Microfluidic chip for dynamic culture of nasal epithelium organs and culture method thereof

Abstract

The invention discloses a microfluidic chip for dynamic culture of nasal epithelium organs and a culture method of the microfluidic chip. The microfluidic chip is of a three-layer structure, the upper layer of the chip is made of PDMS or PMMA, the middle layer of the chip is a microporous membrane made of PET or PDMS, and the lower layer of the chip is made of PDMS or PMMA; a cell culture chamber and a liquid storage tank are arranged in the chip upper layer; a snakelike microfluid channel is arranged in the lower chip layer and is communicated with the cell culture chamber and the liquid storage tank in the upper chip layer. The microfluid organ chip can fill up the deficiency of a current nasal epithelium dynamic in-vitro model, a cilia mucus epithelium structure highly consistent with in-vivo nasal epithelium tissue is constructed by using nasal epithelium organ differentiation, and dynamic stimulation and gas-liquid interface environmental factors on the differentiated nasal epithelium organ are reduced through the microfluid chip. The real in-vivo nasal epithelium tissue is restored to the maximum extent, and a mature nasal epithelium barrier structure can be formed by the nasal epithelium organ in the microfluidic chip at a high success rate.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic organ chips, and in particular to a microfluidic chip for dynamic culture of nasal epithelial organoids and a culture method thereof. Background Art

[0002] The mucosal surfaces of the nasal epithelium and airway epithelium continuously remove various pollutants from the inhaled air through mucociliary clearance, which is an important defense mechanism against lung infection and injury. Although some studies have successfully induced differentiation of nasal epithelial organoids under Transwell or three-dimensional culture conditions for more than 20 days to obtain a complete epithelial barrier and all nasal epithelial cell types, studies have shown that the full maturation of cilia beating and related mucociliary clearance under static conditions in airway epithelium, which is highly similar to the nasal epithelium, may require more than 60 days of differentiation. In addition, these static culture conditions cannot simulate the effects of the fluid environment on the differentiation and maturation of nasal epithelial cells.

[0003] Microfluidic chip technology is an emerging in vitro model construction technology. This technology can reproduce the direct or indirect effects of various fluids or mechanical factors on biological tissues through special fluid channel design, and improve the authenticity and reliability of in vitro models when used to reproduce or predict body responses. The respiratory barrier is one of the important directions for the construction of microfluidic chip models. Since the first alveolar epithelial chip model was proposed in 2010, there have been many studies on reproducing alveoli and airway epithelium on microfluidic chips, as well as various fluids and mechanical factors related to them, and meaningful results have been achieved. However, there are still very few attempts to study microfluidic chips based on nasal epithelium and nasal epithelial organoids, and the nasal cavity and nasal epithelium, as the first barrier to the respiratory tract, also play a very important role. Therefore, it is of great significance to combine nasal epithelial organoids with microfluidic organ chips to construct a nasal epithelial organoid chip model that is closer to the real nasal epithelium environment. Summary of the invention

[0004] In order to solve the above-mentioned existing problems, the present invention provides a microfluidic chip for the dynamic culture of nasal epithelial organoids and a culture method thereof. The microfluidic organ chip combines a microfluidic chip with a nasal epithelial organoid, which can fill the gap in the current dynamic in vitro model of the nasal epithelium. The nasal epithelial organoids are differentiated to construct a ciliary mucus epithelial structure that is highly consistent with the in vivo nasal epithelial tissue, and the dynamic stimulation and air-liquid interface environmental factors of the differentiated nasal epithelial organoids are restored through the microfluidic chip, thereby maximally restoring the real in vivo nasal epithelial tissue. For the amplification and differentiation of nasal epithelial organoids in microfluidic chips, the present invention provides a special amplification and differentiation culture medium and perfects the supporting operation methods, which can achieve a high success rate of nasal epithelial organoids in microfluidic chips to form a mature nasal epithelial barrier structure.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A microfluidic chip for dynamic culture of nasal epithelial organoids, the microfluidic chip having a three-layer structure, wherein the upper layer of the chip is made of PDMS or PMMA, the middle layer of the chip is a microporous membrane made of PET or PDMS, and the lower layer of the chip is made of PDMS or PMMA; a cell culture chamber and a liquid reservoir are arranged in the upper layer of the chip; a serpentine microfluidic channel is arranged in the lower layer of the chip, the microfluidic channel connects the cell culture chamber and the liquid reservoir in the upper layer of the chip, and the channel cross-section of the microfluidic channel is rectangular; the microporous membrane in the middle layer of the chip is used to separate the upper layer of the chip from the lower layer of the chip and can allow liquid to pass through.

[0007] Preferably, in the above-mentioned microfluidic chip for dynamic culture of nasal epithelial organoids, the cell culture chamber is 1-3 independent cylindrical cell culture chambers, the upper end of the culture chamber is an open structure, the culture chamber height is 1.5-5mm, and the diameter is 4-9mm; the liquid reservoir is located on both sides of the chip and is a rectangular liquid reservoir.

[0008] Preferably, in the above-mentioned microfluidic chip for dynamic culture of nasal epithelial organoids, the microporous membrane is uniformly distributed with a large number of micropores penetrating the microporous membrane, the micropore diameter is 0.45-8 μm, and the pore density is 0.5-4×10 7 Pieces / cm 2 The microporous membrane is 8-45mm long, 8-35mm wide and 5-20μm thick.

[0009] Preferably, in the above-mentioned microfluidic chip for dynamic culture of nasal epithelial organoids, a cylindrical chamber is provided in the lower layer of the chip, and the cylindrical chamber is located directly below the upper cell culture chamber, and the diameter of the cylindrical chamber is the same as the diameter of the upper cell culture chamber; the upper end of the cylindrical chamber is separated from the upper cell culture chamber by a microporous membrane, and the lower end is a closed structure, and the cylindrical chambers are connected in series through serpentine flow channels, and the inlet and outlet of the lower microfluidic channel are connected to the liquid storage tanks on both sides of the upper layer of the chip, and the height of the microfluidic channel is 0.4-1.5mm.

[0010] Preferably, in the above-mentioned microfluidic chip for dynamic culture of nasal epithelial organoids, the thickness of the upper layer of PDMS or PMMA material of the chip is 3-10 mm, the thickness of the lower layer of PDMS or PMMA material of the chip is 0.8-5 mm, the overall length of the chip is 34-40 mm, and the width is 25-32 mm.

[0011] Preferably, in the above-mentioned microfluidic chip for dynamic culture of nasal epithelial organoids, the surfaces of the upper cell culture chamber and the lower microfluidic channel need to be smooth; the inlet and outlet of the microfluidic channel are respectively located at the upper left, lower right side or the upper right, lower left side of the cell culture chamber in the horizontal direction, and the horizontal distance between the center of the inlet and outlet and the center of the cell culture chamber is 4-9 mm horizontally and 4-12 mm vertically; the microporous membrane does not block the inlet and outlet of the microfluidic channel. If the microporous membrane covers the inlet and outlet positions, holes are opened at the corresponding positions of the microporous membrane; the edges of the lower cylindrical chamber are 4-9 mm apart; the lower microfluidic channel is straight or arc-shaped, without an angle structure.

[0012] A method for culturing nasal epithelial organoids, using the above-mentioned microfluidic chip for culturing, comprises the following steps:

[0013] (1) Surface treatment of the microfluidic chip: Add 20-300 μg / mL type I collagen or type IV collagen solution to the upper cell culture chamber of the microfluidic chip, incubate at 37°C for 0.5-24 h, aspirate and wash with PBS, and keep the chip moist for later use;

[0014] (2) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at a concentration of 4-20×10 5 The cell density of cells / mL and the cell suspension of 50-130 μL / pore volume are added to the upper cell culture chamber, and 400-800 μL of amplification medium is added to the reservoir to fill the lower microfluidic channel with the amplification medium, and the cells are cultured in an environment with a temperature of 35-38°C, a CO2 concentration of 4-6%, and a culture humidity of 92-98%;

[0015] (3) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper cell culture chamber was replaced every 1-3 days. The cells in the cell culture chamber were expanded for 3-9 days until the cells covered the microporous membrane. The liquid in the upper chamber and the lower flow channel was replaced with nasal epithelial organoid differentiation medium, and then a precision shaker was used to control the flow of the medium in the lower flow channel at a flow rate of 0.2-1.2 μL / min;

[0016] (4) Air-liquid interface (ALI) induced differentiation in microfluidic chips: maintain a constant flow rate of differentiation medium in the lower channel, discard the medium in the upper cell culture chamber and continue to culture under the same conditions for 15-45 days. During this period, the upper chamber is washed with PBS every 4-10 days until the organoids are completely differentiated. An important sign of organoid differentiation is the production of movable cilia.

[0017] Preferably, in the above-mentioned method for culturing nasal epithelial organoids, the internal surface of the microfluidic chip is sterilized by moist heat sterilization or ultraviolet sterilization before treatment, and then the upper chamber and the lower flow channel are cleaned with PBS; when the culture medium is introduced into the lower microfluidic channel, it should be ensured that the liquid completely fills the flow channel without residual bubbles; and the entire process of allowing the lower liquid to flow should prevent bubbles from entering the lower flow channel with the liquid.

[0018] Preferably, in the above-mentioned method for culturing nasal epithelial organoids, the nasal epithelial cell expansion medium in step (2) consists of a basal medium Advanced DMEM / F12, 4-hydroxyethylpiperazineethanesulfonic acid, glutamine, penicillin-streptomycin, recombinant human R-spondin 1, noggin, B27 serum-free additive, N-acetyl-L-cysteine, nicotinamide, Y-27632 dihydrochloride, ALK5 inhibitor, SB202190 hydrochloride, fibroblast growth factor 7, fibroblast growth factor 10 and primary cell antibiotics.

[0019] Preferably, in the above-mentioned method for culturing nasal epithelial organoids, the nasal epithelial organoid differentiation medium in step (3) comprises the basal medium Advanced DMEM / F12, 3,3',5-triiodo-L-thyronine sodium salt, hydrocortisone, (±)-epinephrine hydrochloride, ALK5 inhibitor, aromatic retinoic acid, recombinant human epidermal growth factor, neuregulin 1 and penicillin-streptomycin.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The nasal epithelial organoid microfluidic chip of the present invention combines nasal epithelial organoids that can form a complete differentiated nasal epithelial barrier with a microfluidic chip, introduces a dynamic fluid environment on the basis of the nasal epithelial organoids, and gives the nasal epithelial organoids fluid and mechanical stimulation that is closer to real nasal epithelial tissue;

[0022] (2) The nasal epithelial organoid microfluidic chip of the present invention can simulate the actual trans-epithelial concentration gradient at the local location of the nasal epithelium under a flow environment and simulate the process of the nasal epithelium entering the blood circulation system after drug administration.

[0023] (3) The culture method of the nasal epithelial organoids of the present invention enables the nasal epithelial organoids to stably form a cell composition, distribution, tight junction barrier and mature mucociliary monolayer nasal epithelial structure that are consistent with real nasal epithelial tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the overall structure of the microfluidic organ chip for dynamic culture of nasal epithelial organoids of the present invention, wherein Figure a is the overall appearance of the three-dimensional solid chip; Figure b is a bird's-eye view of the overall chip structure, 1, 2-microfluidic channel inlet and outlet, 3-lower chip microfluidic channel, 4, 5-liquid storage tank, 6-middle layer PET or PDMS microporous membrane, 7, 8, 9-upper cell culture chamber; Figure c is the lower layer PDMS or PMMA of the chip; Figure d is the upper layer PDMS or PMMA of the chip.

[0025] Figure 2 This is a rendering of the present invention constructing a single-layer nasal epithelial organoid in a microfluidic organ chip.

[0026] Figure 3 This is a diagram showing the effect of inducing differentiation of nasal epithelial organoids in the microfluidic organ chip of the present invention. In the figure, green represents goblet cells and red represents the ciliary structure of ciliated cells. Both cells are the main functional cells of the nasal epithelium.

[0027] Figure 4 It is the skeleton structure and tight junction physical barrier formed by the induced differentiation of nasal epithelial organoids in the microfluidic organ chip of the present invention.

[0028] Figure 5 It is the change of transepithelial resistance and permeability of the barrier model during the induced differentiation of nasal epithelial organoids in the microfluidic organ chip of the present invention. DETAILED DESCRIPTION

[0029] The present invention provides a microfluidic chip for dynamic culture of nasal epithelial organoids, see Figure 1The microfluidic chip has a three-layer structure, the upper layer of the chip is made of PDMS or PMMA, the middle layer of the chip is a microporous membrane 6 made of PET or PDMS, and the lower layer of the chip is made of PDMS or PMMA; a cell culture chamber 7 and a liquid reservoir 4 are arranged in the upper layer of the chip; a serpentine microfluidic channel 3 is arranged in the lower layer of the chip, the microfluidic channel 3 connects the cell culture chamber 7 and the liquid reservoir 4 in the upper layer of the chip, and the channel cross-section of the microfluidic channel 3 is rectangular; the microporous membrane 6 in the middle layer of the chip is used to separate the upper layer of the chip from the lower layer of the chip and can allow liquid to pass through.

[0030] In some embodiments, the cell culture chamber is 1-3 independent cylindrical cell culture chambers, the upper end of the culture chamber is an open structure, the height of the culture chamber is 1.5-5mm, and the diameter is 4-9mm; the liquid reservoir is located on both sides of the chip and is a rectangular liquid reservoir.

[0031] In some embodiments, the microporous membrane has a large number of micropores uniformly distributed throughout the microporous membrane, the micropore diameter is 0.45-8 μm, and the pore density is 0.5-4×10 7 Pieces / cm 2 The microporous membrane is 8-45mm long, 8-35mm wide and 5-20μm thick.

[0032] In some embodiments, a cylindrical chamber is provided in the lower layer of the chip, and the cylindrical chamber is located directly below the upper cell culture chamber, and the diameter of the cylindrical chamber is the same as the diameter of the upper cell culture chamber; the upper end of the cylindrical chamber is separated from the upper cell culture chamber by a microporous membrane, and the lower end is a closed structure, and the cylindrical chambers are connected in series through serpentine flow channels, and the inlet and outlet of the lower microfluidic channel are connected to the liquid storage tanks on both sides of the upper layer of the chip, and the height of the microfluidic channel is 0.4-1.5mm.

[0033] In some embodiments, the thickness of the upper layer of PDMS or PMMA material of the chip is 3-10 mm, the thickness of the lower layer of PDMS or PMMA material of the chip is 0.8-5 mm, the overall length of the chip is 34-40 mm, and the width is 25-32 mm.

[0034] In some embodiments, the surfaces of the upper cell culture chamber and the lower microfluidic channel need to be smooth; the inlet and outlet of the microfluidic channel are respectively located at the upper left, lower right side or the upper right, lower left side of the cell culture chamber in the horizontal direction, and the horizontal distance between the center of the inlet and outlet and the center of the cell culture chamber is 4-9 mm horizontally and 4-12 mm vertically; the microporous membrane does not block the inlet and outlet of the microfluidic channel. If the microporous membrane covers the inlet and outlet positions, holes are opened at the corresponding positions of the microporous membrane; the edges of the lower cylindrical chamber are 4-9 mm apart; the lower microfluidic channel is straight or arc-shaped, without a corner structure.

[0035] Example 1: A method for preparing a microfluidic chip for dynamic culture of nasal epithelial organoids, comprising the following steps:

[0036] (A) Microfluidic chip flow channel design: The microfluidic chip has a centrally symmetrical structure in the horizontal direction and three layers in the vertical direction. The upper and lower layers are PDMS, and the middle layer is a microporous membrane made of PET. The upper layer of the chip is equipped with three independent organoid culture chambers and a reservoir for the microfluidic channel. The microporous membrane in the middle layer is used to separate the upper chamber from the lower flow channel and allows liquid to pass through. The lower layer is equipped with a serpentine microfluidic channel that connects the culture chamber and the reservoir. The channel cross-section is rectangular; (B) Design of the upper cell culture chamber of the microfluidic chip: In the middle of the upper layer of the chip are three independent and parallel cylindrical cell culture chambers. The upper end of the chamber is an open structure. The two sides of the chip are rectangular-shaped reservoirs. The lower end is separated from the lower flow channel by a microporous membrane. The chamber height is 1.5 mm and the diameter is 4 mm.

[0037] (C) Design of the microporous membrane in the middle layer of the microfluidic chip: The microporous membrane is evenly distributed with a large number of micropores that penetrate the microporous membrane. The pore diameter is 0.45 μm and the pore density is 4×10 7 Pieces / cm 2 , the microporous membrane is 20 mm long, 8 mm wide and 5 μm thick;

[0038] (D) Design of the lower liquid channel of the microfluidic chip: In the middle of the lower layer of the chip is a cylindrical chamber with the same horizontal position and diameter as the three cell culture chambers in the upper layer. The upper ends of the three chambers are separated from the upper chambers by microporous membranes, and the lower end is a closed structure. The three chambers and the two ends are connected in series through a serpentine flow channel. The inlet and outlet of the lower flow channel are connected to the liquid reservoirs on both sides of the upper chip. The height of the fluid channel is 1.5 mm;

[0039] (E) Surface treatment of the microfluidic chip: Add 50 μg / mL type I collagen solution to the upper chamber of the microfluidic chip, incubate at 37°C for 2 h, aspirate and wash with PBS to keep the chip moist for later use;

[0040] (F) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at 8×10 5 The cell density was 100 μL / well, and the cell suspension was added into the upper chamber. 600 μL of amplification medium was added into the reservoir to fill the lower flow channel with amplification medium. The cells were cultured in an environment with a temperature of 37°C, a CO2 concentration of 4%, and a culture humidity of 96%.

[0041] (G) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper culture chamber was replaced every 1 day. After 5 days of expansion, the organoids in the chamber were covered with the microporous membrane. The liquid in the upper chamber and the lower channel was replaced with nasal epithelial organoid differentiation medium, and then a shaker was used to control the flow of the expansion medium in the lower channel at a flow rate of 0.4 μL / min.

[0042] (H) Air-liquid interface (ALI) induced differentiation of the microfluidic chip: the differentiation medium in the lower channel was maintained at a constant flow rate, and the medium in the upper chamber was discarded and cultured under the same conditions for 27 days. During this period, the upper chamber was washed with PBS every 4 days until the organoids were completely differentiated.

[0043] The obtained microfluidic organ chip was used to construct a single layer of nasal epithelial organoids, and the effect was as follows Figure 2 As shown, an inverted microscope was used to observe the cells at different stages of constructing a single-layer nasal epithelial organoid, and the cells were in good condition. In the late differentiation stage, the cells were observed to be tightly arranged polygonal cells with clear cell boundaries, high cell density and good cell layer coverage. The cell layer was intact without gaps or damage, and cilia were observed to exist and sway regularly under the microscope.

[0044] Effect of induced differentiation of nasal epithelial organoids in microfluidic organ chips Figure 3 As shown in the figure, green represents goblet cells and red represents the ciliary structure of ciliated cells. These two cells are the main functional cells of the nasal epithelium. Figure 3 It can be seen that the entire organoid is stained evenly, and there is no area of ​​over- or under-stained area. The nucleus staining using DAPI shows that the boundary between the nucleus and cytoplasm is clearly visible. An obvious clustered ciliary structure can be seen in the picture, indicating that the induced differentiation of nasal epithelial organoids was successful.

[0045] Figure 4 It is the skeleton structure and tightly connected physical barrier formed by the induced differentiation of nasal epithelial organoids in the microfluidic organ chip of the present invention. The boundary between the cell nucleus and the cytoplasm is clearly visible, and obvious boundaries between cells are visible, indicating that the induced differentiation of nasal epithelial organoids is successful and a good physical barrier function has been formed.

[0046] Figure 5 It is the change of transepithelial resistance and permeability of the barrier model during the induced differentiation of nasal epithelial organoids in the microfluidic organ chip of the present invention. Figure 5 It can be seen that during the model construction process, the transepithelial resistance continued to increase and the permeability gradually decreased; after the model construction was completed, the transepithelial resistance value remained at a high level and the permeability remained at a low level, indicating that the physical barrier performance of the nasal epithelial organoids was good.

[0047] Example 2: A method for preparing a microfluidic chip for dynamic culture of nasal epithelial organoids, comprising the following steps:

[0048] (A) Microfluidic chip flow channel design: The microfluidic chip has a central symmetrical structure in the horizontal direction and three layers in the vertical direction. The upper and lower layers are PMMA, and the middle layer is a microporous membrane made of PET. The upper layer of the chip is equipped with an organoid culture chamber and a reservoir for the microfluidic channel. The microporous membrane in the middle layer is used to separate the upper chamber from the lower flow channel and allows liquid to pass through. The lower layer is equipped with a serpentine microfluidic channel that connects the culture chamber and the reservoir. The channel cross-section is rectangular.

[0049] (B) Design of the upper cell culture chamber of the microfluidic chip: There is a cylindrical cell culture chamber in the middle of the upper layer of the chip. The upper end of the chamber is an open structure. The two sides of the chip are rectangular liquid reservoirs. The lower end is separated from the lower flow channel by a microporous membrane. The chamber height is 5 mm and the diameter is 6 mm.

[0050] (C) Design of the microporous membrane in the middle layer of the microfluidic chip: The microporous membrane is evenly distributed with a large number of micropores that penetrate the microporous membrane. The pore diameter is 3 μm and the pore density is 1×10 7 Pieces / cm 2 , the microporous membrane is 25 mm long, 10 mm wide and 10 μm thick;

[0051] (D) Design of the lower liquid channel of the microfluidic chip: In the middle of the lower layer of the chip is a cylindrical chamber with the same horizontal position and diameter as the upper cell culture chamber. The upper end of the chamber is separated from the upper chamber by a microporous membrane, and the lower end is a closed structure. The chamber and both ends are connected in series through a serpentine flow channel. The inlet and outlet of the lower flow channel are connected to the liquid reservoirs on both sides of the upper chip. The height of the fluid channel is 1 mm.

[0052] (E) Surface treatment of the microfluidic chip: add 80 μg / mL type IV collagen solution to the upper chamber of the microfluidic chip, incubate at 37°C for 3 h, aspirate and wash with PBS, and keep the chip moist for later use;

[0053] (F) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at a concentration of 10 × 10 5 The cell density was 1 / mL, and the cell suspension with a volume of 150 μL / well was added to the upper chamber, and 800 μL of amplification medium was added to the reservoir to fill the lower flow channel with the amplification medium, and the cells were cultured in an environment with a temperature of 37°C, a CO2 concentration of 4%, and a culture humidity of 96%;

[0054] (G) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper culture chamber was replaced every 2 days. After 7 days of expansion, the organoids in the chamber were covered with the microporous membrane. The liquid in the upper chamber and the lower channel was replaced with nasal epithelial organoid differentiation medium, and then a shaker was used to control the flow of the expansion medium in the lower channel at a flow rate of 0.2 μL / min;

[0055] (H) Air-liquid interface (ALI) induced differentiation of the microfluidic chip: the differentiation medium in the lower channel was maintained at a constant flow rate, and the medium in the upper chamber was discarded and cultured under the same conditions for 21 days. During this period, the upper chamber was washed with PBS every 7 days until the organoids were completely differentiated.

[0056] Example 3: A method for preparing a microfluidic chip for dynamic culture of nasal epithelial organoids, comprising the following steps:

[0057] (A) Microfluidic chip flow channel design: The microfluidic chip has a centrally symmetrical structure in the horizontal direction and three layers in the vertical direction. The upper and lower layers are PMMA, and the middle layer is a microporous membrane made of PDMS. The upper layer of the chip is equipped with three independent organoid culture chambers and a reservoir for the microfluidic channel. The microporous membrane in the middle layer is used to separate the upper chamber from the lower flow channel and allows liquid to pass through. The lower layer is equipped with a serpentine microfluidic channel that connects the culture chamber and the reservoir. The channel cross-section is rectangular.

[0058] (B) Design of the upper cell culture chamber of the microfluidic chip: There are three independent parallel cylindrical cell culture chambers in the middle of the upper layer of the chip. The upper end of the chamber is an open structure. The two sides of the chip are rectangular liquid reservoirs. The lower end is separated from the lower flow channel by a microporous membrane. The chamber height is 4 mm and the diameter is 4 mm.

[0059] (C) Design of the microporous membrane in the middle layer of the microfluidic chip: The microporous membrane is evenly distributed with a large number of micropores that penetrate the microporous membrane. The pore diameter is 8 μm and the pore density is 0.8×10 7 Pieces / cm 2 , the microporous membrane is 20 mm long, 12 mm wide and 12 μm thick;

[0060] (D) Design of the lower liquid channel of the microfluidic chip: In the middle of the lower layer of the chip is a cylindrical chamber with the same horizontal position and diameter as the three cell culture chambers in the upper layer. The upper ends of the three chambers are separated from the upper chambers by microporous membranes, and the lower end is a closed structure. The three chambers and the two ends are connected in series through a serpentine flow channel. The inlet and outlet of the lower flow channel are connected to the liquid reservoirs on both sides of the upper chip. The height of the fluid channel is 0.5 mm;

[0061] (E) Surface treatment of the microfluidic chip: Add 100 μg / mL type I collagen solution to the upper chamber of the microfluidic chip, incubate at 37°C for 1 h, aspirate and wash with PBS to keep the chip moist for later use;

[0062] (F) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at 15×10 5 The cell density was 100 μL / well, and the cell suspension was added into the upper chamber. 700 μL of amplification medium was added into the reservoir to fill the lower flow channel with amplification medium. The cells were cultured in an environment with a temperature of 37°C, a CO2 concentration of 4%, and a culture humidity of 98%.

[0063] (G) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper culture chamber was replaced every 3 days. After 4 days of expansion, the organoids in the chamber were covered with the microporous membrane. The liquid in the upper chamber and the lower channel was replaced with nasal epithelial organoid differentiation medium, and then a shaker was used to control the flow of the expansion medium in the lower channel at a flow rate of 0.5 μL / min.

[0064] (H) Air-liquid interface (ALI) induced differentiation of the microfluidic chip: the differentiation medium in the lower channel was maintained at a constant flow rate, and the medium in the upper chamber was discarded and cultured under the same conditions for 25 days. During this period, the upper chamber was washed with PBS every 5 days until the organoids were completely differentiated.

[0065] Example 4: A method for preparing a microfluidic chip for dynamic culture of nasal epithelial organoids, comprising the following steps:

[0066] (A) Microfluidic chip flow channel design: The microfluidic chip has a centrally symmetrical structure in the horizontal direction and three layers in the vertical direction. The upper and lower layers are PMMA, and the middle layer is a microporous membrane made of PET. The upper layer of the chip is equipped with three independent organoid culture chambers and a reservoir for the microfluidic channel. The microporous membrane in the middle layer is used to separate the upper chamber from the lower flow channel and allows liquid to pass through. The lower layer is equipped with a serpentine microfluidic channel that connects the culture chamber and the reservoir. The channel cross-section is rectangular; (B) Design of the upper cell culture chamber of the microfluidic chip: In the middle of the upper layer of the chip are three independent and parallel cylindrical cell culture chambers. The upper end of the chamber is an open structure. The two sides of the chip are rectangular-shaped reservoirs. The lower end is separated from the lower flow channel by a microporous membrane. The chamber height is 5 mm and the diameter is 9 mm.

[0067] (C) Design of the microporous membrane in the middle layer of the microfluidic chip: The microporous membrane is evenly distributed with a large number of micropores that penetrate the microporous membrane. The pore diameter is 8 μm and the pore density is 4×10 7 Pieces / cm 2, the microporous membrane is 16 mm long, 12 mm wide and 20 μm thick;

[0068] (D) Design of the lower liquid channel of the microfluidic chip: In the middle of the lower layer of the chip is a cylindrical chamber with the same horizontal position and diameter as the three cell culture chambers in the upper layer. The upper ends of the three chambers are separated from the upper chambers by microporous membranes, and the lower end is a closed structure. The three chambers and the two ends are connected in series through a serpentine flow channel. The inlet and outlet of the lower flow channel are connected to the liquid reservoirs on both sides of the upper chip. The height of the fluid channel is 1.5 mm;

[0069] (E) Surface treatment of the microfluidic chip: Add 300 μg / mL type IV collagen solution to the upper chamber of the microfluidic chip, incubate at 37°C for 24 h, aspirate and wash with PBS to keep the chip moist for later use;

[0070] (F) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at 20×10 5 The cell density was 1 / mL, and the cell suspension with a volume of 130 μL / well was added to the upper chamber, and 800 μL of amplification medium was added to the reservoir to fill the lower flow channel with the amplification medium, and the cells were cultured in an environment with a temperature of 37°C, a CO2 concentration of 4%, and a culture humidity of 96%;

[0071] (G) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper culture chamber was replaced every 3 days. After 9 days of expansion, the organoids in the chamber were covered with the microporous membrane. The liquid in the upper chamber and the lower channel was replaced with nasal epithelial organoid differentiation medium, and then a shaker was used to control the flow of the expansion medium in the lower channel at a flow rate of 1.2 μL / min;

[0072] (H) Air-liquid interface (ALI) induced differentiation of the microfluidic chip: the differentiation medium in the lower channel was maintained at a constant flow rate, and the medium in the upper chamber was discarded and cultured under the same conditions for 30 days. During this period, the upper chamber was washed with PBS every 5 days until the organoids were completely differentiated.

[0073] Example 5: A method for preparing a microfluidic chip for dynamic culture of nasal epithelial organoids, comprising the following steps:

[0074] (A) Microfluidic chip flow channel design: The microfluidic chip has a centrally symmetrical structure in the horizontal direction and three layers in the vertical direction. The upper and lower layers are PDMS, and the middle layer is a microporous membrane made of PET. The upper layer of the chip is equipped with three independent organoid culture chambers and a reservoir for the microfluidic channel. The microporous membrane in the middle layer is used to separate the upper chamber from the lower flow channel and allows liquid to pass through. The lower layer is equipped with a serpentine microfluidic channel that connects the culture chamber and the reservoir. The channel cross-section is rectangular; (B) Microfluidic chip upper cell culture chamber design: In the middle of the upper layer of the chip are three independent and parallel cylindrical cell culture chambers. The upper end of the chamber is an open structure. The two sides of the chip are rectangular-shaped reservoirs. The lower end is separated from the lower flow channel by a microporous membrane. The chamber height is 5 mm and the diameter is 5 mm.

[0075] (C) Design of the microporous membrane in the middle layer of the microfluidic chip: The microporous membrane is evenly distributed with a large number of micropores that penetrate the microporous membrane. The pore diameter is 0.8 μm and the pore density is 1×10 7 Pieces / cm 2 , the microporous membrane is 10 mm long, 8 mm wide and 9 μm thick;

[0076] (D) Design of the lower liquid channel of the microfluidic chip: In the middle of the lower layer of the chip is a cylindrical chamber with the same horizontal position and diameter as the three cell culture chambers in the upper layer. The upper ends of the three chambers are separated from the upper chambers by microporous membranes, and the lower end is a closed structure. The three chambers and the two ends are connected in series through a serpentine flow channel. The inlet and outlet of the lower flow channel are connected to the liquid reservoirs on both sides of the upper chip. The height of the fluid channel is 0.8 mm;

[0077] (E) Surface treatment of the microfluidic chip: Add 200 μg / mL type I collagen solution to the upper chamber of the microfluidic chip, incubate at 37°C for 8 h, aspirate and wash with PBS to keep the chip moist for later use;

[0078] (F) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at 14×10 5 The cell density was 1 / mL, and the cell suspension with a volume of 120 μL / well was added to the upper chamber, and 400 μL of amplification medium was added to the reservoir to fill the lower flow channel with the amplification medium, and the cells were cultured in an environment with a temperature of 37°C, a CO2 concentration of 4%, and a culture humidity of 97%;

[0079] (G) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper culture chamber was replaced every 1 day. The cells were expanded for 4 days until the organoids in the chamber covered the microporous membrane. The liquid in the upper chamber and the lower channel was replaced with nasal epithelial organoid differentiation medium, and then a shaker was used to control the flow of the expansion medium in the lower channel at a flow rate of 0.6 μL / min.

[0080] (H) Air-liquid interface (ALI) induced differentiation of the microfluidic chip: the differentiation medium in the lower channel was maintained at a constant flow rate, and the medium in the upper chamber was discarded and cultured under the same conditions for 45 days. During this period, the upper chamber was washed with PBS every 10 days until the organoids were completely differentiated.

[0081] Example 6: A method for preparing a microfluidic chip for dynamic culture of nasal epithelial organoids, comprising the following steps:

[0082] (A) Microfluidic chip flow channel design: The microfluidic chip has a centrally symmetrical structure in the horizontal direction and three layers in the vertical direction. The upper and lower layers are PMMA, and the middle layer is a microporous membrane made of PDMS. The upper layer of the chip is equipped with three independent organoid culture chambers and a reservoir for the microfluidic channel. The microporous membrane in the middle layer is used to separate the upper chamber from the lower flow channel and allows liquid to pass through. The lower layer is equipped with a serpentine microfluidic channel that connects the culture chamber and the reservoir. The channel cross-section is rectangular.

[0083] (B) Design of the upper cell culture chamber of the microfluidic chip: There are three independent parallel cylindrical cell culture chambers in the middle of the upper layer of the chip. The upper end of the chamber is an open structure. The two sides of the chip are rectangular liquid reservoirs. The lower end is separated from the lower flow channel by a microporous membrane. The chamber height is 4 mm and the diameter is 6 mm.

[0084] (C) Design of the microporous membrane in the middle layer of the microfluidic chip: The microporous membrane is evenly distributed with a large number of micropores that penetrate the microporous membrane. The pore diameter is 0.45 μm and the pore density is 2×10 7 Pieces / cm 2 , the microporous membrane is 18 mm long, 10 mm wide and 15 μm thick;

[0085] (D) Design of the lower liquid channel of the microfluidic chip: In the middle of the lower layer of the chip is a cylindrical chamber with the same horizontal position and diameter as the three cell culture chambers in the upper layer. The upper ends of the three chambers are separated from the upper chambers by microporous membranes, and the lower end is a closed structure. The three chambers and the two ends are connected in series through serpentine channels. The inlet and outlet of the lower channel are connected to the liquid reservoirs on both sides of the upper chip. The height of the fluid channel is 1 mm.

[0086] (E) Surface treatment of the microfluidic chip: Add 150 μg / mL type I collagen solution to the upper chamber of the microfluidic chip, incubate at 37°C for 6 h, aspirate and wash with PBS to keep the chip moist for later use;

[0087] (F) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at 16×10 5The cell density was 100 μL / well, and the cell suspension was added into the upper chamber. 500 μL of amplification medium was added into the reservoir to fill the lower flow channel with amplification medium. The cells were cultured in an environment with a temperature of 37°C, a CO2 concentration of 4%, and a culture humidity of 98%.

[0088] (G) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper culture chamber was replaced every 2 days. After 6 days of expansion, the organoids in the chamber were covered with the microporous membrane. The liquid in the upper chamber and the lower channel was replaced with nasal epithelial organoid differentiation medium, and then a shaker was used to control the flow of the expansion medium in the lower channel at a flow rate of 0.3 μL / min;

[0089] (H) Air-liquid interface (ALI) induced differentiation of the microfluidic chip: the differentiation medium in the lower channel was maintained at a constant flow rate, and the medium in the upper chamber was discarded and cultured under the same conditions for 24 days. During this period, the upper chamber was washed with PBS every 8 days until the organoids were completely differentiated.

Claims

1. A microfluidic chip for dynamic culture of nasal epithelial organoids, characterized in that: The microfluidic chip has a three-layer structure, the upper layer of the chip is made of PDMS or PMMA, the middle layer of the chip is a microporous membrane made of PET or PDMS, and the lower layer of the chip is made of PDMS or PMMA; a cell culture chamber and a liquid reservoir are arranged in the upper layer of the chip; a serpentine microfluidic channel is arranged in the lower layer of the chip, the microfluidic channel connects the cell culture chamber and the liquid reservoir in the upper layer of the chip, and the channel cross-section of the microfluidic channel is rectangular; the microporous membrane in the middle layer of the chip is used to separate the upper layer of the chip from the lower layer of the chip and can allow liquid to pass through.

2. The microfluidic chip for dynamic culture of nasal epithelial organoids according to claim 1, characterized in that: The cell culture chambers are 1-3 independent cylindrical cell culture chambers, the upper end of the culture chamber is an open structure, the height of the culture chamber is 1.5-5mm, and the diameter is 4-9mm; the liquid storage tanks are located on both sides of the chip and are rectangular liquid storage tanks.

3. The microfluidic chip for dynamic culture of nasal epithelial organoids according to claim 1, characterized in that: The microporous membrane is uniformly distributed with a large number of micropores penetrating the microporous membrane, the micropore diameter is 0.45-8 μm, and the pore density is 0.5-4×10 7 Pieces / cm 2 The microporous membrane is 8-45mm long, 8-35mm wide and 5-20μm thick.

4. The microfluidic chip for dynamic culture of nasal epithelial organoids according to claim 1, characterized in that: A cylindrical chamber is provided in the lower layer of the chip, and the cylindrical chamber is located directly below the upper cell culture chamber, and the diameter of the cylindrical chamber is the same as the diameter of the upper cell culture chamber; the upper end of the cylindrical chamber is separated from the upper cell culture chamber by a microporous membrane, and the lower end is a closed structure, and the cylindrical chambers are connected in series through serpentine flow channels, and the inlet and outlet of the lower microfluidic channel are connected to the liquid storage tanks on both sides of the upper layer of the chip, and the height of the microfluidic channel is 0.4-1.5mm.

5. The microfluidic chip for dynamic culture of nasal epithelial organoids according to claim 1, characterized in that: The thickness of the upper layer of PDMS or PMMA material of the chip is 3-10 mm, the thickness of the lower layer of PDMS or PMMA material of the chip is 0.8-5 mm, the overall length of the chip is 34-40 mm, and the width is 25-32 mm.

6. The microfluidic chip for dynamic culture of nasal epithelial organoids according to claim 1, characterized in that: The surfaces of the upper cell culture chamber and the lower microfluidic channel need to be smooth; the inlet and outlet of the microfluidic channel are respectively located at the upper left and lower right sides or the upper right and lower left sides of the cell culture chamber in the horizontal direction, and the horizontal distance between the center of the inlet and outlet and the center of the cell culture chamber is 4-9 mm horizontally and 4-12 mm vertically; the microporous membrane does not block the inlet and outlet of the microfluidic channel. If the microporous membrane covers the inlet and outlet positions, holes are opened at the corresponding positions of the microporous membrane; the edges of the lower cylindrical chamber are 4-9 mm apart; the lower microfluidic channel is straight or arc-shaped, without a corner structure.

7. A method for culturing nasal epithelial organoids, characterized in that Cultivating using the microfluidic chip according to claim 1 comprises the following steps: (1) Surface treatment of the microfluidic chip: Add 20-300 μg / mL type I collagen or type IV collagen solution to the upper cell culture chamber of the microfluidic chip, incubate at 37°C for 0.5-24 h, aspirate and wash with PBS, and keep the chip moist for later use; (2) Nasal epithelial cell seeding on microfluidic chip: Nasal epithelial cells were digested into single cells and resuspended in nasal epithelial cell expansion medium at a concentration of 4-20×10 5 The cell density of cells / mL and the cell suspension of 50-130 μL / pore volume are added to the upper cell culture chamber, and 400-800 μL of amplification medium is added to the reservoir to fill the lower microfluidic channel with the amplification medium, and the cells are cultured in an environment with a temperature of 35-38°C, a CO2 concentration of 4-6%, and a culture humidity of 92-98%; (3) Continuous expansion and differentiation culture of the microfluidic chip: After inoculating the cells, static culture was performed, and the liquid in the upper cell culture chamber was replaced every 1-3 days. The cells in the cell culture chamber were expanded for 3-9 days until the cells covered the microporous membrane. The liquid in the upper chamber and the lower flow channel was replaced with nasal epithelial organoid differentiation medium, and then a precision shaker was used to control the flow of the medium in the lower flow channel at a flow rate of 0.2-1.2 μL / min; (4) Air-liquid interface induced differentiation of microfluidic chip: maintain a constant flow rate of differentiation medium in the lower channel, discard the medium in the upper cell culture chamber and continue to culture under the same conditions for 15-45 days. During this period, wash the upper chamber with PBS every 4-10 days until the organoids are completely differentiated.

8. The method for culturing nasal epithelial organoids according to claim 7, characterized in that: The microfluidic chip is sterilized by wet heat sterilization or ultraviolet sterilization before internal surface treatment, and then the upper chamber and the lower flow channel are cleaned with PBS; when the culture medium is introduced into the lower microfluidic channel, it should be ensured that the liquid completely fills the flow channel without residual bubbles; during the whole process of making the lower liquid flow, bubbles should be prevented from entering the lower flow channel with the liquid.

9. The method for culturing nasal epithelial organoids according to claim 7, characterized in that: The nasal epithelial cell expansion medium in step (2) consists of a basal medium Advanced DMEM / F12, 4-hydroxyethylpiperazineethanesulfonic acid, glutamine, penicillin-streptomycin, recombinant human R-spondin 1, noggin, B27 serum-free additive, N-acetyl-L-cysteine, nicotinamide, Y-27632 dihydrochloride, ALK5 inhibitor, SB202190 hydrochloride, fibroblast growth factor 7, fibroblast growth factor 10 and primary cell antibiotics.

10. The method for culturing nasal epithelial organoids according to claim 7, characterized in that: The nasal epithelial organoid differentiation culture medium in step (3) comprises the basal culture medium Advanced DMEM / F12, 3,3',5-triiodo-L-thyronine sodium salt, hydrocortisone, (±)-epinephrine hydrochloride, ALK5 inhibitor, aromatic retinoic acid, recombinant human epidermal growth factor, neuregulin 1 and penicillin-streptomycin.

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