Preparation method of airway organoid

Abstract

The invention discloses a preparation method of an airway organ, which comprises the following steps: dissociating and digesting airway tissues into single cells, and performing 2D multiplication culture to P3-P4 generations; uniformly mixing the cells subjected to 2D multiplication culture to P3-P4 generations with matrigel to obtain a cell-matrigel mixture; shearing the cell-matrigel mixture into monodisperse liquid drops by using a microfluidic technology, and curing the monodisperse liquid drops at 37 DEG C to obtain cell microspheres after curing; and inoculating the cell microspheres into an airway organoid culture medium, and culturing in a 5% CO2 incubator at 37 DEG C to obtain the airway organoid after the culture is finished. According to the method for constructing the airway organoid, the period is shortened, cell components such as airway epithelial cells and cilia cells are provided, and high-efficiency construction of the airway organoid can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of tumor organoid preparation, and particularly relates to a method for preparing airway organoids. Background Art

[0002] The airway (respiratory tract) includes the nose, pharynx, larynx (upper respiratory tract) and the trachea, bronchi and their branches in the lungs (lower respiratory tract). As the respiratory tract continuously branches, a series of changes occur in its structure and function. The number of airways increases, the caliber decreases, the total cross-sectional area increases, and the wall thickness thins. These changes have important functional significance. By adjusting airway resistance, the amount, speed, and respiratory function of the airways entering and leaving the lungs can be adjusted. The environmental temperature and humidity are not constant, and they may contain dust particles and harmful gases, all of which are harmful to the health of the body.

[0003] Traditional models for airway research are mainly 2D cell lines and xenograft models, but both have certain limitations. Organoids are tissue analogs with a certain spatial structure formed by in vitro three-dimensional (3D) culture of adult stem cells or pluripotent stem cells. They can simulate the in vivo tissue structure and function to the greatest extent in terms of structure and function and can be passaged in culture stably for a long time. They have the characteristics of a relatively short construction period, easy preservation, and retention of donor heterogeneity. Airway organoids have great application value in basic research, new drug development, personalized treatment, etc.

[0004] The current organoid technology has limitations. By manually encapsulating cells with Matrigel to culture organoids, it takes a lot of time and effort, there are easily differences between batches, and the throughput is low, which cannot meet the current research needs. The current culture protocol takes more than a month to obtain large organoids, increasing the time and cost of drug screening based on organoids. At the same time, the artificially prepared airway organoid model does not have a true 3D stereostructure and cannot simulate airway-related functions, hindering the research and application of airway models. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects existing in the prior art, and thus provide a method for preparing airway organoids. The present invention uses microfluidic technology to construct a three-dimensional structure that can reproduce the phenotypic and functional characteristics of differentiated airway epithelium. It is a powerful tool for studying the pathological modeling of respiratory viruses, and realizes the preparation of organoids with high efficiency, high throughput, and uniformity. The prepared airway organoids are a useful tool for drug screening and personalized treatment of patients with airway-related diseases.

[0006] The specific technical solution is as follows:

[0007] The present invention provides a method for preparing airway organoids, comprising the following steps:

[0008] 1) Dissociate and digest airway tissue into single cells, and perform 2D expansion culture to the P3-P4 generation, preferably passaging once every 3-7 days; the 2D expansion culture includes primary culture and subculture, and the primary culture and subculture are carried out using airway medium;

[0009] The airway medium is prepared according to the following formula: After mixing the components PneumaCult-Ex Plus Basal Medium and PneumaCult-Ex Plus 50X Supplement in the STEMCELL airway complete medium kit, add Hydrocotisone Stock Solution, and the ratio of Hydrocotisone Stock Solution to the medium is 1:1000. Among them, 10 mM Y-27632 is added to the airway medium on the first day of primary culture and subculture;

[0010] 2) Mix the cells obtained by 2D expansion culture to the P3-P4 generation in step 1) evenly with Matrigel to obtain a cell-Matrigel mixture;

[0011] 3) Use the cell-Matrigel mixture obtained in step 2) as the dispersed phase and the oil-phase reagent as the continuous phase, and use microfluidic technology to shear the cell-Matrigel mixture into monodisperse droplets, and cure the obtained monodisperse droplets at 37°C to obtain cell microspheres after curing;

[0012] 4) Inoculate the cell microspheres obtained in step 3) into the airway organoid medium and culture them in an incubator at 37°C and 5% CO2. After the culture is completed, airway organs are obtained;

[0013] The airway organoid medium is prepared according to the following formula: After mixing the components PneumaCult-Ex Plus Basal Medium and PneumaCult-Ex Plus 50X Supplement in the STEMCELL airway complete medium kit, add Hydrocotisone Stock Solution, and the ratio of Hydrocotisone Stock Solution to the medium is 1:1000.

[0014] Furthermore, the airway tissue in step 1) is human airway tissue.

[0015] Further, the method for dissociating and digesting airway tissue into single cells in step 1) includes the steps of: taking airway tissue, washing it with PBS containing 1% double antibody (P / S) by volume percentage concentration, cutting it into pieces, adding digestive juice to the tissue pieces for digestion until the dissociation standard is met, terminating digestion, centrifuging to collect the precipitate, resuspending the precipitate with DMEM containing 10% FBS by volume percentage concentration, filtering through a 100 μm cell sieve, collecting the filtrate, centrifuging the filtrate to discard the supernatant, then adding erythrocyte lysate, terminating the erythrolysis reaction after lysis, centrifuging to discard the supernatant, and collecting the precipitate to obtain;

[0016] Preferably, the digestive juice is prepared according to the following method: dissolving 2 mg / mL collagenase, 2 mg / mL DNase, and 2 mg / mL dispase in DMEM / F12 medium and filtering through a 22 μm filter membrane.

[0017] Further, the detailed steps for dissociating and digesting airway tissue into single cells in step 1) include: taking out the airway tissue in a culture dish, washing the tissue with PBS containing 1% P / S (double antibody) by percentage concentration until the washing solution is clear. Cutting the tissue into 2 mm pieces, adding 5 mL of digestive juice to a centrifuge tube containing the tissue pieces, incubating at 37 °C on a shaker at 200 rpm for 10 - 30 min (the digestion time is determined according to the size of the tissue block) until the dissociation standard is met, adding 5 mL of medium containing 10% serum to terminate digestion. Centrifuging the obtained tissue suspension at 500 g at 4 °C for 5 min, discarding the supernatant, adding 5 mL of medium containing 10% serum to resuspend the precipitate, filtering through a 100 μm cell sieve, examining the filtrate under a microscope to observe the number of crypts. Centrifuging the collected tissue filtrate at 500 g at 4 °C for 5 min, discarding the supernatant. Adding 1 - 3 mL of erythrocyte lysate, standing at room temperature for lysis for 3 min, adding an equal volume of medium containing 10% serum to terminate the erythrolysis reaction, centrifuging in a centrifuge at 500 g at 4 °C for 5 min, discarding the supernatant, and collecting the precipitate to obtain.

[0018] Further, in step 2), every (5 - 10) × 10 5Cells were seeded in 3.5 cm culture dishes and amplified in 2D. After resuspending the cells with 2 mL of airway medium, the cell suspension was transferred to a 3.5 cm culture dish for cultivation. When the number of cells was less than 500,000, 3D culture could also be performed using 48-well plates / 96-well plates. On the first day of plating, airway medium containing 10 mM Y-27632 (1:500) was added, and the medium was changed every 2-3 days. When the cells had amplified to cover the dish and the centers of the clusters turned yellow or black, passage was carried out. Take out the cell culture dish, discard the culture medium in the dish, add digestive solution to cover the cells, incubate at 37 °C for 5-8 min. When the adherent cells floated up, gently pipette several times and then add digestive neutralization solution to terminate digestion. Transfer to a 15 mL centrifuge tube, centrifuge at 4 °C, 500 g for 5 min. After centrifugation, discard the supernatant, add airway medium to resuspend the cells, and count. Select the number of dishes for inoculation according to the cell count. Similarly, on the first day of passage, airway medium containing 10 μM Y-27632 (1:500) was added, and subsequent medium changes did not use airway medium containing Y-27632.

[0019] Further, at a concentration of 1×10 4 / μL Matrigel, the cells amplified in 2D to passages P3-P4 were mixed evenly with Matrigel.

[0020] Further, the oil-phase reagent in step 3) is fluorinated oil.

[0021] Further, the microfluidic technology in step 3) is a microfluidic technology based on the T-channel method;

[0022] Preferably, the flow rate of the cell-Matrigel mixture is controlled at 10-15 μL / min, and the flow rate of the oil-phase reagent is controlled at 100-110 μL / min.

[0023] Further, the curing time at 37 °C in step 3) is 30 min.

[0024] Further, the cultivation time in a 37 °C, 5% CO2 incubator in step 4) is 7-30 days. Solid organoid structures can grow in 7-10 days, and airway organoids with ciliated structures can grow in 30 days.

[0025] Further, the particle size of the airway organoids in step 4) is 600-650 μm.

[0026] The present invention also provides airway organs prepared by the above preparation method.

[0027] The beneficial effects of the present invention are:

[0028] The present invention prepares airway organoids through microfluidic technology, with reduced usage of Matrigel, uniform organoid size, shortened culturing and maturation time, enabling high-fidelity and high-throughput construction. Moreover, the prepared airway organoids can mimic airway-related functions. Specifically: 1) The size of the airway organoids prepared by the present invention is all within 600-650 μm, and their morphology and structure are uniform; 2) The culturing time of the airway organoids of the present invention is shortened. Compared with the traditional manual preparation of organoids, the culturing time of the organoids is shortened from 30-60 days to 7-30 days; 3) The present invention can achieve high-efficiency and high-throughput preparation of airway organoids. Under the same cell number and time, the speed of constructing organoids using the method of the present invention can be increased by 2-3 times; 4) The present invention achieves high-fidelity construction of airway organoids. The prepared airway organs retain the morphology and characteristics of the parental tissue and express airway organoid markers. They not only possess the characteristics of the parental tissue but also include ciliated cells, goblet cells, basal cells, etc. Description of the Drawings

[0029] Figure 1 Bright field of human airway cells;

[0030] Figure 2 Bright field of human airway organoids;

[0031] Figure 3 HE staining of human airway parental tissue and its organoids;

[0032] Figure 4 IF detection diagram of engineered airway organoids prepared from culture medium droplets after 2D amplification of airway cells (a goblet cell marker, b club cell marker, c basal cell and ciliated cell marker). Detailed Embodiments

[0033] To better understand the present invention, the present invention will be further described with reference to the following embodiments and drawings. The embodiments are only for explanation and do not limit the present invention in any way. In the embodiments, all the original reagent materials can be obtained commercially. The experimental methods without specific conditions are the conventional methods and conventional conditions well-known in the art, or the conditions recommended by the instrument manufacturers.

[0034] The culture media and reagents involved in the embodiments of the present invention are shown in Table 1 below:

[0035] Table 1

[0036]

[0037] Example 1: Preparation Method of Human Airway Organoids

[0038] This embodiment provides a method for preparing human airway organoids, which includes the following steps:

[0039] Step 1) Tissue processing and digestion

[0040] Take out the human airway tissue in a 6 cm culture dish, aspirate the tissue preservation solution, add 3 mL of PBS containing 1% P / S (double antibody) to resuspend the tissue, wash the tissue 3 times until the washing solution is clear, and remove the washing solution. Fix the tissue with forceps and cut it into 2 mm fragments. Transfer the tissue and suspension to a 15 mL centrifuge tube using a pipette or Pasteur pipette, and remove the PBS. Prepare the digestion solution: Dissolve 20 mg of type IV collagenase, 20 mg of type I DNase, and 20 mg of dispase in 10 mL of DMEM / F12 solution, prepare it immediately before use, and filter it through a 22 μm filter membrane. Add 5 mL of the digestion solution to the centrifuge tube containing the tissue fragments, seal the tube cap with parafilm, place it in a shaker at 37 °C, place it obliquely, and incubate at 200 rpm for 15 minutes. Observe the dissociation state of the tissue blocks every 5 minutes until it meets the dissociation standard, and add 5 mL (equal volume to the digestion solution) of DMEM solution containing 10% FBS by volume percentage to terminate the digestion. Obtain the tissue suspension. Place a 100 μm cell sieve on a 50 mL centrifuge tube with forceps, add 2 mL of DMEM containing 10% FBS by volume percentage to rinse the sieve, transfer the resuspended tissue fluid to the cell sieve with a Pasteur pipette for filtration, gently tap the outer wall of the test tube to make the filtrate flow down slowly, wash the inner wall of the centrifuge tube used for digestion and the filter residue on the sieve with an appropriate amount of DMEM containing 10% FBS by volume percentage, gently grind the fragments remaining on the sieve with the piston shaft of a syringe, add an appropriate amount of complete medium to rinse the sieve, and centrifuge the obtained cell suspension at 500 g for 5 min at 4 °C. Discard the supernatant, observe the number of red blood cells, add 1 - 3 mL of red blood cell lysate, let it stand at room temperature for 3 min for lysis, add an equal volume of DMEM containing 10% FBS by volume percentage to terminate the red blood cell lysis reaction, centrifuge it at 300 g for 5 min at 4 °C in a centrifuge, and discard the supernatant. Add 1 mL of DMEM containing 10% FBS by volume percentage to resuspend the cell pellet, take 10 μL of the cell suspension and add it to 10 μL of AOPI, mix well, and inject 20 μL of the mixed solution onto a clean counting plate for cell counting.

[0041] Step 2) 2D cell expansion culture

[0042] Every 5×10 5Add cells to a 3.5-cm culture dish for 2D expansion culture. After resuspending the cells with 2 mL of airway medium, transfer the cell suspension to the 3.5-cm culture dish for culture. When the number of cells is less than 500,000, 48-well plates / 96-well plates can also be used for 3D culture. On the first day of both 2D expansion culture and 3D culture, add 10 mM Y-27632. Change the medium every 2 - 3 days. When the cells have expanded to cover the dish and the center of the cluster turns yellow or black (such as Figure 1 ), passage the cells. Take out the cell culture dish, discard the culture medium in the dish, add digestive solution to cover the cells, incubate at 37 °C for 5 - 8 min. When the adherent cells float up, gently pipette several times and then add digestive neutralization solution to terminate digestion. Transfer to a 15-mL centrifuge tube, centrifuge at 500 g at 4 °C for 5 min. After centrifugation, discard the supernatant, add airway medium to resuspend the cells, and count. Select the number of dishes for inoculation according to the cell count. Similarly, add 10 mM Y-27632 on the first day of passage, and do not add Y-27632 for subsequent medium changes.

[0043] The airway medium is prepared according to the following formula: Mix the components PneumaCult-Ex Plus Basal Medium and PneumaCult-Ex Plus 50X Supplement in the STEMCELL airway complete medium kit, and then add Hydrocotisone Stock Solution. The ratio of Hydrocotisone Stock Solution to the medium is 1:1000. Among them, add 10 mM Y-27632 to the airway medium on the first day of primary culture and passage culture.

[0044] Step 3) Preparation of cell microspheres

[0045] Pre-cool a sterilized 100-μL pipette tip in a -20 °C refrigerator 1 day in advance, and pre-cool a 1-mL syringe containing 0.5 mL of fluorinated oil on ice or in a -20 °C refrigerator 30 min in advance. Aspirate 5 mL of fluorinated oil with a 10-mL syringe, connect the needle and about 20 cm of polytetrafluoroethylene (PTFE) tube, install it in the right hole of the three-way joint. Install about 10 m of PTFE tube in the left hole of the three-way joint and install it in an injection pump outside the refrigerator (flow rate: 110 μL / min). Adjust the injection pump to fill the fluorinated oil in the 10-mL syringe into the 20-cm PTFE tube connected to its needle. Transfer the cell suspension to a 1.5-mL centrifuge tube, centrifuge at 300 g at room temperature for 3 min, discard the supernatant. According to the cell count result, at a density of 1×10 4The volume of Matrigel required for calculating the Matrigel concentration of cells / μL was added to the cell pellet using a pre-chilled pipette tip. After gently stirring several times on ice and then pipetting up and down to ensure no air bubbles were generated, and mixing until there were no cell clumps, the cell-Matrigel mixture was injected into a pre-chilled 1 mL syringe filled with fluorinated oil. A needle and approximately 10 cm PTFE tube were connected, and then it was installed in an injection pump (flow rate: 15 μL / min) inside the refrigerator. It was placed in the middle hole of a three-way joint, and the injection pump was adjusted so that the cell-Matrigel mixture appeared in this 10 cm PTFE tube. First, start the injection pump outside the refrigerator, and then start the injection pump inside the refrigerator. The cell-Matrigel mixture was sheared into monodisperse droplets through the three-way tube and then conducted to a PTFE tube of approximately 10 m. When the PTFE tube connected to the 1 mL syringe no longer contained the cell-Matrigel mixture, stop both injection pumps. The PTFE tube carrying the dispersed droplets of the cell-Matrigel mixture was placed in an incubator at 37 °C for 30 min to fix, and cell microspheres were obtained.

[0046] Step 4) Organoid culture

[0047] The cell microspheres were blown into a petri dish containing human airway organoid medium using a 10 mL syringe. The fluorinated oil on the surface of the petri dish was gently aspirated using a 1 mL syringe with a needle. A low-attachment flat-bottom 96-well plate was taken, and 100 - 150 μL of airway medium was added to each well. The airway organoid microspheres were individually picked into the wells of the plate. It was placed in an incubator at 37 °C with 5.0% CO2 for culture for 40 days. Fresh medium was replaced every 2 - 4 days. Observation and photography were carried out daily under bright field microscopy to record whether organoid structures grew. After 14 days of culture, the airway organoid model matured, with the organoids being of uniform size, forming a symmetrical cystic morphology, and obvious structures appearing inside the spheres. With the growth of ciliated epithelial cells and other absorptive cells, solid organoid structures could grow in 7 - 10 days, and airway organoids with ciliated structures could grow in 30 days. ( Figure 2 )

[0048] The airway organoid medium was prepared according to the following formula: The components PneumaCult-Ex Plus Basal Medium and PneumaCult-Ex Plus 50X Supplement in the STEMCELL airway complete medium kit were mixed, and then Hydrocotisone Stock Solution hydrocortisone (with a ratio of 1:1000 to the medium) was added.

[0049] Example 2: HE staining of human airway organoids

[0050] HE staining was performed on the human airway organoids prepared in Example 1, specifically including:

[0051] Step 1) Paraffin embedding of organoids

[0052] Turn on the embedding machine in advance and preheat it to 65°C.

[0053] Fixation: Add 10 times the volume of the organoids of 4% PFA (about 200 μL) to the EP tube and fix at room temperature for 20 min.

[0054] Pre-staining of organoids: Carefully aspirate the PFA with a 100 μL pipette, add 20 μL of eosin solution, and stain at room temperature for 5 min.

[0055] Dehydration of organoids: Add 10 times the volume of 75% ethanol and dehydrate at room temperature for 5 min; aspirate the 75% ethanol, add 10 times the volume of 90% ethanol, and dehydrate at room temperature for 5 min.

[0056] Re-staining of organoids: Add 20 μL of eosin solution and stain at room temperature for 5 min. (Re-staining can be performed if the decolorization is severe).

[0057] Dehydration of organoids: Add 10 times the volume of absolute ethanol and dehydrate at room temperature for 5 min, repeating three times.

[0058] Clearing of organoids: Add 10 times the volume of xylene and soak at room temperature for 5 min, repeating three times.

[0059] Infiltration of organoids with wax: Take a white embedding cassette, remove the lid, and label the organoid batch number on the cassette. Wait for the organoids to sink to the bottom, carefully aspirate the upper layer of xylene (leave 50 μL), use a 1 mL pipette tip with the tip cut off to aspirate the sample, transfer the sample to a paraffin mold, add paraffin, soak at 65°C for 10 min; tilt the mold to discard the paraffin, re-add paraffin and soak for 10 min, gently place the white embedding cassette on the paraffin mold, let it stand at 65°C for 10 min, and store the wax block at room temperature after the paraffin solidifies.

[0060] Step 2) Sectioning and HE staining

[0061] Section the paraffin-embedded block on a microtome with a thickness of 5 μm.

[0062] Deparaffinize and hydrate the sections: Place the sections in xylene I for 5 min, xylene II for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and wash with distilled water in sequence.

[0063] Stain the cell nuclei with hematoxylin: Turn on the water bath in advance and preheat it to 60°C. Immerse the sections in hematoxylin and stain at 60°C for 3 - 5 min. Soak in tap water for a while to remove the excess hematoxylin, differentiate with 1% acidic ethanol differentiation solution for 20 s, wash with tap water, blue with 1% ammonia water for 20 s, and wash with tap water.

[0064] Stain the cytoplasm with eosin: Immerse the sections in eosin staining solution and stain for 30 s.

[0065] Dehydration and sealing: Put the slices into 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to dehydrate and make them transparent. Take the slices out of xylene, put a clean absorbent paper on the table, take the slide out of xylene and put it on the paper (with the slice side facing up), quickly drop a drop of gum in the center of the slice, hold the right side of the cover glass gently with tweezers, tilt it slightly so that its left side touches the sealing medium, and then slowly put the cover glass down to reduce or avoid the generation of bubbles. Then perform microscopic examination, image acquisition and analysis (such as Figure 3 ).

[0066] Example 3: IF detection of human airway organoids

[0067] The human airway organoids prepared in Example 1 were subjected to IF testing, specifically including:

[0068] Step 1) Organoid paraffin embedding

[0069] The specific steps are the same as step 1 of Example 2).

[0070] Step 2) Slice IF detection

[0071] The paraffin sections were placed in a 65°C oven for 30 min-1 h until the wax dissolved.

[0072] Dewax the paraffin sections to water: sequentially place the sections in xylene I for 20 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol for 10 min, 95% ethanol for 10 min, 70% ethanol for 10 min, and wash with distilled water for 5 min.

[0073] Antigen repair: Place the slides in a repair box filled with EDTA antigen repair buffer (pH 8.0) in a microwave oven for antigen repair. Heat at high heat for 5 minutes, medium heat for 20 minutes, and then switch to low heat for 2 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After cooling naturally, place the slides in PBS (pH 7.4) and wash them twice on a decolorizing shaker, 5 minutes each time.

[0074] Blocking: After the slices were taken out of PBS and dried, a circle was drawn around the tissue with an immunohistochemistry pen, and 100 μL of blocking solution (the blocking solution was PBS containing 5% BSA and 0.3% Triton X-100) was added into the circle for blocking for 1 hour.

[0075] Primary antibody incubation: dilute the primary antibody to an appropriate concentration with blocking solution, add 50 μL of primary antibody to each slice, place the slices flat in a humidified box, and incubate at 4°C overnight.

[0076] Secondary antibody incubation: Take out the sections from the refrigerator and warm them up for 20 - 30 minutes. Wash them 3 times with PBST for 10 minutes each time. Dilute the fluorescently labeled secondary antibody with PBS, and add 50 μL of the secondary antibody dropwise to each section. Incubate at room temperature for half an hour.

[0077] Counterstain with DAPI: Wash 3 times with PBST for 10 minutes each time. Add the diluted DAPI and incubate at room temperature for 10 minutes. Wash with PBS for 5 minutes.

[0078] Mounting and microscopy: Add about 30 μL of anti - fluorescence quencher dropwise to each section, cover with a coverslip, and observe under a fluorescence microscope and take pictures for record.

[0079] IF was used to detect the expression of club cell, goblet cell, basal cell, and ciliated cell epithelial cell markers in airway parental tissue and its organoids. CC10 (clara cell 10 - kD protein, CC10) is a club cell marker, distributed in the non - ciliated, non - serous secretory epithelial cells of the terminal bronchioles in the lung. The IF results showed that both the airway parental tissue and its organoids expressed CC10, and the expression locations were the same ( Figure 4 ). The expression level was basically the same as that of the parental tissue, with no significant difference, proving that the organoids were consistent with the parental tissue. MUC5AC is a goblet cell marker, distributed in the mucus - secreting cells between the columnar epithelial cells of the airway mucosa. The IF results showed that MUC5AC was expressed in both the parental tissue and the organoids ( Figure 4 ). The expression level was basically the same as that of the parental tissue, with no significant difference, proving that the organoids were consistent with the parental tissue. P63 is a basal cell marker, which, like club cells, belongs to the most common epithelial cell type in the airway. The IF results showed that P63 was expressed in both the parental tissue and the organoids ( Figure 4 ) ACCTUB - 1 is a marker of airway ciliated epithelial cells. The expression level was basically the same as that of the parental tissue, with no significant difference, proving that the organoids were consistent with the parental tissue. The above results proved that this human airway organoid had the expression of club cell, goblet cell, basal cell, and ciliated cell epithelial cell markers.

Claims

1. A method for preparing airway organoids, characterized in that: The steps include: 1) Dissociate and digest the airway tissue into single cells, and perform 2D expansion culture to P3-P4 generations; 2D expansion culture includes primary culture and subculture, and primary culture and subculture are cultured in airway culture medium; The airway culture medium is prepared according to the following formula: PneumaCult-Ex Plus Basal Medium and PneumaCult-Ex Plus 50X Supplement in the STEMCELL airway complete culture medium kit are mixed, and Hydrocotisone Stock Solution is added, and the ratio of Hydrocotisone Stock Solution to culture medium is 1:1000, wherein 10mM Y-27632 is added to the airway culture medium of the first day of primary culture and subculture; 2) mixing the cells cultured to the P3-P4 generation in the 2D expansion in step 1) with the matrix gel to obtain a cell-matrix gel mixture; 3) using the cell-matrix gel mixture obtained in step 2) as the dispersed phase and the oil phase reagent as the continuous phase, shearing the cell-matrix gel mixture into monodisperse droplets using microfluidics technology, and solidifying the monodisperse droplets at 37° C. to obtain cell microspheres after solidification; 4) inoculating the cell microspheres obtained in step 3) into airway organoid culture medium, and culturing in a 37° C., 5% CO 2 incubator, to obtain airway organoids after the culture is completed; The airway organoid culture medium is prepared according to the following formula: after mixing the components PneumaCult-Ex Plus Basal Medium and PneumaCult-Ex Plus 50XSupplement in the STEMCELL Airway Complete Culture Medium Kit, Hydrocotisone Stock Solution is added, and the ratio of Hydrocotisone Stock Solution to culture medium is 1:1000.

2. The preparation method according to claim 1, characterized in that The airway tissue in step 1) is human airway tissue; Preferably, the method for dissociating and digesting the airway tissue into single cells in step 1) comprises the steps of: taking airway tissue, washing it with PBS containing 1% double antibody P / S and then cutting it into pieces, adding digestion solution to the tissue fragments for digestion until the dissociation standard is met, terminating digestion, collecting the precipitate by centrifugation, adding DMEM containing 10% FBS by volume to resuspend the precipitate, filtering with a 100 μm cell mesh, collecting the filtrate, centrifuging the filtrate to discard the supernatant, then adding red blood cell lysis solution, terminating the red blood cell lysis reaction after the lysis is completed, centrifuging to discard the supernatant, and collecting the precipitate; Preferably, the digestion solution is prepared as follows: 2 mg / mL collagenase, 2 mg / mL DNase, and 2 mg / mL dispase are dissolved in DMEM / F12 culture medium and filtered through a 22 μm filter membrane.

3. The preparation method according to claim 1, characterized in that: In step 1), the method for 2D expansion culture to P3-P4 generation is: every 5×10 5 The cells were added into 3 cm culture dishes for 2D expansion culture and expanded to P3-P4 generations.

4. The preparation method according to claim 1, characterized in that: 1×10 4 The cells cultured in 2D to P3-P4 were mixed evenly with the matrix gel.

5. The preparation method according to claim 1, characterized in that: The oil phase reagent in step 3) is fluorinated oil.

6. The preparation method according to claim 1, characterized in that: In step 3), the microfluidic technology is a microfluidic technology based on the T-channel method; Preferably, the flow rate of the cell-matrix gel mixture is controlled to be 10-15 μL / min, and the flow rate of the oil phase reagent is controlled to be 100-110 μL / min.

7. The preparation method according to claim 1, characterized in that: The curing time at 37° C. in step 3) is 30 minutes.

8. The preparation method according to claim 1, characterized in that: In step 4), the culture time in a 37° C., 5% CO 2 incubator is 7-30 days.

9. The preparation method according to claim 1, characterized in that: The particle size of the airway organoids described in step 4) is 600-650 μm.

10. Airway organoids prepared by the preparation method according to any one of claims 1-9.

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