发明的微流体装置的第一较佳实施例是于培养层13上设有两凹沟123、且软层132设有一透孔1321。
在无菌操作台内牺牲4周龄Balb/c小鼠，先将小鼠大腿骨取出后，浸泡在无菌4℃培养液中暂存，之后以无菌操作技术取出小鼠大腿骨中的骨髓细胞(mice bone marrow cells，MBM cells)进行培养(如图11A所示)。接着将骨髓细胞混入胶原蛋白溶液中，以此含有骨髓细胞的胶原蛋白溶液注满培养层12的其中一凹沟123，使其形成凝胶并作为3D培养组；培养层12的另一凹沟123则注满不含骨髓细胞的胶原蛋白溶液，使其形成凝胶并作为对照组。并在3D培养组以及对照组分别覆满以红色荧光(living dye)预先染色的人类脐静脉内皮细胞(HUVEC)，将此含细胞的玻片以细胞培养液培养待用，如图11B所示。
在无菌操作台将微流体装置的管道注满细胞培养液，之后依序将盖层11、含细胞的培养层12及流体层13组装成流体室10，接着将组装好的流体室10分别与蠕动泵40及不残留细胞的曝气槽20以连接管30相连接，以形成在无菌环境下细胞培养液可以循环流动的管道，再将整个微流体装置放入细胞培养箱中培养。由红色荧光可确定内皮细胞仍然保持完整，将流体室10于37℃及5％CO2细胞培养箱中培养24小时后，由曝气槽20加入10000颗以绿色荧光预先活染色(living dye)的前列腺癌细胞株PC3或 LNCaP
Figure PCTCN2016092343-appb-000006
随时可以以荧光显微镜或共轭焦显微镜观测癌细胞在内皮细胞上滚动、黏着、移动及穿过内皮细胞的过程、相位差、红色荧光(内皮细胞)及绿色荧光(前列腺癌细胞)的影像，进以检测本发明的微流体装置用于评估癌细胞远端转移能力。其中PC3细胞株是由人类前列腺癌骨转移癌细胞中取得，普遍被认为是比较恶性(aggressive)转移(metastatic)能力较强的前列腺癌细胞株，LNCaP是由人类前列腺癌淋巴转移癌细胞中取得，普遍被认为是比较良性(indolent)转移能力较弱的前列腺癌细胞株。将PC3细胞注入小鼠的循环系统内，可以成功形成骨转移，而原始的LNCaP细胞则无法形成远端转移。
如图12A及图13A所示，前列腺癌细胞加入24小时后，可观察红色荧光的内皮细胞影像以分别确定内皮细胞仍然保持完整；如图12B及图13B所示，由绿色荧光的前列腺癌细胞影像可分别比较对照组及实验组的癌细胞黏着的数目及癌细胞外型变化。
分别观察后，在各凹槽依序灌入磷酸缓冲溶液(Phosphate buffered saline，PBS)及5％多聚甲醛(paraformaldehyde)各3分钟，待细胞及胶原蛋白凝胶固定后，将3D培养组以及对照组的基底层121(即玻片)由流体室10取出进行免疫荧光染色，染色完成后使用共轭焦显微镜观测内皮细胞与癌细胞，以确定癌细胞是否有穿过内皮细胞层。
结果显示，利用骨髓组织细胞3D培养并在其上覆盖一层血管内皮细胞，组成模拟骨髓组织间微血管类似的微环境(即3D培养组)，与单纯只有内皮细胞的对照组(如图12B所示)比较，3D培养组成功的吸引了较多转移能力较强前列腺癌细胞PC3贴附(如图13B所示)。且对照组的前列腺癌细胞PC3的外型呈圆形颗粒状，而3D培养组的前列腺癌细胞PC3的外型呈现不规则的变形虫样外型，故3D培养组的癌细胞明显较为活化。如图14A至图14D是以共轭焦显微镜观察活化的前列腺癌细胞PC3，其中是以图14A的深度位置为基点[0微米(μm)]图14B的深度是图14A下方3μm(即为-3μm)，图14C的深度是图14A下方5μm(即为-5μm)，图14D的深度是图14A下方8μm(即为-8μm)，该图14A至图14D的图组显示前列腺癌细胞PC3的位置在内皮细胞的下方，发现大多数活化的癌细胞都已经穿过内皮细胞层。如图15A与图15B(皆为对照组)及图15C与图15D(皆为3D培养组)所示，以转移能力较弱的LNCaP细胞进行相同的实验，则3D培养组的LNCaP细胞贴附及活化都明显比PC3细胞少。
经由本实施例可证实本发明所述的微流体装置的流体室10具有以下几个功效：
1.本发明可用于模拟器官微环境的3D组织培养，且可于其中一凹槽进行3D组织培养，且不影响对照组的细胞。
2.本发明所述的微流体装置可在细胞仍然存活、培养液仍然持续循环流动的状态下，不需要拆解即可在荧光显微镜下观测到清楚的细胞荧光影像。
3.本发明可以模拟出组织与血管内细胞间的交互作用。在相同的条件下，比较3D培养组与对照组的前列腺癌细胞PC3在外型及黏着与穿过内皮细胞层数量有明显的差异。且以前列腺癌细胞LNCaP进行相同的实验，则发现与对照组比较3D培养组的贴附及活化的癌细胞都明显比PC3细胞少。
4.流体室10的培养层12的基底层121(玻片)可在实验后轻易拆卸，并可再次进行免疫染色及共轭焦显微镜观察。
第二实施例：模拟器官微环境进以检测癌细胞由组织进入血管的能力
在无菌操作台将两种待测的癌细胞分别以红色或绿色的荧光染色，分别将以染色的两种癌细胞分别混入胶原蛋白溶液中，并将此胶原蛋白溶液分别注满培养层12的两凹沟123并使其形成凝胶，最后在3D培养玻片及凝胶上覆满人类脐静脉内皮细胞，将此含细胞的3D培养玻片以细胞培养液培养待用。
在无菌操作台将微流体装置的管道注满细胞培养液，之后依序将盖层11、含细胞的培养层12及流体层13组装成流体室10，接着将组装好的流体室10分别与蠕动泵40及不会残留细胞的曝气槽20以连接管30相连接，以形成在无菌环境下细胞培养液可以循环流动的管道，再将整个微流体装置放入细胞培养箱中培养24小时。24小时后收集此微流体装置中的培养液，将培养液离心后在荧光显微镜下计算收集到的细胞，分别计算带有红色与绿色荧光细胞的数目及两种细胞的比例，若在溶液中测得比例较高的癌细胞，代表该细胞由组织进入血管的能力也较高，藉以此结果预测不同癌细胞的远端转移能力。
第三实施例：模拟器官微环境进以检测癌细胞由组织进入血管后再进入另一组织的能力
本实施例中，采用两个流体室10，分别为第一流体室10A以及第二流体室10B，且各培养层13A,13B上分别设有两凹沟123A,123B、软层132A,132B分别设有一透孔1321A,1321B。
在无菌操作台将两种待测的癌细胞分别以红色及绿色的荧光染色，分别将这两种癌细胞分别混入胶原蛋白溶液中，以此溶液分别注满培养层12A的两凹沟123A内并使其形成凝胶，并在3D培养玻片及凝胶上覆满人类脐静脉内皮细胞，将此含细胞的3D培养玻片以细胞培养液培养待用。
由动物特定器官组织分离出细胞，将此细胞混入另一胶原蛋白溶液，以此溶液注满培养层12B的其中一凹沟123B，并使其形成凝胶；另一凹沟123B则注满不含细胞的胶原蛋白溶液，同样使其形成凝胶；并在3D培养玻片及凝胶上覆满以人类脐静脉内皮细 胞，将此含细胞的3D培养玻片以细胞培养液培养待用。
在无菌操作台依序将盖层11A、含细胞的培养层12A及流体层13A组装成第一流体室10A，将盖层11B、含细胞的培养层12B及流体层13B组装成第二流体室10B，接着将组装好的第一流体室10A、第二流体室10B、蠕动泵40及不残留细胞的曝气槽20以连接管30相连接(如图16所示)，以形成在无菌环境下细胞培养液可以循环流动的管道，再将整个微流体装置放入细胞培养箱中培养24小时。原本位于第一流体室10A的癌细胞会由培养层12A的凝胶移动进入循环的培养液中，再沿连接管30流到第二流体室10B的培养层12B的内皮细胞上黏着，最终癌细胞转移到第二流体室10B含细胞的胶原蛋白凝胶中(即模拟进入动物组织)，如此得以一次完整在体外模拟癌细胞远端转的每个步骤，并进行观测。在此同时，可依实验需要，随时以荧光显微镜或共轭焦显微镜观测第二培养层12B细胞穿过内皮细胞的过程。48小时后将第二流体室10B放在荧光显微镜下分别观察不含组织细胞的对照组培养层及3D培养侧的相位差、红色荧光及绿色荧光影像，比较癌细胞黏着的数目及癌细胞外型变化。
实验结束后将含有组织细胞的培养层灌入磷酸缓冲溶液及5％多聚甲醛各3分钟，等细胞及胶原蛋白凝胶固定后，将培养层12A,12B分别由流体室10A,10B取出进行免疫荧光染色，染色完成后使用共轭焦显微镜观测内皮细胞与癌细胞，以确定癌细胞是否有穿过内皮细胞层。
以上所述仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本领域的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (20)

  1. 一种微流体装置，其特征在于，其包括：
    至少一流体室，其包括：
    一盖层，其设有一孔洞；
    一培养层，其与盖层可分离地相连接，该培养层包括：
    一基底层；
    一夹层，其叠置于基底层上，该夹层还设有至少一穿透于夹层的透孔，使得于培养层形成至少一凹沟；以及，
    一流体层，其与培养层可分离地相连接，并使培养层介于盖层与流体层之间，该流体层包括：
    一硬层，其靠近两端缘处分别形成一通道，且硬层还设有一贯孔，该贯孔贯穿硬层并设于两通道之间；
    一软层，该软层的一面设有至少一透孔，另一面还设有一凹槽，该凹槽与至少一透孔相连通，且至少一透孔的相对两侧朝向远离彼此方向延伸分别形成一沟槽，所述沟槽分别与硬层的各通道相连通；至少一透孔的位置对应于培养层的各凹沟的位置；以及
    一透光层，其可分离地置于软层的凹槽内；
    一曝气槽，其顶部设有一可透气的防菌盖，该曝气槽的底部为圆锥状；
    至少一连通管，该至少一连通管的两端可分别插入流体室的流体层的硬层的各通道；以及，
    一蠕动泵，其中一端与曝气槽相连接，另一端藉由至少一连接管与流体室相连接。
  2. 根据权利要求1所述的微流体装置，其特征在于，所述的硬层的各通道是有一穿孔及一通孔相连通所形成，其中两穿孔分别形成于硬层的表面靠近两端缘处，其中两通孔分别形成于硬层的相对两侧壁。
  3. 根据权利要求1所述的微流体装置，其特征在于，所述的流体层的透光层的外缘形状大于软层的透孔的外缘形状，且透光层的外缘形状等于软层的凹槽的外缘形状而使透光层可置于凹槽内。
  4. 根据权利要求1所述的微流体装置，其特征在于，所述的盖层还包含有多数磁铁，所述磁铁等间距并围绕孔洞且设于盖层中。
  5. 根据权利要求1所述的微流体装置，其特征在于，所述的流体层还包含多数磁铁，所述多数磁铁等间距设于硬层中且围绕软层的透孔及凹槽。
  6. 根据权利要求4或5所述的微流体装置，其特征在于，所述的多数磁铁的材质是钕铁硼磁铁。
  7. 根据权利要求1所述的微流体装置，其特征在于，所述的盖层的材质与流体层的硬层的材质是硬性材质，包括硬塑胶、压克力、环氧树脂、不锈钢或铝。
  8. 根据权利要求1所述的微流体装置，其特征在于，所述的培养层的夹层的材质与流体层的软层的材质是可塑形且具有弹性，包括硅胶、乳胶、橡胶、氟硅橡胶(fluorosilicone，FVMQ)或顺丁橡胶(butadiene rubber，BR)。
  9. 根据权利要求1所述的微流体装置，其特征在于，所述的软层的各通道的内壁可涂覆抗细胞黏附物质包括聚(甲基丙烯酸羟乙酯)[poly(2-hydroxyethyl methacrylate)，poly(HEMA)]、有机硅、聚四氟乙烯聚合物(polytetrafluoroethene，PTFE)或硅油。
  10. 根据权利要求1所述的微流体装置，其特征在于，所述的曝气槽与各连接管的内壁可涂覆抗细胞黏附物质包括聚(甲基丙烯酸羟乙酯)、有机硅、聚四氟乙烯聚合物或硅油。
  11. 根据权利要求1所述的微流体装置，其特征在于，所述的培养层的基底层及流体层的透光层的材料为可透光的材质，包括透明玻片。
  12. 根据权利要求1所述的微流体装置，其特征在于，所述的至少一凹沟可填入生物相容性凝胶，该生物相容性凝胶包括胶原蛋白(collagen)、明胶(gelatin)、玻尿酸(hyaluronic acid)、聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid)，PLGA]、基质胶(matrigelTM)或组织脱细胞细胞外间质(tissue decellularized extracellular matrix)。
  13. 根据权利要求1、4或5中任一项所述的微流体装置，其特征在于，所述的盖层、培养层以及流体层之间可藉由多数磁铁、螺丝螺帽、夹具、真空吸引、黏胶或其他结合方式将盖层、培养层以及流体层紧密相连接。
  14. 根据权利要求1所述的微流体装置，其特征在于，所述的流体层的至少一透孔的数量为一个，培养层的至少一凹沟的数量为一个。
  15. 根据权利要求1所述的微流体装置，其特征在于，所述的流体层的至少一透孔的数量为一个，培养层的至少一凹沟的数量为两个。
  16. 根据权利要求1所述的微流体装置，其特征在于，所述的至少一流体室的数量为两个。
  17. 一种根据权利要求1至16中任一项所述的微流体装置的用途，其特征在于，所述的微流体装置可同时提供细胞培养、细胞观察、细胞转移、细胞入侵或细胞附着的用途。
  18. 一种根据权利要求1至16任一项中所述的微流体装置的使用方法，其特征在于，包括以下步骤：
    齐备一细胞；
    将该细胞培养于至少一凹沟内；以及，
    藉由显微镜透过盖层的孔洞及培养层的基层观察至少一凹沟内的细胞，或透过流体层的贯孔及透光层观察至少一凹沟内的细胞。
  19. 根据权利要求18所述的使用方法，其特征在于，齐备一细胞的步骤还包括将细胞与生物相容性凝胶混合后再培养于至少一凹沟内。
  20. 根据权利要求19所述的使用方法，其特征在于，将该细胞培养于至少一凹沟内的步骤后，还包括添加药物或具有移动特性的细胞株。
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