Method for rapidly and efficiently inducing multiple human airway epithelium in-vitro models for long time

Abstract

A method for rapidly and efficiently inducing multiple human airway epithelium in vitro models for a long time relates to the technical field of organoid culture.The method comprises the steps that firstly, a clinical lung tissue sample is induced and differentiated to form a cilia inward lung airway organoid, the cilia inward lung airway organoid is digested into single cells, and multiplication culture is conducted through a basal cell culture medium; the obtained basal cells are subjected to induced differentiation to form cilia outward lung airway organs and a gas-liquid interface culture model. The two types of models can successfully simulate the functional cellular morphology of the human airway epithelium in vitro, can be used as experimental models for disease modeling, drug screening, toxicity testing and the like, and can simulate biological models of the human airway structure and function. The method can solve the problems that clinical samples are difficult to obtain and primary lung epithelial cells have limited passage times in the construction process of lung airway organs, and realizes long-term rapid and efficient induction of various human airway epithelium in-vitro models.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a method for long-term, rapid and efficient induction of multiple human airway epithelial in vitro models. Background Art

[0002] Pulmonary airway organoids can be established from cells of various origins, including trachea, airway basal cells, alveoli, human induced pluripotent stem cells, and embryonic lung cells. Typical pulmonary airway organoids include ciliated cells, goblet cells, club cells, and basal cells, which have the phenotypic and functional characteristics of airway epithelium. The movement of airway cilia can be observed under a microscope, and they are a powerful tool for modeling respiratory virus pathology.

[0003] Air-liquid interface (ALI) culture is often used in lung research, but it requires a large number of cells, which are not easy to obtain from patients. In addition, the success of ALI culture is affected by primary bronchial epithelial cells, so it is necessary to use primary cells with an early generation. The most common lung airway organoids are implanted in Matrigel, with ciliated cells located in the cavity and basal cells located on the outside. When this type of organoid is used for toxicity testing, the lung airway organoids are exposed to toxic substances in a different way from the human airway. Therefore, lung airway organoids with cilia facing outward will be a good physiological model for toxicity testing, and its culture model that does not rely on Matrigel is easy to achieve high throughput.

[0004] Currently, the efficiency of constructing ALI and outward-facing lung airway organoids is limited by the cell source and cell quality, and the induction time is relatively long. Summary of the invention

[0005] In view of the shortcomings of the current construction of human airway epithelial in vitro models, the present invention provides a method for inducing multiple human airway epithelial in vitro models quickly and efficiently over a long period of time, which can induce human airway ALI and lung airway organoids with cilia facing outwards over a long period of time, thereby providing the possibility for the widespread application of ALI and the use of lung airway organoids with cilia facing outwards for high-throughput drug screening, and therefore has good value for practical promotion and application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for inducing multiple human airway epithelial in vitro models in a long-term, rapid and efficient manner. First, clinical lung tissue samples are used to induce differentiation to form inward-facing cilia-facing lung airway organoids, which are then digested into single cells and expanded and cultured using basal cell culture medium. The resulting basal cells are induced to differentiate into outward-facing cilia-facing lung airway organoids and air-liquid interface culture models.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0009] 1. The present invention can successfully complete the construction of an ALI model within 14 days using basal cells derived from lung airway organoids with inward-facing cilia. The obtained ALI culture showed obvious cilia beating under a microscope, AB-PAS staining test proved that it had mucus secretion function, and immunofluorescence staining showed that it contained basal cells, goblet cells, club cells and ciliated cells.

[0010] 2. The present invention can successfully construct outward-facing cilia lung airway organoids within 14 days using basal cells derived from inward-facing cilia lung airway organoids, with an efficiency of up to 100%.

[0011] 3. The basal cells derived from the inward-facing lung airway organoids obtained by the present invention can be cryopreserved, and after thawing, they still maintain the activity of differentiating into ALI and outward-facing lung airway organoids.

[0012] 4. The present invention has successfully developed a simple and repeatable method to establish two human airway epithelial in vitro models. These two types of models can successfully simulate the functional cell morphology of human airway epithelium in vitro. In addition, the problem of limited cell source and quality in the construction of traditional airway epithelial in vitro models is solved, making the model construction more efficient.

[0013] 5. The lung airway organoids constructed by the present invention can be used as experimental models for disease modeling, drug screening, and toxicity testing, etc. They can simulate biological models of human airway structure and function, and can solve the problems of difficulty in obtaining clinical samples and limited number of passages of primary lung epithelial cells during the construction of lung airway organoids, thereby achieving long-term, rapid, and efficient induction of a variety of human airway epithelial in vitro models. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of a method for generating ALI and cilia-facing-inward lung airway organoids using cilia-facing-outward lung airway organoids in an embodiment of the present invention.

[0015] Figure 2 This is the bright field image and identification image of the amplified basal cells in Example 3 of the present invention.

[0016] Figure 3 This is a bright field image of the ALI culture process in Example 4 of the present invention.

[0017] Figure 4 This is a bright field image of the culture process of lung airway organoids with cilia facing outward in Example 5 of the present invention.

[0018] Figure 5 HE staining, AB-PAS staining and immunofluorescence identification of paraffin sections of the ALI model in Example 6 of the present invention.

[0019] Figure 6These are HE staining, AB-PAS staining, and immunofluorescence identification images of paraffin sections of lung airway organoids with cilia facing outward in Example 6 of the present invention.

[0020] Figure 7 This is an immunofluorescence identification diagram of ALI induced by resuscitation and culture of frozen cilia-facing inward lung airway organoid-derived basal cells in Example 7 of the present invention.

[0021] Figure 8 This is an immunofluorescence identification diagram of the basal cells derived from the frozen lung airway organoids with inward-facing cilia induced into lung airway organoids with outward-facing cilia after resuscitation and culture in Example 7 of the present invention. DETAILED DESCRIPTION

[0022] See also Figure 1 As shown, the present invention proposes a method for long-term, rapid and efficient induction of multiple human airway epithelial in vitro models, firstly using clinical lung tissue samples to induce differentiation to form lung airway organoids with cilia facing inward, which are digested into single cells and expanded and cultured using basal cell culture medium, and the obtained basal cells are induced to differentiate into lung airway organoids with cilia facing outward and an air-liquid interface culture model.

[0023] The present invention is further described in detail below in conjunction with embodiments and drawings.

[0024] Example 1 Experimental Materials

[0025] 1. Cell and organoid culture medium

[0026]

[0027] 2. ALI and Cilia-Facing-Outward Lung Airway Organoid Culture Materials

[0028]

[0029] 3. Immunofluorescence Antibodies

[0030]

[0031] Example 2 Construction of lung airway organoids with inward-facing cilia

[0032] (1) Clinical lung tissue samples were analyzed by AdDF +++ The lung tissue was washed with culture medium (DMEM / F12, 10 mM HEPES, 1×GlutaMax, 1×Penicillin-streptomycin) and centrifuged to remove blood cells; the lung tissue was transferred to a 35 mm culture dish and 500 μL of digestion solution (AdDF containing 2 mg / mL collagenase I) was added. +++ ), mince the lung tissue into 1 mm pieces with a blade 3, transfer the chopped lung tissue into a 15 mL centrifuge tube, add 6 mL of digestion solution, and digest at 37 °C for 2 h.

[0033] (2) After digestion, the tissue was blown with a Pasteur pipette to separate the cell clusters from the tissue. The digested tissue was filtered through a 40 μm cell sieve into a 50 mL centrifuge tube. AdDF was added to the cell suspension. +++ When the volume reaches 10 mL, transfer all of it into a 15 mL centrifuge tube and centrifuge at 500 rpm for 5 min to precipitate the cell pellet. Take out the centrifuge tube after centrifugation, discard the supernatant, and add 1 mL of red blood cell lysis buffer to mix well.

[0034] (3) After centrifugation, discard the supernatant, add a small amount of cilia-facing lung airway organoid culture medium to resuspend and count. Mix the cells with 40% cell suspension and 60% matrix gel, and seed the cells in a 48-well plate at 30 μL per well, with approximately 300 cell clusters per well; place the 48-well plate in a 37°C incubator to gel for 10 minutes, then add 300 μL of cilia-facing lung airway organoid culture medium to each well and place the plate in a 37°C incubator for culture.

[0035] Example 3: Basal cell expansion, culture and cryopreservation

[0036] The cilia-oriented lung airway organoids obtained in Example 2 were isolated using the cell dissociation reagent TrypLE TM Express enzyme digestion at 37℃ for 6-8min, with AdDF +++ The cells were transferred to a 15 mL centrifuge tube and centrifuged at 850 rpm for 5 min. The cells were resuspended in 1 mL of basal cell expansion medium. The single cell suspension was seeded in a six-well plate and 2 mL of basal cell expansion medium was added. The cells were cultured at 37°C and 5% CO2 for 3-4 days. Cells cultured in basal cell medium widely expressed basal cell markers ( Figure 2 shown).

[0037] When the cell confluence reaches 70%-80%, it can be frozen. The specific freezing steps are as follows: add 300 μL of cell dissociation reagent TrypLE to each well of the six-well plate. TM Express enzyme, digest at 37℃ for 6min, transfer to 15mL centrifuge tube after digestion, add 4mL AdDF +++ , centrifuge at 850 rpm for 5 min, discard the supernatant, add 1 mL of freezing solution (basal cell expansion medium + 8% DMSO), put it in a gradient freezing box at -80°C overnight, and transfer it to a liquid nitrogen tank for storage the next day.

[0038] Example 4 Air-Liquid Interface (ALI) Model Construction

[0039] When the basal cell confluence of Example 3 reaches 70%-80%, the cell dissociation reagent TrypLE TM The cells were digested with Express enzyme and seeded in Transwell chambers, with 3.3×10 cells per well. 4 cells, 200 μL and 500 μL of basal cell expansion medium were added to the upper and lower chambers of the Transwell chamber, respectively. When the cell confluence reached 100% (approximately 3 days), the cells were cultured in the basal cell expansion medium and then switched to air-liquid interface culture, i.e., the upper chamber medium of the Transwell was discarded, and 500 μL of ALI differentiation medium was added to the lower chamber for 14 days, with the lower chamber medium replaced every 2 days.

[0040] Figure 3 This is a bright field image of the ALI culture process. Day 0 in the figure shows basal cells cultured in a six-well plate. Day 2-14 is a bright field image of the culture process using ALI differentiation medium. The cells change from a single layer of cells to a multilayer of cells, and the cilia can be seen beating under the microscope.

[0041] Example 5 Construction of lung airway organoids with cilia facing outwards

[0042] When the basal cell confluence of Example 3 reaches 70%-80%, the cell dissociation reagent TrypLE TM Express enzyme, digest at 37°C for 6-8 minutes, and add 2 mL of AdDF when the cells are completely detached +++ The cells were then transferred to a 15 mL centrifuge tube and centrifuged at 850 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 mL of lung airway organoid culture medium with cilia facing outwards and seeded in AggreWell treated with an anti-adsorption reagent. TM 400 wells, 1.2×10 per well 5 cells. TM 400well was centrifuged at 100×g for 3min. Incubated at 37℃, 5% CO2 for 6 days to aggregate the cells. 24-well flat-bottom plates were pretreated with anti-adhesion reagents and then transferred to AggreWell TM Add 1 mL of lung airway organoid culture medium with cilia facing outward to each well of 400 wells, use a Pasteur pipette to resuspend the cell pellet, and transfer 1 mL of the suspension to the pretreated 24-well plate. Incubate at 37°C, 5% CO2 for 8 days, changing the culture medium every two days.

[0043] Figure 4 Bright field image of the construction process of lung airway organoids with cilia facing outwards, in the basal cell AggreWell TMThe cells aggregated in 400-well plates to form dense cell clusters with smooth edges. After the cell clusters were suspended and cultured in 24-well plates for 8 days, cilia were observed beating in the outer circle of the cell clusters.

[0044] Example 6 In vitro model identification

[0045] 1. Sample Collection

[0046] (1) Example 4 The air-liquid interface culture was cultured for 14 days, the culture medium was discarded, and 4% paraformaldehyde was used for fixation. 200 μL was added to the upper chamber and 500 μL was added to the lower chamber. After fixation at room temperature for 30 minutes, PBS was used to wash 3 times, each time for 5 minutes. After washing, the PET membrane containing the air-liquid interface culture was peeled off from the Transwell and cut in half with surgical scissors, and then placed vertically in a 48-well plate and embedded with 2% agarose.

[0047] (2) The cilia of the lung airway organoids obtained by culture in Example 5 are facing outwards, and the culture medium is transferred to a 1.5 mL centrifuge tube. After natural precipitation at room temperature for 10 min, the culture medium is discarded, 100 μL of 4% paraformaldehyde is added and fixed at room temperature for 30 min, and then 1 mL of PBS is added for washing. The tube is centrifuged at 300 rpm for 3 min, and washed 3 times in total. After washing is completed, 50 μL of 2% agarose is added for embedding.

[0048] 2. Paraffin Embedded Sections

[0049] The agarose-embedded samples were dehydrated using a tissue dehydrator and then embedded in paraffin, and then sliced ​​using a microtome. The slice thickness of the air-liquid interface culture was 6 μm, and the slice thickness of the lung airway organoid with cilia facing outward was 7 μm. The slices were stored at room temperature.

[0050] 3. HE staining

[0051] (1) Dewaxing sections into water (xylene I, xylene II, 100% ethanol I, 100% ethanol II, 90% ethanol, 80% ethanol, ddH2O, 5 min each);

[0052] (2) Hematoxylin staining for 30 seconds, followed by rinsing with tap water;

[0053] (3) Differentiate with 0.2% HCl in ethanol solution for 3-5 seconds and rinse with tap water;

[0054] (4) Immerse the sections in 80% ethanol for 5 min;

[0055] (5) Eosin staining for 15 seconds;

[0056] (6) The sections were quickly immersed in 100% ethanol three times;

[0057] (7) After being transparentized with xylene, the slides were sealed with neutral gum.

[0058] like Figure 5 , 6 The HE staining result in the figure shows that the slices obtained using the air-liquid interface culture prepared in Example 4 and the lung airway organoids with cilia facing outwards prepared in Example 5 have complete morphological structures.

[0059] 4. AB-PAS staining

[0060] (1) Dewax the sections into water (xylene I, xylene II, 100% ethanol I, 100% ethanol II, 90% ethanol, 80% ethanol, ddH2O for 5 min each), and wash with ddH2O for 2 min;

[0061] (2) Stain with Alcian blue solution for 20 min and wash with ddH2O three times, 2 min each time;

[0062] (3) Add oxidant and oxidize for 8 min, then wash twice with ddH2O, each time for 2 min;

[0063] (4) Add Schiff staining solution to cover the sections and stain for 20 min, then rinse twice with ddH2O, 5 min each time;

[0064] (5) Stain the nucleus with hematoxylin solution for 1 min and wash with ddH2O;

[0065] (6) Differentiate with acidic differentiation solution for 2-5 seconds and wash with ddH2O;

[0066] (7) Use Scott bluing solution to reverse blue for 3 min, and wash with ddH2O for 3 min;

[0067] (8) Stepwise ethanol dehydration (80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, 5 min each);

[0068] (9) After being transparentized with xylene, the slides were sealed with neutral gum.

[0069] like Figure 5 , 6 The AB-PAS staining result graph shows that the air-liquid interface culture prepared in Example 4 and the lung airway organoids with cilia facing outward prepared in Example 5 both have the function of mucin secretion, blue represents acidic mucin, and red represents neutral mucin.

[0070] 5. Immunofluorescence on Paraffin Sections

[0071] (1) The obtained paraffin sections were first baked at 60°C for 30 min;

[0072] (2) hydration, specifically: 100% xylene for 5 min, 100% xylene for 5 min, 100% ethanol for 5 min, 100% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, ddH2O for 5 min;

[0073] (3) Antigen retrieval: Antigen retrieval was performed using 0.1 M, pH 6.2 citrate buffer followed by immunofluorescence staining;

[0074] (4) Use 3% BSA and 0.5% TritonX-100 to block the membrane at room temperature for 1 h;

[0075] (5) Primary antibody incubation: Dilute P63, MUC5AC, FOXJ1, Acetylated αTubulin, E-Cadherin, CC10, and KRT5 antibodies according to the corresponding proportions using 3% BSA;

[0076] (6) Wash away the primary antibody with PBS for 3 times, 15 min each time.

[0077] (7) Add fluorescently labeled secondary antibody working solution and incubate at room temperature for 2 h;

[0078] (8) After washing with PBS three times, add 1 μg / mL Hoechst 33342 dye solution and incubate for 10 min;

[0079] (9) After sealing with an anti-fluorescence quencher, take photos using an inverted fluorescence microscope or a high-content imaging system.

[0080] like Figure 5 , 6 The immunofluorescence identification result graph shows that the air-liquid interface culture obtained in Example 4 expresses the epithelial cell marker E-Cadherin and contains basal cells (P63 + ), ciliated cells (FOXJ1 + ), Club Cell (CC10 + ), goblet cells (MUC5AC + The outward-facing cilia lung airway organoid constructed in Example 5 contains basal cells (KRT5 + ), ciliated cells (FOXJ1 + ), goblet cells (MUC5AC + ).

[0081] Example 7: In vitro model induction and immunofluorescence identification after cryopreserved basal cells were revived and cultured

[0082] The frozen cells of Example 3 were thawed at 37°C, centrifuged at 850 rpm for 5 min, and seeded in a six-well plate. When the confluence reached 70%-80%, the in vitro model was induced according to the methods shown in Examples 4 and 5, and then paraffin section immunofluorescence was performed by the methods shown in Example 6.

[0083] like Figure 7 , 8 The immunofluorescence identification results in the figure show that the air-liquid interface culture model constructed by cryopreserved thawed basal cells contains basal cells (P63 + ), ciliated cells (FOXJ1 + ), Club Cell (CC10 + ) and goblet cells (MUC5AC + The constructed lung airway organoids with cilia facing outwards contain basal cells (KRT5 + ), ciliated cells (FOXJ1 + ), goblet cells (MUC5AC + ). This indicates that the basal cells after cryopreservation and resuscitation can still maintain good differentiation ability.

[0084] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A method for long-term, rapid and efficient induction of multiple human airway epithelial in vitro models, characterized in that: First, clinical lung tissue samples were used to induce differentiation to form lung airway organoids with cilia facing inward, which were digested into single cells and expanded and cultured using basal cell culture medium. The obtained basal cells were induced to differentiate into lung airway organoids with cilia facing outward and an air-liquid interface culture model.

2. The method according to claim 1, characterized in that The method to induce differentiation to form inward-facing cilia-oriented lung airway organoids is: (1) Clinical lung tissue samples were analyzed by AdDF +++ The lung tissue was washed with culture medium and centrifuged to remove blood cells; the lung tissue was transferred to a 35 mm culture dish, 500 μL of digestion solution was added, and the lung tissue was minced into 1 mm pieces with a blade. 3 , transfer the chopped lung tissue into a 15 mL centrifuge tube, add 6 mL of digestion solution, and digest at 37 °C for 2 h; (2) After digestion, the tissue was blown with a Pasteur pipette to separate the cell clusters from the tissue. The digested tissue was filtered through a 40 μm cell sieve into a 50 mL centrifuge tube. AdDF was added to the cell suspension. +++ When the volume is 10 mL, transfer all of it into a 15 mL centrifuge tube and centrifuge at 500 rpm for 5 min to precipitate the cell pellet. Remove the centrifuge tube after centrifugation, discard the supernatant, and add 1 mL of red blood cell lysis buffer to mix well. (3) After centrifugation, discard the supernatant, add a small amount of lung airway organoid culture medium with cilia facing inward, resuspend and count; Mix cells with 40% cell suspension and 60% matrix gel, and seed 30 μL of cells into each well of a 48-well plate. Place the 48-well plate in a 37°C incubator to gel for 10 minutes, then add 300 μL of lung airway organoid culture medium with cilia facing inward to each well, and culture the plate in a 37°C incubator.

3. The method according to claim 2, characterized in that The method for digesting the inward-facing lung airway organoids into single cells and expanding and culturing them in basal cell culture medium to obtain basal cells is as follows: The obtained lung airway organoids with cilia facing inward were dissociated using the cell dissociation reagent TrypLE TM Express enzyme digestion at 37℃ for 6-8min, with AdDF +++ The cells were transferred to a 15 mL centrifuge tube after being blown down and centrifuged at 850 rpm for 5 min. The cells were resuspended in 1 mL of basal cell expansion medium and the obtained single cell suspension was seeded in a six-well plate. 2 mL of basal cell expansion medium was added for culture.

4. The method according to claim 3, characterized in that The method for inducing basal cells to differentiate into lung airway organoids with cilia facing outwards is: When the confluence of cultured basal cells reached 70%-80%, the cell dissociation reagent TrypLE TM Express enzyme, digest at 37°C for 6-8 minutes, and add 2 mL of AdDF when the cells are completely detached +++ The cells were then transferred to a 15 mL centrifuge tube and centrifuged at 850 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 mL of lung airway organoid culture medium with cilia facing outwards and then seeded in AggreWell treated with an anti-adsorption reagent. TM 400 wells, 1.2×10 per well 5 cells; AggreWell TM 400well was centrifuged at 100×g for 3min; incubated at 37℃, 5% CO2 for 6 days to aggregate the cells; the 24-well flat-bottom plate was pretreated with anti-adhesion reagent and then transferred to AggreWell TM Add 1 mL of lung airway organoid culture medium with cilia facing outward to each well of the 400-well plate, use a Pasteur pipette to pipette to resuspend the cell pellet, and transfer 1 mL of the suspension to the pretreated 24-well plate; The cells were cultured at 37°C, 5% CO2 for 8 days, with the culture medium replaced every two days.

5. The method according to claim 3, characterized in that The method for inducing differentiation of basal cells to form an air-liquid interface culture model is as follows: When the confluence of cultured basal cells reached 70%-80%, the cell dissociation reagent TrypLE TM The cells were digested with Express enzyme and seeded in Transwell chambers. Basal cell expansion medium was used to culture the cells until the cell confluence reached 100%, and then the cells were switched to air-liquid interface culture. The medium in the upper chamber of Transwell was discarded, and 500 μL ALI differentiation medium was added to the lower chamber for culture. The culture was continued for 14 days, and the medium in the lower chamber was replaced every 2 days.

6. The method according to claim 3, characterized in that The basal cells obtained can still be used to construct lung airway organoids with cilia facing outward and air-liquid interface culture models after being frozen and thawed.

7. The in vitro model of human airway epithelium induced and differentiated by the method according to any one of claims 1 to 6, characterized in that: Includes lung airway organoids with cilia facing outward and air-liquid interface culture models.

8. The use of the human airway epithelial in vitro model as claimed in claim 7, characterized in that: Used as an experimental model for disease modeling, drug screening, and toxicity testing.

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