WO2019019526A1 - Puce microfluidique et application correspondante - Google Patents

Puce microfluidique et application correspondante Download PDF

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WO2019019526A1
WO2019019526A1 PCT/CN2017/115760 CN2017115760W WO2019019526A1 WO 2019019526 A1 WO2019019526 A1 WO 2019019526A1 CN 2017115760 W CN2017115760 W CN 2017115760W WO 2019019526 A1 WO2019019526 A1 WO 2019019526A1
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elastic porous
substrate
channel
cells
porous membrane
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PCT/CN2017/115760
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Chinese (zh)
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罗勇
杜昱光
孙明
邓权锋
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中科芯瑞(苏州)生物科技有限公司
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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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture

Definitions

  • the invention belongs to the technical field of microfluidics, and in particular relates to a microfluidic chip and an application thereof.
  • Microfluidics also known as Lab-on-a-chip, is a science and technology that manipulates fluids in the micron-scale space, minimizing the basic functions of biological and chemical laboratories. On the chip of several square centimeters, it is one of the most important cutting-edge technologies in the 21st century. It is considered to be a key technology to solve the key problems of innovative drugs, cosmetics and health products, such as excessive cost and long cycle, and to innovate the original technology system. It is facing major development opportunities and challenges.
  • Organ on a chip is a sub-class of microfluidic chips, which is a biomimetic technique for simulating organs in a few square centimetres of slices, simulating organs.
  • Organ chips The "organ” in it is very small, but it has the basic physiological functions of real organs.
  • the reason why the organ chip can simulate the organ artificially is that: (1) it not only simultaneously cultivates a variety of cells contained in the organ, but also the spatial arrangement of the cells can mimic the physiological structure of the organ; (2) it can reconstruct the physiological environment of the organ in the body, Such as fluid shear force, signal molecule concentration gradient. It can be said that organ chips simulate organs from three aspects of "composition”, "structure” and "environment”, and the degree of simulation is very high.
  • Each kind of human organ corresponds to an organ chip, such as a liver chip that simulates the liver, a kidney chip that simulates the kidney, an islet chip that simulates the pancreas, a heart chip that simulates the heart, etc., and the same organ chip can also Contains a variety of "organs” such as the intestine-liver chip, kidney-heart chip and so on.
  • organ chips The use of organ chips is to test chemicals in place of real human or animal organs. Common chemicals include drugs, health products, cosmetics and environmental toxicants. It can determine the drug's efficacy, toxicity and pharmacokinetics. It can measure the absorption of health products in the intestine, the metabolism in the liver and the protection of the intestinal flora. It can measure the absorption of cosmetics in the skin and the stimulation of the skin. Sex, it is also possible to determine the damage of environmental poisons to a particular organ.
  • the intestine has a lumen
  • the blood vessel has a lumen
  • the kidney has a renal tubular lumen, a glomerular lumen, and a collecting duct lumen
  • the heart has an atrium and a ventricle
  • the uterus has a uterine cavity.
  • the gum has a gingival sulcus
  • the esophagus has an esophagus
  • the stomach has a gastric cavity
  • the lymph has a lymphatic vessel
  • the lung Has a trachea
  • the eye has a lacrimal gland, and so on.
  • luminal structure Even organs that do not contain a luminal structure, such as muscle, fat, tumor, brain, etc., have a luminal structure because they carry blood vessels.
  • These lumen structures have two characteristics: one is that they have a certain degree of deformability, they are soft, not rigid; the other is that there are functional cells on the wall of the lumen, and sometimes there are more than one layer of functional cells.
  • the existing organ chips that simulate the lumen structure are mainly divided into three categories: one is to simulate the wall structure by using a single layer of elastic porous membrane combined with cell culture, for example, in "Remi Villenave, Donald E. Ingber. Human Gut-On-A -Chip Supports Polarized Infection of Coxsackie B1Virus In Vitro.PLOS ONE.2017” simulates the intestinal wall by technically culturing intestinal epithelial cells on a single layer of elastic PDMS porous membrane - the simulated intestinal wall is soft and variable
  • the structure of the intestinal lumen is not formed, so the function is incomplete, such as less substance exchange, less cell metabolism products, and it is difficult to reach the detection limit.
  • the single-layer hard porous membrane is combined with cell culture to simulate the wall structure, for example, "Dong Jin, Tingjiao Liu.
  • the technical means is hard in a single layer Cultured vascular endothelial cells on a porous membrane, in the lower three-dimensional Tissue cells - due to the irreversible deformation of the hard porous membrane under pressure, it will cause cell damage, affect cell function, and the cell stress is not consistent with the stress environment in the body;
  • the third is the use of double-layer hard porous Thin film-bound cell culture mimics the lumen structure, for example, in "Young Bok Kang, Moses Noh. Liver Sinusoid on a Chip: Long-Term Layered Co-Culture of Primary Rat Hepatocytes and Endothelial Cells in Microfluidic Platforms. Biotechnology and Bioengineering.
  • the hepatic sinusoid is simulated by the technique of culturing stellate cells on the upper side of the upper hard microporous membrane, culturing vascular endothelial cells on the lower side of the upper hard microporous membrane, and culturing Kupffer cells in the middle lumen of the chip.
  • the biliary epithelial cells are cultured on the upper side of the hard porous membrane of the lower layer of the chip, and the parenchymal cells are cultured on the lower side of the hard porous membrane of the lower layer of the chip.
  • the structure is a luminal structure composed of a hard porous membrane, and the cavity is not Soft, irreversible deformation under pressure, not available
  • the mechanical and physical environment, and simulation of the body is not high, and the material exchange does not match the real situation.
  • the three lumen structure designs are unreasonable, and the technical problem to be solved by the present invention is to provide an organ for simulating a soft lumen in a human body and a lumen structure. And organized microfluidic chips and their applications.
  • the organ chip simulating the lumen structure of the invention has a double-layer flexible membrane structure, and can generate deformation on both sides of the lumen under the action of pressure, and simulates the lumen structure in the body with high simulation degree. With different cells (bacteria), you can simulate different lumens.
  • the intestinal epithelial cells can be cultured in an organ chip to simulate the intestinal lumen; the vascular endothelial cells can be cultured to simulate blood vessels, and the simulated blood vessels can be combined with other cells to simulate a blood vessel tissue or organ.
  • microfluidic chip In order to achieve the above object, a microfluidic chip is provided, and the present invention adopts the following technical solutions:
  • a microfluidic chip the chip being configured as a multilayer structure comprising a double-layered elastic porous membrane forming a flexible lumen structure, wherein upper and lower sides of the upper elastic porous membrane and the lower elastic porous membrane Material and energy exchange between the upper and lower sides.
  • the multilayer structure of the chip comprises: an upper substrate, an upper elastic porous film, a middle substrate, a lower elastic porous film, and a lower substrate in order from top to bottom;
  • the upper surface of the upper substrate is provided with an upper groove, and the upper groove forms a closed upper channel with the upper elastic porous film, and each inner wall of the channel is used for cultivating different cells or bacteria;
  • the middle substrate portion is hollowed out, and the hollow portion thereof is surrounded by the upper elastic porous membrane and the lower elastic porous membrane to form a closed intermediate layer passage, and each inner wall of the passage is used for cultivating different cells or bacteria;
  • the lower surface of the lower substrate is provided with a lower groove, and the lower groove forms a closed lower channel with the lower elastic porous membrane, and the inner walls of the channel are used for cultivating different cells or bacteria.
  • the upper channel, the lower channel, and the intermediate layer channel respectively form a soft lumen structure with the upper elastic porous membrane and the lower elastic porous membrane.
  • the upper and lower elastic porous membranes are deformed by the pressure difference, thereby simulating the soft lumen in the human body.
  • the upper groove has a rectangular, semi-circular or semi-elliptical cross section.
  • the lower groove has a rectangular, semi-circular or semi-elliptical cross section.
  • the multilayer structure of the chip an upper substrate, an upper elastic porous film, a middle substrate, The lower elastic porous membrane and the lower substrate are detachably connected. Therefore, the technical solution has the advantages of facilitating disassembly and assembly between the multi-layer structures, and convenient application and maintenance.
  • the material of the lower substrate, the intermediate substrate and the upper substrate is selected from any one of quartz, glass, PMMA, PDMS polymer, polycarbonate, polyester, agarose, chitosan or sodium alginate.
  • the material of the elastic porous film is PDMS or polyvinylidene fluoride.
  • the invention also provides an application of the above microfluidic chip for simulating a flexible lumen in a human body.
  • the cells implanted in the chip include the intestine, heart, liver, kidney, islets, skin, mouth, stomach, uterus, ovary, eyes, bones, blood vessels, lungs, muscles, fat, tumors, lymph and brain organs.
  • the cells contained in the chip; the bacteria grown in the chip include the intestinal flora and the stomach flora; thus the chip can be applied to simulate organs and tissues with a lumen structure.
  • the technical solution enables the invention to be further applied to test biomarkers to achieve effective evaluation of drugs, cosmetics, health products, and environmental poisons.
  • the invention forms an elastic, soft, cell-loaded (bacterial) chamber on the chip, which can deform under both sides of the lumen under the action of pressure, thereby enabling high simulation degree. Simulate the luminal structure in the real human body, or the tissue or organ with the luminal structure, and then test the biomarkers to achieve effective evaluation of drugs, cosmetics, health products, and environmental toxicants.
  • Figure 1 is a schematic view of the structure of the present invention.
  • Figure 2 is a HUVEC permeability profile on a soft elastic film, from left to right, vascular endothelial cell permeability of sodium fluorescein, fennelol, 40 kD dextran and 70 kD dextran.
  • Figure 2' is a HUVEC permeability profile on the hard membrane. From left to right is the vascular endothelial cell permeability of sodium fluorescein, fennelol, 40 kD dextran and 70 kD dextran.
  • Figure 3 is a diagram showing the effect of a single drug on HUVEC, PTX (0.5 ⁇ g/ml); CDDP (5 ⁇ g/ml); 5-FU (400 ⁇ g/ml).
  • Scale bar 50 ⁇ m.
  • Figure 4 is a graph showing the effect of HUVEC on the combination drug reaction, PTX (0.5 ⁇ g/ml) in combination with CDDP (5 ⁇ g/ml); 5-FU (400 ⁇ g/ml) in combination with CDDP (5 ⁇ g/ml).
  • Scale bar 50 ⁇ m.
  • Figure 5 is a graphical representation of the amount of glucose reabsorption by renal tubular cells on a soft elastic membrane.
  • Figure 5' is a graphical representation of the amount of glucose reabsorption by renal tubular cells on the hard membrane.
  • Figure 6 is a graphical representation of the amount of amino horse uric acid secreted by renal tubular cells on a soft elastic membrane.
  • Figure 6' is a graphical representation of the amount of amino horse uric acid secreted by renal tubular cells on the hard membrane.
  • Figure 7 is a waveform diagram of fluid pressure in the intermediate channel when the artery is simulated using the present invention.
  • Figure 8 is a schematic illustration of the simulated diastolic state (left) and tension (right) of an artery using the present invention.
  • Fig. 9 is a waveform diagram showing the pressure of the intermediate passage bacterial culture solution when the large intestine (or esophagus) is simulated by the present invention.
  • Figure 10 is a schematic illustration of the simulated intestinal wall (or esophageal wall) under different pressures when simulating the large intestine (or esophagus) using the present invention.
  • the chip is provided in a multilayer structure comprising a double-layered elastic porous film forming a flexible lumen, wherein substance and energy are carried out between the upper side of the upper elastic porous membrane 2b and the lower side of the lower elastic porous membrane 2a exchange.
  • the structure is a luminal structure composed of a soft porous membrane. Because the cavity is soft, reversible deformation occurs under pressure, and has a mechanical and physical environment in vivo, which is highly simulated in vivo, and the substance and energy exchange are in accordance with the actual situation. .
  • the multilayer structure of the chip includes an upper substrate 1c, an upper elastic porous film 2b, a middle substrate 1b, a lower elastic porous film 2a, and a lower substrate 1a in order from top to bottom;
  • the upper surface of the upper substrate 1c is provided with an upper groove, and the upper groove forms a closed upper channel 3 with the upper elastic porous film 2b, and the inner walls of the channel are used for cultivating different cells or bacteria;
  • the middle substrate 1b is partially hollowed out, and the hollow portion thereof is surrounded by the upper elastic porous membrane 2b and the lower elastic porous membrane 2a to form a closed intermediate layer passage 3a, and the inner walls of the passage are used for cultivating different cells or bacteria;
  • the upper surface of the lower substrate 1a is provided with a lower groove, and the lower groove forms a closed lower channel 3b with the lower elastic porous film 2a, and the respective inner walls of the channel are used to culture different cells or bacteria.
  • the sections of the upper and lower grooves may be rectangular, semi-circular or semi-elliptical or other required shapes.
  • the multilayer structure of the chip is detachably connected between the upper substrate 1c, the upper elastic porous film 2b, the intermediate substrate 1b, the lower elastic porous film 2a, and the lower substrate 1a.
  • the materials of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c are selected from quartz, glass, PMMA, PDMS polymer, polycarbonate, polyester, agarose, chitosan or sodium alginate. Any of the materials; the material of the elastic porous film is PDMS or polyvinylidene fluoride.
  • the cells implanted in the chip include cells contained in the intestine, heart, liver, kidney, islets, skin, mouth, stomach, uterus, ovaries, eyes, bones, blood vessels, lungs, muscles, fat, tumors, lymph and brain organs;
  • the bacteria grown in the chip include the intestinal flora and the stomach flora; thus the chip is applied to simulate organs and tissues with a lumen structure.
  • the material of the lower substrate 1a, the intermediate substrate 1b, and the upper substrate 1c is PDMS; the upper and lower grooves have a rectangular cross section; the upper channel 3, the intermediate channel 3a, and the lower channel 3b have a width of 50 ⁇ m.
  • the height is 50 microns; human umbilical cord blood endothelial cells (HUVEC) are cultured on the lower surface of the upper elastic porous membrane 2b of PDMS, and the lower PDMS is made of PDMS.
  • Human umbilical cord blood endothelial cells (HUVEC) are cultured on the upper surface of the porous membrane 2a.
  • the vascular endothelial cells of the upper and lower layers grow into a dense cell membrane, and the cells form a tight connection with the cells, which does not leak, and the upper and lower layers are PDMS.
  • the thickness of the elastic porous membrane is 8 ⁇ m
  • the lumen structure composed of the upper and lower elastic porous membranes and the hollow structure of the intermediate substrate simulates human capillaries.
  • the pressure of the fluid in the upper and lower channels is 10 mbar
  • the pressure of the simulated blood in the middle channel is 20 mbar.
  • the PDMS porous membrane undergoes elastic deformation and stretches the vascular endothelial cells, but does not destroy the tight junction between the vascular endothelial cells, and does not affect the barrier function and physiology of the vascular endothelial cell membrane.
  • the simulated capillaries have the permeability characteristics of real capillaries, and the larger the molecular weight, the slower the permeation rate (sodium fluorescein>Punain>40 kD dextran>70 kD dextran ).
  • the capillaries simulated in this example were used for the study of drug vascular toxicity.
  • high concentrations of CDDP (f channel) and 5-FU (d, e, f channels) resulted in gaps between HUVEC cells.
  • the effect of PTX on HUVEC is not obvious. This result suggests that high concentrations of CDDP and 5-FU can cause damage to the HUVEC barrier, which may cause phlebitis in part when administered in vivo.
  • This embodiment has the advantage that the conventional technique only simulates the capillary wall on one side, and the substance exchange occurs only on the side, and the present embodiment simulates the capillary lumen, the substance exchange occurs on both sides, and the exchange area is larger. Since the simulated capillary wall is soft, the vascular endothelial cell layer can maintain a dense cell layer under pressure, thereby maintaining its barrier function and physiological function. The simulated blood vessel can be used to evaluate the toxic effects of the drug on the vessel wall.
  • Example 2 Simulation of renal tubular and perivascular vessels
  • the material of the lower substrate 1a, the intermediate substrate 1b, and the upper substrate 1c is PDMS; the upper and lower grooves have a rectangular cross section; the upper, middle, and lower channels have a width of 100 ⁇ m and a height of 100 ⁇ m.
  • Renal tubular epithelial cells were cultured on the lower surface of the upper PDMS elastic porous membrane, vascular endothelial cells were cultured on the upper surface, renal tubular epithelial cells were cultured on the upper surface of the lower PDMS elastic porous membrane, and vascular endothelial cells were cultured on the lower surface, similar to the body, up and down
  • the layers of vascular endothelial cells and renal tubular epithelial cells grow into a dense cell membrane, and the cells form a tight connection with the cells without leakage.
  • the thickness of the upper and lower PDMS elastic porous membrane is 15 micrometers, and the upper and lower elastic porous membranes
  • the lumen structure composed of the hollow structure of the middle substrate simulates the human renal tubules, while the upper channel 3 and the lower channel 3b simulate the perivascular vessels.
  • the simulated blood pressure in the upper channel 3 and the lower channel 3b was 10 mbar, and the simulated urine pressure in the middle channel 3a was 40 mbar. Since the simulated renal tubules and perivascular vessels are soft, the PDMS porous membrane undergoes elastic deformation under tension of 30 mbar, stretching vascular endothelial cells and renal tubular epithelial cells, but does not destroy vascular endothelial cells and renal tubular epithelial cells.
  • the tight junctions do not affect the barrier and physiological functions of the vascular endothelial cell layer and the renal tubular epithelial cell layer. If it is a traditional hard membrane, under the pressure, the hard membrane will undergo irreversible deformation, and there will be fissures between the vascular endothelial cells or between the renal tubular cells, affecting the barrier function and physiological function of the vascular endothelial cell membrane or muscle cells.
  • the simulated renal tubules and perivascular vessels have reabsorption and secretion of true tubular and perivascular vessels.
  • the concentration of glucose in the upper channel 3 and the lower channel 3b was 16.88 mM
  • the glucose concentration in the middle channel 3a was 3.12 mM.
  • the advantage of this embodiment is that the physiological structures of the renal tubules and perivascular vessels (including the vascular endothelial cell layer and the renal tubular epithelial cell layer) are simulated. Since the walls of the simulated renal tubules and perivascular vessels are soft, the vascular endothelial cell layer and the tubular epithelial cell layer can maintain a dense cell layer under pressure, thereby maintaining its barrier function and physiology. The function, which lays a solid foundation for the molecular biology and cell biology of tubular and perivascular vessels in vitro. The simulated renal tubules and perivascular vessels have reabsorption and secretion of true tubular and perivascular vessels.
  • the material of the lower substrate 1a, the intermediate substrate 1b, and the upper substrate 1c is PDMS; the upper and lower grooves have a rectangular cross section; the upper channel 3, the intermediate channel 3a, and the lower channel 3b have a width of 2 mm.
  • the height is 500 microns.
  • Human umbilical cord blood endothelial cells (HUVEC) were cultured on the lower surface of the upper PDMS elastic porous membrane, muscle cells were cultured on the upper surface, human umbilical cord blood endothelial cells (HUVEC) were cultured on the upper surface of the lower PDMS elastic porous membrane, and muscle cells were cultured on the lower surface.
  • the vascular endothelial cells and muscle cells in the upper and lower layers grow into a dense cell membrane, and the cells form a tight connection with the cells without leakage.
  • the thickness of the upper and lower PDMS elastic porous membrane is 30 microns, and the upper and lower layers are
  • the luminal structure composed of the elastic porous membrane and the hollow structure of the middle substrate simulates the small arteries of the human.
  • the pressure of the fluid in the upper channel 3 and the lower channel 3b is 20 mbar
  • the pressure of the simulated blood in the middle channel 3a is a square wave, switching back and forth between 20 mbar and 50 mbar, as shown in FIG.
  • the upper and lower elastic porous membranes generate periodic pulsations due to pressure differences on both sides, simulating the pulsation of small arterial blood vessels.
  • the state of relaxation and the state of tension of the elastic porous membrane are as shown in Fig. 8, respectively simulating the diastolic state (left) and the tension state (right) of the small arteries, and in the diastolic state, the upper, middle and lower layers.
  • the fluid pressure is 20 mbar.
  • the pore diameter of the elastic porous membrane is 10 ⁇ m.
  • the fluid pressures of the upper, middle and lower layers are 20, 50, 20 mbar, respectively, and the pore diameter of the elastic porous membrane is expanded to 14 ⁇ m. .
  • the simulated arterioles are soft and elastic, they can produce pulsation under pressure, and under the pressure of 30 mbar, the PDMS porous membrane undergoes elastic deformation, stretching vascular endothelial cells and muscle cells, but not It will destroy the tight junction between vascular endothelial cells and muscle cells, and will not affect the barrier function and physiological function of vascular endothelial cell membrane and muscle cells.
  • the hard membrane will undergo irreversible deformation, and there will be fissures between the vascular endothelial cells or between the muscle cells, affecting the barrier function and physiological function of the vascular endothelial cell membrane or muscle cells.
  • This example has the advantage of simulating the physiological structure of the arterioles (including the vascular endothelial cell layer and the muscle cell layer) for simulation, since the simulated arteriolar wall is soft and elastic, thus simulating the pulsation of the artery. Also because the simulated small arterial wall is soft, the arterial endothelial cell layer and the muscle layer can maintain a dense cell layer under pressure, thereby maintaining its barrier function and physiological function. Furthermore, it lays a solid foundation for studying the molecular biology and cell biology of small arteries in vitro.
  • the material of the lower substrate 1a, the intermediate substrate 1b, and the upper substrate 1c is PDMS; the upper and lower grooves have a rectangular cross section; the upper channel 3, the intermediate channel 3a, and the lower channel 3b have a width of 750 micrometers.
  • the height is 1 mm.
  • Intestinal epithelial cells are cultured on the lower surface of the upper PDMS elastic porous membrane 2b, muscle cells are cultured on the upper surface, intestinal epithelial cells are cultured on the upper surface of the lower PDMS elastic porous membrane 2a, muscle cells are cultured on the lower surface, and intestinal epithelial cells are cultured on the upper and lower layers.
  • the surface is inoculated with intestinal flora, similar to the body.
  • the thickness of the upper and lower PDMS elastic porous membrane is At 70 microns, the lumen structure consisting of the upper and lower elastic porous membranes and the hollowed-out structure of the intermediate substrate simulates the human large intestine.
  • the pressure of the fluid in the upper channel 3 and the lower channel 3b was 20 mbar, and the pressure of the bacterial culture solution in the middle channel 3a was a sinusoidal waveform with an amplitude of 120 mbar, as shown in FIG.
  • the upper and lower elastic porous membranes generate periodic pulsations due to pressure differences on both sides, simulating The peristalsis of the intestines.
  • the state of the elastic porous membrane under different pressures respectively simulates the state of the intestinal wall when the large intestine is peristaltic. Because the simulated arterioles are soft and elastic, the state of peristalsis can be simulated. Under the pressure of 100 mbar, the PDMS porous membrane undergoes elastic deformation, stretching intestinal epithelial cells and muscle cells, but does not break the intestinal epithelium. The tight junction between cells and muscle cells does not affect the barrier function and physiological function of the intestinal cell layer and muscle cell layer.
  • the hard membrane will undergo irreversible deformation, and there will be fissures between the intestinal epithelial cells or between the muscle cells, affecting the barrier function and physiological function of the intestinal epithelial cell layer or muscle cell layer.
  • the physiological structure of the large intestine (including the intestinal epithelial cell layer, the muscle cell layer, and the intestinal flora layer) is simulated, and since the simulated large intestine wall is soft and elastic, the peristalsis of the large intestine can be simulated. Also, since the simulated large intestine wall is soft, the intestinal epithelial cell layer and the muscle layer can maintain a dense cell layer under pressure, thereby maintaining its barrier function and physiological function. Furthermore, it lays a solid foundation for studying intestinal absorption and metabolism in vitro.
  • Example 5 Simulation of uterus and in vitro culture of fertilized eggs
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3 and the lower channel 3b are both 500 ⁇ m and the height is 1 mm, and the width and height of the middle channel 3a are both 5 mm, the thickness of the upper and lower PDMS elastic porous membrane is 60 ⁇ m, and the endometrial epithelial cells are cultured on the lower surface of the upper elastic porous membrane 2a, and the endometrial epithelial cells are cultured on the upper surface of the lower elastic porous membrane 2a.
  • the luminal structure composed of the layered elastic porous membrane and the hollow portion of the intermediate substrate simulates the human uterus.
  • the artificially fertilized egg is introduced into the simulated uterus, that is, the middle channel 3a in Fig. 1, so that it adheres to the endometrial epithelial cells, and the nutrients required for the development of the artificially fertilized egg flow from the upper channel 3 and the lower channel 3b.
  • the culture medium is supplied, the pressure of the fluid is 5 mbar, and the pressure of the culture medium in the middle channel 3a is also 5 mbar. Since the simulated uterus has endometrial epithelial cells, the artificial fertilized egg grows better, and the simulated uterus has the characteristics of easy disassembly. The artificial fertilized egg is easily taken out.
  • the artificial fertilized egg of the uterus culture based on the invention has a survival rate of 70% before being implanted back to the mother body, and the traditional micro-droplet culture method is carried out before returning to the mother body.
  • the rate is only 20%.
  • the advantage of this embodiment is that the physiological structure of the uterus is simulated, and the culture of the artificial fertilized egg is realized. Because the simulated uterus has endometrial epithelial cells and is easy to disassemble, the adhered artificial fertilized eggs are easily removed, which greatly improves the success rate of artificial fertilized eggs in vitro, thereby improving the success rate of IVF and reducing The cost of IVF.
  • Example 6 Simulation of esophagus and swallowing
  • the upper channel 3, the intermediate channel 3a and the lower channel 3b each have a width of 500 ⁇ m; the height is 1 mm, and the epithelial cells of the esophagus are cultured on the lower surface of the PDMS upper elastic porous membrane 2b, and the smooth muscle is cultured on the upper surface.
  • the cells culture the epithelial cells of the esophagus on the upper surface of the PDMS lower elastic porous membrane 2a, and the smooth muscle cells are cultured on the lower surface.
  • the thickness of the upper and lower PDMS elastic porous membrane is 30 micrometers, and the upper and lower elastic porous membranes and the intermediate substrate hollow structure are composed.
  • the lumen structure mimics the human esophageal lumen.
  • the pressure of the fluid in the upper channel 3 and the lower channel 3b was 20 mbar, and the pressure of the culture solution in the middle channel 3a was changed in the form of a sinusoidal waveform with an amplitude of 120 mbar as shown in FIG.
  • the upper and lower elastic porous membranes generate periodic pulsations due to pressure differences on both sides, simulating the swallowing process of the esophagus.
  • the PDMS porous membrane Under the pressure of 100 mbar, the PDMS porous membrane undergoes elastic deformation, stretching esophageal epithelial cells and smooth muscle cells, and promoting esophageal epithelial cells to secrete esophageal mucosa. If it is a traditional hard membrane, under the pressure, the hard membrane will undergo irreversible deformation, affecting the function and physiological function of the secretory mucosa of the esophageal epithelial cell layer or smooth muscle cell layer.
  • the advantage of this embodiment is that it simulates the physiological structure of the esophagus (including the esophageal epithelial cell layer, the smooth muscle cell layer and the intestinal flora layer), and because the simulated esophageal wall is soft and elastic, it can simulate the peristalsis of the esophagus. . Also, since the simulated esophageal wall is soft, the esophageal epithelial cell layer and the smooth muscle layer can promote the secretion of mucosa by the esophageal epithelial cells under the action of pressure, and promote the food swallowing process and protect the food from esophageal epithelial cells during swallowing. damage. This lays a solid foundation for studying the esophageal swallowing process in vitro.
  • the material of the lower substrate 1a and the intermediate substrate 1b is PDMS
  • the material of the upper substrate 1c is glass
  • the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 750 micrometers and the height is 1 mm, respectively.
  • the skin horny layer cells are cultured on the upper surface of the PDMS upper elastic porous membrane 2b, and the dermal layer cells are cultured on the lower surface, in PDMS
  • the lower surface of the lower elastic porous membrane 2a is cultured with vascular endothelial cells, and a hydrogel is filled in the lumen structure composed of the upper and lower elastic porous membranes and the hollow structure of the intermediate substrate, and the PDMS upper elastic porous membrane 2b and the hydrogel are loaded.
  • the combination simulates the skin
  • the lower channel 3b simulates the subcutaneous blood vessels.
  • the culture medium flowing in the subcutaneous blood vessels 3b provides nutrients to the dermal cells and the stratum corneum cells through the vascular endothelial cells and the hydrogel to maintain the physiological activity of the skin.
  • the upper channel 3 is connected to a high-pressure air stream, and the soft simulated skin can sense the high-pressure air flow, thereby promoting multi-layer differentiation of the stratum corneum cells.
  • the advantage of this embodiment is that it simulates the physiological structure of the skin (including the stratum corneum, the dermis layer and the subcutaneous blood vessels), and since the simulated skin is soft and elastic, it can sense the high-pressure air flow, thereby promoting the differentiation of the stratum corneum. Simulated subcutaneous blood vessels provide nutrients to skin cells and maintain the physiological properties of the skin.
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 750 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • the trigeminal ganglion cells are cultured on the upper surface of the PDMS upper elastic porous membrane 2b, the vascular endothelial cells are cultured on the lower surface, the trigeminal ganglion cells are cultured on the lower surface of the PDMS lower elastic porous membrane 2a, and the upper surface cultures the vascular endothelial cells in the upper layer.
  • the channel 3 and the lower channel 3b are passed through a culture medium suitable for the trigeminal ganglion cells to maintain the activity and morphological function of the cells, and are pulsating in the luminal structure composed of the upper and lower elastic porous membranes and the hollow structure of the intermediate substrate.
  • the culture medium due to the flexibility of the porous PDMS film (2b and 2a in Fig. 1), the pulsating culture solution causes the elastic porous membrane to contract and relax, mimicking the contractile relaxation of the blood vessel, which changes the permeability of the vascular endothelial cell membrane.
  • the advantages of this embodiment are: by the softness characteristics of the elastic porous film, applying different pressures, simulating the mechanical environment of the blood vessel during contraction and relaxation, and more simulating the microenvironment of the brain trigeminal nerve and blood vessel, by adding different Stimulating factors, convenient study of migraine factors caused by trigeminal vasculature, more simulation than existing cell models, real response to the body Environment, compared with animal models, the time period is shorter and more convenient, providing a good research tool for the establishment of migraine pathological model.
  • Example 9 Simulation of a muscle stretching model
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • Micron, smooth muscle cells were cultured on the upper surface of the PDMS upper elastic porous membrane 2b, vascular endothelial cells were cultured on the lower surface, smooth muscle cells were cultured on the lower surface of the PDMS lower elastic porous membrane 2a, and vascular endothelial cells were cultured on the upper surface, in the upper channel 3 and the lower layer.
  • the channel 3b is provided with a culture medium suitable for smooth muscle cells to maintain the activity and morphological function of the cells, and the pulsating culture medium is filled in the lumen composed of the upper and lower elastic porous membranes and the hollow structure of the intermediate substrate, that is, 3a in FIG. .
  • the pulsating culture solution Due to the flexibility of the porous PDMS film (2b and 2a in Fig. 1), the pulsating culture solution causes the film to contract and relax, simulating the stretching process of the muscle, and controlling the force of the smooth muscle cells by controlling the amplitude of the pulsating culture medium. Size, when the smooth muscle cells are stressed too much, smooth muscle cell damage occurs, and the relationship between the stress of smooth muscle cells and cell damage is determined by measuring the specific protein expression of smooth muscle cells, thereby simulating the cell model of muscle damage.
  • the advantage of this embodiment is that, by the softness characteristics of the elastic porous film, different pressures are applied to simulate the mechanical mechanical environment during muscle stretching. Unlike the existing cell model, the PDMS can be quantitatively controlled by changing the pressure. The deformation of the elastic porous film in the vertical direction can conveniently control the force of the smooth muscle cells.
  • Example 10 Simulation of a measurement model of cardiac cell mechanics characteristics
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • the cardiomyocytes are cultured on the upper surface of the PDMS upper elastic membrane 2b, the cardiomyocytes are cultured on the lower surface of the PDMS lower elastic membrane 2a, and the culture medium suitable for the cardiomyocytes is introduced into the upper channel 3 and the lower channel 3b to maintain the cells.
  • the activity and morphological function are filled in the culture medium in a lumen composed of the upper and lower elastic membranes and the hollow structure of the intermediate substrate, that is, 3a in Fig. 1 . Due to the softness of the PDMS film (ie, 2b and 2a in Fig. 1), the beating state of the cardiomyocytes can be transmitted, thereby visually determining the change of the culture medium of the lumen in the lumen level to determine the heart. The beating frequency and the amplitude of the beating of muscle cells.
  • the advantage of this embodiment is that: by the softness characteristics of the PDMS elastic film, the cardiomyocytes are planted in the elastic film, and the cardiomyocytes themselves will beat, thereby driving the elastic film to beat, and measuring the change of the lumen liquid surface, the ingenious reaction The beating state of the cardiomyocytes.
  • Example 11 Simulation of the effect of tumor microenvironmental mechanical characteristics on tumor metastasis
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • the tumor cells are cultured on the upper surface of the PDMS upper elastic porous membrane 2b, the vascular endothelial cells are cultured on the lower surface, the tumor cells are cultured on the lower surface of the PDMS lower elastic porous membrane 2a, and the upper surface cultures the vascular endothelial cells in the upper channel 3 and the lower layer.
  • the channel 3b is passed through a culture medium suitable for tumor cells to maintain the activity and morphological function of the cells, and a pulsating culture solution is filled in the lumen structure composed of the upper and lower elastic porous membranes and the hollow structure of the intermediate substrate. Due to the flexibility of the porous PDMS film, the pulsating culture solution causes the film to contract and relax. By controlling the amplitude of the pulsating culture medium, the force of the tumor cells is controlled, thereby observing the tumor cell transfer under the force of the tumor cells. The effect of rate changes.
  • the advantage of this embodiment is that: by the softness characteristics of the elastic porous film, different pressures can be applied, the deformation of the film can be changed, and the force of the tumor cells can be controlled by the control of the pressure, thereby observing the force of the tumor cells. The effect on the rate of tumor cell metastasis.
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • Micron, hepatocytes and hepatic stellate cells are cultured on the upper surface of the PDMS upper elastic porous membrane 2b, vascular endothelial cells are cultured on the lower surface, and hepatocytes and hepatic stellate cells are cultured on the lower surface of the PDMS lower elastic porous membrane 2a.
  • the surface cultures vascular endothelial cells, and the culture medium suitable for hepatocytes and hepatic stellate cells is introduced into the upper channel 3 and the lower channel 3b to maintain the activity and morphological function of the cells, and is hollowed out by the upper and lower elastic porous membranes and the middle substrate.
  • the advantage of this embodiment is that the physiological structure of the hepatic sinusoid is simulated, which is more simulated than the existing cell model, and the device is easy to disassemble, and it is easy to take out the cells inside to detect relevant indicators, which greatly reduces the cost of drug testing. .
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • vascular endothelial cells were cultured on the upper surface of the PDMS upper elastic porous membrane 2b, lung epithelial cells were cultured on the lower surface, vascular endothelial cells were cultured on the lower surface of the PDMS lower elastic porous membrane 2a, and the upper surface cultured lung epithelial cells were in the upper passage 3 a culture medium suitable for vascular endothelial cells is introduced into the lower channel 3b to maintain the activity and morphological function of the cells, and pulsating sterile air is filled into the lumen composed of the upper and lower elastic porous membranes and the hollow structure of the intermediate substrate. Simulate the physiological structure of the bronchi. The passage of pulsating sterile air simulates the process of air entering the bronchus during breathing.
  • the advantage of this embodiment is that the physiological structure of the bronchus is simulated. Due to the softness of the PDMS elastic porous membrane, the pulsating sterile air can simulate the breathing process, and the medicine for treating asthma can be added in the sterile air. The test drug can pass through the lung epithelial cells into the blood to determine the dosage form of the drug.
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • the lymphatic endothelial cells are cultured on the upper surface of the PDMS upper elastic porous membrane 2b, the vascular endothelial cells are cultured on the lower surface of the PDMS lower elastic porous membrane 2a, the culture medium with lymphocytes is introduced into the upper channel 3, and the lower channel 3b is passed.
  • a culture medium suitable for vascular endothelial cells and an inflammatory factor is added to the culture solution of the lower channel 3b, and the tissue cells and the three-dimensional gel are introduced into the luminal structure composed of the upper and lower elastic porous membranes and the hollow structure of the intermediate substrate.
  • the mixture mimics the lymphatic-tissue-vascular system.
  • the advantage of this embodiment is that the lymphatic-tissue-vessel micro-environment is simulated, the chip is easy to disassemble, the pore size of the porous PDMS elastic membrane is controllable, the substance exchange is easy, and the lymphocyte metastasis process is easily observed, and the lymphatic system in vitro is realized. Dynamic visualization.
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 1 mm and 150, respectively.
  • the vascular endothelial cells are cultured on the upper surface of the PDMS upper elastic porous membrane 2b, the osteoblasts are cultured on the upper surface of the PDMS lower elastic porous membrane 2a, and the culture medium suitable for the vascular endothelial cells is introduced into the upper passage 3, and on the upper layer.
  • Macrophage is added to the culture medium of channel 3, and a culture medium suitable for osteoblasts is introduced into the lower channel 3b, and a lumen composed of an upper and lower elastic porous membrane and a hollow structure of the intermediate substrate (ie, 3a in Fig. 1)
  • the culture medium with LPS is introduced to simulate the microenvironment of the gingival sulcus.
  • the advantages of this embodiment are: simulating the microenvironment of the gingival sulcus, for the study of periodontal disease, the chip is easy to disassemble, and it is convenient to evaluate the activity and morphology of the cells in each layer.
  • the aperture of the porous PDMS elastic membrane is controllable and easy to exchange substances. And easy to observe the macrophage transfer process.
  • Example 16 Simulation of a fatty diabetes model
  • the material of the lower substrate 1a, the intermediate substrate 1b and the upper substrate 1c is PDMS, and the widths of the upper channel 3, the intermediate channel 3a and the lower channel 3b are respectively 500 ⁇ m and the heights are 150 ⁇ m, 150 ⁇ m and 150, respectively.
  • the lower elastic porous membrane 2a and the upper elastic porous membrane 2b have a thickness of 30 micrometers, and a co-culture solution suitable for the fat cells and the islet cells is introduced into the upper channel 3, and is composed of the upper and lower elastic porous membranes and the intermediate substrate hollow structure.
  • the lumen i.e., at 3a in Fig.
  • the advantage of this embodiment is that the fat diabetes model is simulated. Due to the softness and porosity of the PDMS elastic porous membrane, the adhesion between the three-dimensional adhesive and the membrane is tight, which is beneficial to material exchange, and the experimental phenomenon is obvious, and the chip is easy to disassemble. It is convenient to evaluate the activity and morphology of cells in each layer, as well as the protein expression pathway of islet cells.

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Abstract

L'invention concerne une puce microfluidique et une application correspondante. La puce est une structure multicouche, la structure multicouche correspondante comprenant en particulier : un substrat de couche supérieure (1c), une membrane poreuse élastique de couche supérieure (2b), un substrat de couche intermédiaire (1b), une membrane poreuse élastique de couche inférieure (2a) et un substrat de couche inférieure (1a) étant compris en séquence de haut en bas, une structure de lumière flexible étant formée par un canal de couche supérieure (3), un canal de couche inférieure (3b) et un canal de couche intermédiaire (3a) respectivement avec la membrane poreuse élastique de couche supérieure (2b) et la membrane poreuse élastique de couche inférieure (2a). Lorsque la pression fluidique dans les canaux de couche supérieure et inférieure est incompatible avec la pression fluidique dans le canal de couche intermédiaire, les membranes poreuses élastiques de couche supérieure et inférieure se déforment en raison de la différence de pression, de telle sorte que la puce microfluidique peut simuler une lumière flexible ou un organe et un tissu avec une structure de lumière dans le corps humain à un degré de simulation élevé et ainsi, un biomarqueur peut être testé pour réaliser l'évaluation efficace d'un médicament, d'un produit cosmétique, d'un produit de soin de santé et d'une toxine environnementale.
PCT/CN2017/115760 2017-07-28 2017-12-12 Puce microfluidique et application correspondante WO2019019526A1 (fr)

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KR102534432B1 (ko) * 2022-09-13 2023-05-26 텐드바이오 주식회사 생체 모사 장기 칩

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