CN112553142B - 3D organ of nasal mucosa epithelial cells and culture method and application thereof - Google Patents

3D organ of nasal mucosa epithelial cells and culture method and application thereof Download PDF

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CN112553142B
CN112553142B CN202011443034.XA CN202011443034A CN112553142B CN 112553142 B CN112553142 B CN 112553142B CN 202011443034 A CN202011443034 A CN 202011443034A CN 112553142 B CN112553142 B CN 112553142B
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organoid
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organoids
epithelial cells
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CN112553142A (en
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史丽
赵莉
金鹏
訾晓雪
张红萍
张海令
于克娜
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Shandong University
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Abstract

The invention provides a 3D organoid of nasal mucosa epithelial cells and a culture method and application thereof, belonging to the technical field of organoid culture. The method for culturing the 3D organoid of the nasal mucosa epithelial cells comprises the step of culturing the nasal mucosa epithelial cells by adopting a U-shaped bottom organoid culture method or a gelatin-vapor-liquid horizontal organoid culture method. The invention successfully constructs and obtains the 3D type organ of the nasal mucosa epithelial cells by optimizing the culture method, and simultaneously, further optimizes the analysis method, thereby providing possibility for high-flux drug screening, and having good practical popularization and application values.

Description

3D organ of nasal mucosa epithelial cells and culture method and application thereof
Technical Field
The invention belongs to the technical field of organoid culture, and particularly relates to a 3D organoid of nasal mucosa epithelial cells, and a culture method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Chronic inflammatory diseases of the Nasal mucosa are common and multiple diseases of the ear-nose-throat department, including Allergic Rhinitis (AR) and Chronic Rhinosinusitis (CRS) (with or without Nasal Polyps (NP)) and the like. Not only are the incidences of the AR and the CRS closely related to each other, but also the occurrence of lower respiratory diseases such as Asthma (athma) and Chronic Obstructive Pulmonary Disease (COPD) is closely related to each other, which seriously impairs the quality of life of patients, causes high medical expenses, and imposes a great medical burden on society. At present, the pathogenesis of chronic inflammatory diseases of nasal mucosa is very complex and still not completely clear, and the conventional treatment method still has the problems of improvement of curative effect, lack of radical treatment means and the like, so that continuous and deep basic research is urgently needed, clinical conversion is carried out on the basis of elucidation of the pathogenesis, and a more effective and targeted treatment strategy is explored.
The in vitro culture of the epithelial cells of the human nasal mucosa plays an important role in the basic research of respiratory biology, disease pathogenesis and the development of novel diagnosis and treatment technologies. The air-liquid interface culture method (ALI) is the most commonly used respiratory epithelium culture technology at present, and provides a good in-vitro research platform for researching airway epithelial cell morphology and function. The embedded culture dish based on the Tranwell is widely applied to the culture of the cell ALI and can support the in-vitro detection of the medicine, such as the measurement of the transportation and the metabolism of the medicine crossing a cell layer. However, the Transwell-ALI-bound culture mode can only be used for laboratory tests with small sample size, and cannot be applied to high-throughput tests such as in vitro drug screening for respiratory diseases. Therefore, the Transwell-based ALI culture method greatly limits the development of drugs for the upper respiratory tract. In recent years, with the maturation of 3D organoid culture technology, in vitro drug high throughput screening has become a better choice. The 3D organoid models can be cultured on high throughput platforms using small numbers of cells, and can be combined with existing fluidic chips. 3D organoid culture systems have been successfully applied to certain types of epithelial cells, such as prostate, bowel and lung, however, the inventors have discovered that 3D organoid models of the upper respiratory epithelium have been rarely reported.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a 3D organoid of nasal mucosa epithelial cells, a culture method and application thereof.
Specifically, the invention relates to the following technical scheme:
according to the first aspect of the invention, the invention provides a method for culturing 3D organoids of nasal mucosal epithelial cells, which comprises the step of culturing the nasal mucosal epithelial cells by adopting a U-shaped bottom organoid culture method or a gelatin vapor liquid level organoid culture method.
It is noted that nasal mucosal epithelial cells can be obtained by referring to ZHao, xuening, et al, "The use of nasal epithelial stem/producer cells to produced functional cells in vitro," American Journal of Rhinology & Allergy26.5 (2012): 345-350; and will not be described in detail herein.
In a second aspect of the invention, the nasal mucosa epithelial cell 3D organoid obtained by the above culture method is provided.
When a U-shaped bottom organoid culture method is selected for culture, the size of the obtained 3D organoid of the nasal mucosa epithelial cells is 100-200 mu m, and the outer layer of the 3D organoid has swinging cilia;
when a glue-vapor-liquid flat organoid culture method is selected for culture, the size of the obtained 3D organoid of the nasal mucosa epithelial cells is 300-400 mu m, and a cavity which is slightly transparent and uniform in size is formed inside the organ; inside the cavity there are ciliated cells that oscillate.
In a third aspect of the invention, the application of the nasal mucosa epithelial cell 3D organoid in-vitro screening and/or detection of drugs is provided.
In a fourth aspect of the invention, an in vitro detection platform is provided, wherein the in vitro detection platform comprises the nasal mucosa epithelial cell 3D type organ, so that the in vitro detection platform which can simulate the in vivo environment, is stable and has low cost is provided for high-throughput drug screening.
In a fifth aspect of the present invention, there is provided a method of high-throughput drug screening in vitro, the method comprising: and applying the drug to be detected to the nasal mucosa epithelial cell 3D organoid and/or the in-vitro detection platform, and detecting and analyzing the nasal mucosa epithelial cell 3D organoid and/or the in-vitro detection platform.
The beneficial technical effects of one or more of the above technical solutions are as follows:
two simple and reproducible methods were successfully developed in the above-described protocol to establish human nasal mucosal organoids. This 3D model successfully mimics the functional cell morphology of nasal epithelium in vitro. In addition, the technical scheme also establishes and optimizes a downstream detection method of the 3D organoid.
Particularly in the U-bottom method, the maturation rate of nasal mucosal epithelial cells was faster (only 7 days), and the number of seeded cells was reduced by 100-fold compared to the transwell method (the number of seeded cells was from 100k to 1 k). The culture technology provides another economic and efficient method for modeling human nasal mucosa in vitro, and provides possibility for high-throughput drug screening and individual drug optimization of nasal mucosa epithelium, so that the culture technology has good value of practical popularization and application.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of three methods for organoid generation using primary cells of human nasal mucosal epithelium in accordance with an embodiment of the present invention.
FIG. 2 is a diagram of the morphology of nasal mucosa organoids formed by three methods in accordance with an embodiment of the present invention; the scale bar is 100 μm. FIG. 3 is a diagram illustrating organoid differentiation in a U-bottom organoid culture method according to an embodiment of the present invention; the scale bar is 100 μm.
FIG. 4 is a GelALI-derived organoid differentiation map in an embodiment of the present invention, wherein A is a schematic diagram of the differentiation process, and B is a diagram of the actual organoids at different time points during the differentiation process; the scale bar is 100 μm.
FIG. 5 is a graph showing the effect of cell seeding number on organoid formation in an example of the present invention. A. Organoid morphology at different time points, different cell seeding numbers. B. And comparing the roundness of the organoids at different time points and different cell seeding numbers. C. Different time points, different cell seeding numbers and organoid diameters. D. Different time points, different cell seeding numbers and organoid transparency comparison.
FIG. 6 shows a nasal mucosal epithelial cell model formed by the ALItranswell culture system and a staining pattern thereof according to an embodiment of the present invention.
FIG. 7 shows an iSpace sheet sealing step in the embodiment of the present invention. A. Schematic of sealing the sample in an isepactor. B. The specific steps of sealing 3D organoids using iSpacer: 1. peeling the protective liner of the iospacers; 2. place the gasket (exposed, sticky side down) on the dry slide or coverslip surface and press gently to seal; 3. placing the specimen in the hole, adding proper amount of the mixture
Figure BDA0002830699150000031
Placing a cover glass to seal; 4. excess solution was removed from the wells and the peripheral area of the wells was sealed with clear nail polish to form a hard film.
FIG. 8 is a comparison of imaging of two nasal mucosa organoids (before differentiation) in an example of the invention; olympus BX51 fluorescence microscope is the first row (200X) and Zeiss LSM700 is the second row (200X).
FIG. 9 is a 3D organoid image of nasal mucosa at different magnifications in an embodiment of the invention.
FIG. 10 is a comparison between two batches in an example of the present invention. A. Comparison of organoid structures between two batches; B. comparison of cilia density between two batches.
FIG. 11 is an organoid culture of primary nasal mucosal epithelial cells in an example of the invention. A. Organoid culture and passaging schematic of primary nasal mucosal epithelial cells. B. Morphology of primary nasal mucosal epithelial organoids. C. Organoid morphology after first passage. D. Organoid morphology after two passages. E. IF staining of 2 nd organoids.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention is further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.
As mentioned above, the establishment of the nasal mucosa epithelium organoid model can better simulate the in vivo growth environment of nasal mucosa stem cells, provide more accurate and reliable experimental data for the research of tissue engineering, and assist in the in vitro screening and development of nasal local drugs.
In view of the above, in an exemplary embodiment of the present invention, a method for culturing 3D organoids of nasal mucosal epithelial cells is provided, wherein the method comprises culturing nasal mucosal epithelial cells (hNECs) by using U-bottom organoid culture or gel-vapor-liquid-level organoid culture;
wherein the content of the first and second substances,
the U-shaped bottom organoid culture method specifically comprises the following steps:
resuspending the nasal mucosa epithelial cells in an organoid culture medium, inoculating the organoid culture medium in a cell culture vessel with a U-shaped bottom, and culturing after centrifugation.
In yet another embodiment of the present invention, the organoid culture medium comprises cell expansion medium, B27 cell culture additives and recombinant human noggin.
In yet another embodiment of the present invention, the cell expansion medium comprises: DMEM/F12, human epidermal growth factor, insulin, cholera toxin, cortisol, 3', 5-triiodo-L-thyronine, N-2 cell culture additives, and antibiotic-antifungal solutions.
In yet another embodiment of the present invention, the organoid culture medium comprises the following components in amounts: DMEM/F12, 1-5 x; 5-20 ng/mL of human epidermal growth factor; 1-10 ng/mL of insulin; cholera toxin, 0.05-0.2 nM; 0.1-1 mug/mL of cortisol; 3,3', 5-triiodo-L-thyronine, 1-5 nM; n-2 cell culture additive, 5-15 mul/mL; antibiotic-antifungal solution (100 x), 50-200 IU/mL; b27 cell culture additive (50 ×), 1 to 5 ×; the recombinant human noggin is 50-200 ng/mL.
In yet another embodiment of the present invention, the hNECs are resuspended in organoid medium at a concentration of 1-5X 10 3 ；
In another embodiment of the present invention, the cell culture vessel with a U-shaped bottom is a cell culture plate, preferably a 96-well culture plate;
in another embodiment of the present invention, the centrifugation control condition is centrifugation at 1000 to 2000rpm for 1 to 5 minutes, preferably at 1500rpm for 2 minutes;
culturing after centrifugation can be carried out by conventional cell culture method, such as CO 2 Culturing in a humidifying incubator; preferably, a portion (e.g., 50%) of the organoid medium is replaced every 3 to 5 days (e.g., 4 days) to ensure proper nutrient supply to the tissue cells.
By adopting the culture method, organoid formation is very rapid, a good organoid structure can be formed basically in the next day, and the boundary is clear;
in still another embodiment of the present invention, when organoids grow to more than 200 μm, differentiation is induced by adding a differentiation medium on day 7 of culture; on day 6 of differentiation, i.e., the oscillating cilia were observed in the outer layers of the organoids, and the organoids were reduced in size (size reduced to 100-200 μm) earlier, thereby obtaining nasal mucosal epithelial cell 3D organoids.
In another embodiment of the present invention, the differentiation medium is human bronchial epithelial cell gas-liquid interface medium (Pneuma Cult) TM ALI Medium)，1～5×。
In another embodiment of the present invention, the gel vapor-liquid flat organoid culture method specifically comprises:
diluting Matrigel (Matrigel) by using organoid culture medium, coating the Matrigel in a cell culture vessel and incubating to obtain a culture medium A; resuspending the nasal mucosal epithelial cells in organoid culture medium containing matrigel, and then inoculating in the culture medium A for culture.
In yet another embodiment of the present invention, the organoid medium comprises cell expansion medium, B27 cell culture additives and recombinant human noggin.
In yet another embodiment of the present invention, the cell expansion medium comprises: DMEM/F12, human epidermal growth factor, insulin, cholera toxin, cortisol, 3', 5-triiodo-L-thyronine, N-2 cell culture additives, and antibiotic-antifungal solution.
In yet another embodiment of the present invention, the organoid medium comprises the following components and amounts: DMEM/F12, 1-5 ×; 5-20 ng/mL of human epidermal growth factor; 1-10 ng/mL of insulin; cholera toxin, 0.05-0.2 nM; 0.1-1 mug/mL of cortisol; 3,3', 5-triiodo-L-thyronine, 1-5 nM; n-2 cell culture additive, 5-15 mul/mL; antibiotic-antifungal solution (100 x), 50-200 IU/mL; b27 cell culture additive (50 ×), 1 to 5 ×; 50-200 ng/mL of recombinant human noggin.
The culture medium A is specifically an organoid culture medium containing 30-50% (preferably 40%) of matrigel;
in yet another embodiment of the present invention, the cell culture vessel is a cell culture plate, preferably a 24-well culture plate;
in another embodiment of the present invention, the incubation is specifically incubation at 35-40 ℃ for 10-30 minutes, preferably at 37 ℃ for 20 minutes;
in another embodiment of the present invention, the step of resuspending the nasal mucosal epithelial cells in organoid culture medium containing matrigel, the content of matrigel is controlled to be 1-10%, preferably 5%;
in still another embodiment of the present invention, the step of "inoculating into the above-mentioned medium A and culturing" may be carried out by a conventional cell culture method, for example, CO may be used 2 Culturing in a humidifying incubator; preferably, a portion (e.g., 50%) of the organoid medium is replaced every 3 to 5 days (e.g., 4 days) to ensure proper nutrient supply to the tissue cells.
With the above culture method, organoid structures can be observed substantially on the third day of culture; on the 7 th day of culture, the size of organoid in GelALI reaches 200-300 μm; but because the organoids grow too densely, differentiation cannot be initiated; therefore, the organoids can be mechanically separated by enzymolysis and repeated beating (in this case, the size of the organoids is about 100 μm); adding organoid culture medium, centrifuging, re-suspending in organoid culture medium containing 50-70% matrigel, adding differentiation culture medium to induce differentiation, subculturing organoid by gel embedding, diluting, re-suspending and inoculating in cell culture container, adding differentiation culture medium, and completely covering the solidified matrigel liquid drop.
After culture using the above method, organoids grew gradually and reached 300-400 μm on day 21, at which point a large number of ciliated cells with ciliary beat appeared. At the same time, the inside of the organoid becomes slightly transparent and forms a cavity of uniform size. The beating ciliated cells were observed at the inner edge of the cavity.
The differentiation medium is a human bronchial epithelial cell gas-liquid interface medium (Pneuma Cult) TM ALI Medium)，1～5×。
In another embodiment of the present invention, there is provided a 3D organoid of nasal mucosal epithelial cells obtained by the above culture method, wherein the 3D organoid of nasal mucosal epithelial cells has a size of 100-200 μm, and the outer layer thereof has oscillating cilia; or the size of the 3D organoid of the nasal mucosa epithelial cells is 300-400 mu m, and a cavity which is slightly transparent and uniform in size is formed inside the nasal mucosa epithelial cells; inside the cavity there are ciliated cells that oscillate.
In another embodiment of the present invention, the application of the nasal mucosal epithelial cell 3D organoid in vitro screening and/or detection of drugs is provided.
The in vitro screening of the medicament is in particular in vitro medicament high-flux screening.
In another embodiment of the present invention, an in vitro detection platform is provided, which comprises the above-mentioned nasal mucosa epithelial cell 3D organoid, thereby providing a stable and low-cost in vitro detection platform capable of simulating an in vivo environment for high-throughput drug screening.
In another embodiment of the present invention, there is provided a method for high-throughput drug screening in vitro, the method comprising: and applying the drug to be detected to the nasal mucosa epithelial cell 3D organoid and/or the in-vitro detection platform, and detecting and analyzing the nasal mucosa epithelial cell 3D organoid and/or the in-vitro detection platform.
In yet another embodiment of the present invention, the detection and analysis methods include, but are not limited to, fluorescent staining, RNA extraction, image capture, and biomarker analysis.
In still another embodiment of the present invention, tritonX-100 is added to the selected reagent in the fluorescent staining method, thereby further facilitating the permeation of the reagent.
The reagent comprises washing liquor, confining liquid and antibody diluent;
wherein the washing liquid comprises the following components: PBS +0.3% TritonX-100;
the confining liquid comprises the following components: PBS +10% of FBS +0.3% TritonX-100;
the antibody diluent comprises the following components: PBS +1% FBS +0.3% TritonX-100.
In still another embodiment of the present invention, the fluorescent staining method comprises:
1) Nasal mucosal organoids were collected in 1.5mL Eppendorf tubes (EP tubes).
2) Wash twice with Wash buffer.
3) Organoids were fixed with 4% pfa for 10 min.
4) Wash twice with Wash buffer.
5) Blocking with Blocking buffer for 1 hour.
6) Organoids were pelleted using a mini centrifuge spindown ep tube and observed under a microscope.
7) The Blocking buffer was aspirated, taking care not to disturb the organoids at the bottom of the EP tube.
8) An anti-diluent is added to the EP tube to submerge the organoids in the bottom of the EP tube.
9) Incubate on shaker at 4 ℃ for 2 days to allow the primary antibody to fully penetrate the organoids.
10 After primary antibody incubation, organoids were pelleted using a mini centrifuge spin down ep tube and washed twice with Wash buffer.
11 ) on a shaker at 4 ℃ for two days in a Wash buffer.
12 Using a mini centrifuge spindown ep tube, organoids were pelleted and Wash buffer was removed.
13 Secondary antibody and DAPI were diluted into antibody dilution buffer and added to the EP tube to allow submergence of organoids at the bottom of the EP tube.
14 ) were incubated at 4 ℃ for 2 days on a shaker.
15 Spin down EP tubes were centrifuged using a mini centrifuge, organoids were pelleted and washed twice with a Wash buffer.
16 ) on a shaker at 4 ℃ for two days in a Wash buffer.
17 Using a mini centrifuge spin down EP tube, precipitating the organoid, removing the Wash buffer, and resuspending the organoid in the anti-fluorescence quenching mounting media.
18 Use an iseparer seal.
In still another embodiment of the present invention, in the RNA extraction method, it is preferable to perform cleavage using TRIzol.
In yet another embodiment of the present invention, the image capturing and biomarker analysis method preferably uses a confocal Z-step imaging method, thereby facilitating the capture of clearer 3D structures of nasal organoids.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
1. Material
1. The source of the cells.
Human nasal mucosal epithelial cells (hNECs) were supplied by university of shandong. 3T3 mouse fibroblasts obtained from ATCC (American Type Culture Collection, manassas, va.) were used as the cytotrophoblasts.
2. Cell culture fluid and 3D culture material.
Figure BDA0002830699150000091
Figure BDA0002830699150000101
3. Immunofluorescent staining antibody and reagent
Figure BDA0002830699150000102
2. Method for establishing human nasal mucosa epithelium 3D organoid model, exploration and comparison
(I) Experimental method
Three methods of organoid generation using human nasal mucosal epithelial primary cells were explored, as shown in figure 1. In this process, a previously established, currently well-established Transwell culture was used as a parallel control for monitoring the activity and differentiation capacity of hNECs (human nasal mucosal epithelial cells). The detailed steps are as follows:
(1) Isolation of hNECs from nasal mucosal tissue: nasal polyp tissue and inferior turbinate mucosa collected during nasal endoscopic surgery were washed in cold PBS within 1 hour ex vivo and placed in Hanks' salt equilibrium solution containing 100IU/ml antibiotic-antifungal. The tissue was then cut into small pieces using sterile scissors in a fume hood, added with 10mg/ml Dispase II enzyme and digested overnight on a shaker at 4 ℃. Centrifuging, removing supernatant, adding 0.05% pancreatin (containing EDTA) and digesting in water bath at 37 deg.C for 15min. The digested tissue was repeatedly blown and the separated epithelial cells were collected through a 100 μm sieve. The digestion process was terminated after the addition of the culture medium, and the isolated single cell (primary cell, P0) suspension was washed with PBS and kept ready for use.
(2) Preparation of trophoblast cells: 3T3 mouse fibroblasts were used as trophoblast cells. Mitomycin C was added to the medium at a concentration of 10. Mu.g/ml, and after overnight treatment, the cells were grown in fresh medium for 2 to 3 days to restore the cells to good condition.
(3) In vitro amplification of hNECs: in the amplification medium, 2X 10 3 Per cm 2 Cell density of (2) seeding of hNECs on 3T3 cells. After 80% confluence of hNECs proliferation, cells were harvested for subsequent organoid and Transwell culture: cells were digested for 1 minute using cell digest (Accutase, invitrogen) and gently pipetted repeatedly to separate 3T3 mouse fibroblasts (i.e., trophoblast cells) from hNECs cells. After removal of 3T3 cells, the cells were washed twice with PBS. Fresh cell digest (Accutase, invitrogen) was then added and incubated at 37 ℃ for 7 minutes to completely separate the 3T3 cells from the hNECs. (the specific culture conditions are shown in Table 1).
(4) Transwell culture: a24-well plate was inserted using a Transwell of 0.4 μm. Will be 5X 10 4 The hNECs were resuspended in 100. Mu.l of amplification medium, added to the Transwell, and 350. Mu.l of amplification medium was added to the wells of a 24-well plate below the Transwell.
The above steps are published, and the references are: ZHao, xuening, et al, "The use of a nasal epithelial stem/producer cell to a product functional cell in vitro," American Journal of Rhinology & Allergy26.5 (2012): 345-350.
The following are the innovative explorations of the invention:
(5) U-bottom (U-bottom) organoid culture:
(1) the hNECs were resuspended in 100. Mu.l organoid medium at 5X 10 concentrations 3 、2.5×10 3 、1×10 3 (ii) a (2) The seeds were plated in the lower liquid level of 96-well plates.
(3) The 96-well plate was centrifuged at 1500rpm for 2 minutes.
(4) Transferring 96 well plates to 37 ℃ C. And 5% CO 2 In a humidified incubator. Every 4 days 50% of the medium was changed.
(6) Gel vapor-liquid level (GelALI) organoid culture method:
(1) matrigel was diluted to 40% concentration using organoid medium and coated on 24-well plates.
(2) Incubate at 37 ℃ for 20 minutes.
(3) Will be 1 × 10 4 The hNECs were resuspended in 500. Mu.l of organoid medium containing 5% matrigel.
(4) The cells were plated on the prepared 24-well plate.
(5) Transfer of 24 well plates to 37 ℃ C. And 5% CO 2 In a humidified incubator. Every 4 days 50% of the medium was changed.
(7) Gel embedding (GelEmbedding) organoid culture method:
(1) matrigel was diluted to 60% concentration using organoid medium.
(2) Will be 1 × 10 3 The hNECs were resuspended in Matrigel at a concentration of 20. Mu.l 60%.
(3) The hNECs were seeded in small droplets on a 24-well plate.
(4) The gel was cured at 37 ℃ for 10 minutes.
(5) Add 500. Mu.l organoid medium per well and cover the gel.
(6) Transfer of 24 well plates to 37 ℃ C. And 5% CO 2 In a humidified incubator. The medium was changed every 4 days.
TABLE 1 specific conditions for transwell and organoid culture
Figure BDA0002830699150000121
(II) results
1. Observing the growth of cells/organoids with a microscope for 0-7 days
After seeding of the hNECs, the cells were continuously monitored for 7 days using a microscope to observe the morphology and aggregation of the cells (fig. 2).
(1) U-bottom: a good organoid structure was formed on the next day of culture with clear borders.
(2) GelALI: organoid structures were observed on the third day of culture.
(3) Gel Embedding: obvious organoid structures were shown on the fourth day of culture.
The cellular organoids in Gel ALI and Gel Embedding were smaller in size during the initial phase and grew into more compact structures after four days in culture compared to U-bottom.
2. After 7 days, the differentiation of the cells/organoids was observed using a microscope
①U-bottom：
When the organoids grew to over 200 μm, differentiation medium was added on day 7 of culture. The organoids do not change much in size during the differentiation process thereafter. On day 6 of differentiation, oscillating cilia were observed in the outer layers of the organoids, while the size of the organoids decreased earlier (size decreased to 100-200 μm), as shown in figure 3.
②Gel ALI：
On day 7 of culture, organoids in Gel ALI reached a size of 200
Figure BDA0002830699150000131
300 μm. However, organoids grow too densely to initiate differentiation. Dense organoids were digested using 1ml TrypLE Express (Invitrogen-12605036) and incubated at 37 ℃ for 5 minutes, followed by repeated pipetting to mechanically detach organoids for passage. 10ml organoid medium was then added and centrifuged for 5 minutes at 300 rcf. It was then resuspended in 60% Matrigel matrix and differentiation medium was added, after which organoid spheres were passaged using gelmbedding, as per 1: the seeds were re-sown in 24-well plates after dilution at a ratio of 6. The wells were then topped up with differentiation medium to completely cover the solidified Matrigel droplets.
As shown in FIG. 4, the organoids decreased in size to around 100 μm after separation from each other, then gradually increased, and increased to 300-400 μm on day 21, at which point a large number of ciliated cells with ciliary beat appeared. At the same time, the inside of the organoid becomes slightly transparent and forms a cavity of uniform size. The oscillating ciliated cells were observed at the inner edge of the lumen.
③Gel Embedding：
After 7 days of culture, organoids began to attach to the bottom of the plate and subsequently expand into a monolayer structure. After 9 days of culture, more and more primary cell organoids lost 3D structure, thus stopping the culture process.
3. Optimization and exploration of the number of cells seeded
Since the aggregation of cells in Gel ALI and Gel Embedding is random, organoid size cannot be controlled by optimizing the number of cells seeded. Therefore, the U-bottom method was used to explore the effect of cell seeding number on organoid formation and image analysis of organoids using imageJ.
5X 10 wells compared to wells seeded with a smaller number of cells 3 The number of organoids generated from the individual hNECs seeded wells was high (fig. 5A), but the number of seeded cells had no effect on the roundness of the organoids (fig. 5B). The larger the number of cells seeded, the slightly larger the organoid diameter. However, there were no significant differences in organoid diameters observed at day 12 of differentiation for the three different cell seeding densities (FIG. 5C). Furthermore, assessment of organoid transparency found that cell density in each organoid was positively correlated with the number of cell inoculations (fig. 5D).
4. Comparison of cell morphology and differentiation Processes in organoid culture and traditional ALI gas-liquid culture
In the traditional ALI culture, the Transwell bottom surface with air holes can form an air-liquid interface culture environment. hNECs were seeded at high density (100 kcells) in Transwell chambers and cultured to expand and fuse with each other for 3 days. The media from the upper and bottom layers is then removed and differentiation media is added to the bottom layer. Oscillating cilia were observed for about 21 days of differentiation. As shown in fig. 6, using Transwell, a parallel control procedure successfully produced differentiated nasal mucosal epithelium with positive staining of ciliated cells (β -IV tubulin, green) and goblet cells (MUC 5AC, red) 21 days after differentiation.
GelALI culture derived organoids produced differentiated wobbled ciliated cells on a similar number of days as Transwell. The swinging cilia were observed in the cavity formed inside the Gel ALI organoid when the organoids were cultured in a differentiated environment for about 21 days. While cilia wobble was observed in organoids cultured using the U-bottom method after 5 days of differentiation.
5. Optimized downstream analysis method for nasal organoids
1. Method optimization of organoid immunofluorescence staining
The most difficult part of the staining of 3D organoids is antibody penetration. Thus, the incubation time of the antibody was extended and TritonX-100 was added at each step to aid permeation of the reagents (reagents used for staining are listed in Table 2).
TABLE 2 reagents for fluorescent staining
Figure BDA0002830699150000141
Figure BDA0002830699150000151
The optimization method comprises the following steps:
1) Nasal mucosal organoids were collected in 1.5mL Eppendorf tubes (EP tubes).
2) Wash twice with Wash buffer.
3) Organoids were fixed with 4% pfa for 10 min.
4) Wash twice with Wash buffer.
5) Blocking with Blocking buffer for 1 hour.
6) Organoids were pelleted using a mini centrifuge spindown ep tube and observed under a microscope.
7) The Blocking buffer was aspirated, taking care not to disturb the organoids at the bottom of the EP tube.
8) Primary antibody was added to the EP tube to submerge the organoids in the bottom of the EP tube.
9) Incubate on a shaker at 4 ℃ for 2 days to allow the primary antibody to fully penetrate the organoids.
10 After primary antibody incubation, organoids were pelleted using a mini centrifuge spin down ep tube and washed twice with Wash buffer.
11 ) on a shaker at 4 ℃ for two days in a Wash buffer.
12 Using a mini centrifuge spindown ep tube, organoids were pelleted and Wash buffer was removed.
13 Secondary antibody and DAPI were diluted into antibody dilution buffer and added to the EP tube to allow submergence of organoids at the bottom of the EP tube.
14 ) were incubated at 4 ℃ for 2 days on a shaker.
15 Spin down EP tubes were centrifuged using a mini centrifuge, organoids were pelleted and washed twice with a Wash buffer.
16 ) on a shaker at 4 ℃ for two days in a Wash buffer.
17 Using a mini centrifuge spin down EP tube, precipitating the organoid, removing the Wash buffer, and resuspending the organoid in the anti-fluorescence quenching mounting media.
18 An iseparer seal (fig. 7).
2. Optimization of imaging
In this study, two methods of obtaining 3D nasal organoid images were explored. As shown in fig. 8, olympus BX51 fluorescence microscopy was unable to image the 3D structures of nasal organoids well compared to the confocal Z-step imaging method (Zeiss LSM 700).
The image quality at different magnifications was further evaluated using a Zeiss LSM700 confocal system (fig. 9). An image magnified 400 times may show more structural detail of the organoid.
Meanwhile, 3D imaging graphs of different methods show that different culture methods can influence the formation of the internal structure of the nasal mucosa organoid.
Consistent with the observations made under phase contrast microscopy, cilia (green staining) developed from the U-bottom cultured nasal mucosal organoids covered the outer layers of the organoids, while cilia (green staining) of the GelALI cultured source organoids were located at the edges of the cavities within them. (FIGS. 3, 4, and 9)
3. Optimization of RNA extraction method
Organoids of both U-bottom and Gel ALI culture were collected.
Two lysates were used: TRK lysis buffer and TRIzol from e.z.n.a. Total RNA kit (Omega), whereas organoids can only be completely lysed by TRIzol. Therefore, extraction of nasal mucosal organoids RNA was performed using the TRIzol RNA extraction procedure (Invitrogen).
In addition, for the U-bottom culture mode, 167ng of total RNA was harvested from four organoids and only 40ng of total RNA was harvested from three organoids (OD 260/280 of 1.4 and 1.6, respectively). Therefore, for RNA extraction and gene analysis of nasal mucosa organoids, it is suggested that at least four or more organoids should be collected for RNA extraction to ensure quality and quantity of RNA. In addition, commercial kits using RNA extraction for small numbers of cells (cell numbers less than 500 k) can be used to improve the quality of organoid RNA extraction. If organoids are used for sequencing analysis, it is recommended to use a one-step PCR or third-generation sequencing method to reduce the number of process steps, thereby minimizing RNA loss and improving the utilization rate of the nasal mucosa organoids.
6. Comparison of culture Process stability (comparison between batches)
To evaluate the stability of the culture method, both U-bottom and GelALI methods were repeated. According to the results in FIG. 5C, the size of the nasal mucosal organoids was not affected by the number of cells seeded by the U-bottom method. Thus, 60 wells were seeded at 1K cells per well on a 96 well plate for the first batch validation and differentiation was initiated after cell spheroid formation (day 5). After 7 days of differentiation, oscillating cilia were observed in 73% of the wells, with organoids slightly smaller in size than batch 1 (fig. 10A). Cilia density was evaluated using the imageJ3D object counting plug-in (https:// imageJ. Net/3d _objects _counter). The cilia density of organoids in lot 2 was lower compared to the data in lot 1.
In addition, gelALI cultures in the second batch showed oscillating cilia around 20 days after differentiation, which is consistent with the experimental results in the first batch.
7. Other findings of nasal mucosal epithelial stem cell culture into nasal mucosal organoid
On the next day of culturing primary nasal mucosal epithelial cells, it was surprisingly found that primary organoids could form spontaneously in the supernatant in a 2D culture environment. These primary organoids were captured and seeded by Gel Embedding (fig. 11A). After 2 days of gel embedding culture, three main organoid phenotypes were observed: transparent organoids, solid organoids, and tubular structural organoids (fig. 11B). In addition, there are adherent, monolayer-growing cells and cells expressing a nasal epithelial progenitor marker (KRT 5). The oscillating cilia were observed in transparent organoids, whereas they were not observed in solid and tubular structures. In the GelALI study, it was verified that those solid organoids were nasal progenitors with positive KRT5 staining (fig. 8). These three phenotypes can be passaged in organoid mode in gel-embedded culture (fig. 11C and D). However, as the number of passages increased, the number of clear organoids gradually decreased, while the number of solid organoids increased accordingly. This may be due to the fact that the nasal mucosa organoid medium has a certain selective effect on the cell type.
In FIG. 11E, immunofluorescent staining of P2 organoids indicates that a mixed population of cells from primary organoids can be maintained in a gel-embedded culture and passaging system.
In this example, two simple and reproducible methods were successfully developed to establish human nasal mucosal organoids. This 3D model successfully mimics the functional cell morphology of nasal epithelium in vitro. In addition, downstream detection methods for 3D organoids, such as fluorescent staining, RNA extraction, image capture, and biomarker analysis, are also established and optimized.
In the GelALI method, nasal epithelial progenitor cells proliferate and differentiate in a 3D environment. Differentiation took 21 days, which is the same as the transwell culture method. However, the luminal structure generated inside the organoid suggests that this differentiation process may be physiologically related to nasal cavity development. This provides a new tool for understanding nasal diseases from the developmental biology point of view. However, due to the high cost of Matrigel and the long incubation time of this method, it is not recommended to use this method for high-throughput drug screening.
In the U-bottom method, the maturation rate of nasal mucosal epithelial cells was faster (only 7 days), and the number of seeded cells was reduced by 100-fold compared to the transwell method (the number of seeded cells ranged from 100k to 1 k). The culture technology provides another economic and efficient method for modeling human nasal mucosa in vitro, and provides possibility for high-throughput drug screening and individual drug optimization of nasal mucosa epithelium.
But at present, the 3D nasal mucosa organoid also has some limiting factors: first, only two samples are used to optimize the method, and later the stability of the method still needs to be verified in a larger sample size. Second, image acquisition and analysis of organoids takes a relatively long time, requiring only a single sample of one hour. Therefore, it is essential to develop an automated method for image capture and 3D organoid analysis. Finally, due to the small number of cells, the quality of RNA isolated from each organoid is poor. Therefore, in order to avoid sample loss during RNA analysis, it is suggested to apply a one-step PCR method or a third generation sequencing method to the gene expression analysis of 3D nasal mucosa organoids.
The generation of organoids from nasal epithelial progenitor cells and the tracking of the 3D structures generated by their differentiation can provide a new idea for the basic research of nasal diseases. These 3D nasal organoids can also be used to predict drug response in an individual (e.g., the degree of sensitivity of different nasal polyps, sinusitis, or allergic rhinitis patients to nasal glucocorticoids, nasal antihistamines). The nasal organoid established by the method only needs 1/100 cell number of Transwell, and the culture method with simple operation and low cost can be suitable for nasal mucosa samples taken by a nasal brush, so that individual diagnosis and drug sensitivity test of chronic inflammatory diseases of the nasal mucosa become possible. In addition, the 3D model with low cost and high benefit can be used for drug research and development, can be applied to in vitro models before clinic as a drug, and can be used for high-throughput screening of new drugs for treating nasal diseases.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (12)

1. A method for culturing 3D organoids of nasal mucosa epithelial cells is characterized in that the method comprises culturing nasal mucosa epithelial cells by adopting a U-shaped bottom organoid culture method or a gelatin vapor liquid flat organoid culture method;
the U-shaped bottom organoid culture method specifically comprises the following steps:
resuspending the nasal mucosa epithelial cells in an organoid culture medium, inoculating the nasal mucosa epithelial cells in a cell culture vessel with a U-shaped bottom, and culturing after centrifuging;
the gelatin vapor-liquid level organoid culture method specifically comprises the following steps:
diluting the matrigel by using an organoid culture medium, coating the matrigel in a cell culture vessel, and incubating to obtain a culture medium A; resuspending the nasal mucosa epithelial cells in an organoid culture medium containing matrigel, and then inoculating the organoid culture medium in the culture medium A for culture;
the organoid culture medium comprises the following components in percentage by weight: DMEM/F12,1 to 5X; 5 to 20ng/mL of human epidermal growth factor; 1 to 10ng/mL of insulin; cholera toxin, 0.05 to 0.2nM; 0.1 to 1 mu g/mL of cortisol; 3,3', 5-triiodo-L-thyronine, 1 to 5nM; n-2 cell culture additive, 5 to 15 mu L/mL; antibiotic-antifungal solution, 50 to 200IU/mL; b27 cell culture additive, 1 to 5 ×; 50 to 200ng/mL of recombinant human noggin;
in the U-shaped bottom organoid culture method:
resuspending the nasal mucosa epithelial cells in organoid culture medium, and controlling the concentration to be 1 to 5 × 10 3 (ii) a The cell culture vessel with the U-shaped bottom is a 96-hole culture plate; centrifuging for 1 to 5 minutes under the centrifugal control condition of 1000 to 2000 rpm; when organoids grow to more than 200 μm, add the components on day 7 of cultureInducing differentiation by using a culture medium; the differentiation medium is a human bronchial epithelial cell gas-liquid interface medium, and the culture medium is 1 to 5 times;
in the gel vapor-liquid level organoid culture method:
the culture medium A is organoid culture medium containing 40% matrigel; the cell culture vessel is a 24-hole culture plate; the incubation is carried out for 10 to 30 minutes at the temperature of 35 to 40 ℃; resuspending the nasal mucosa epithelial cells in an organoid culture medium containing matrigel, wherein the content of the matrigel is controlled to be 1 to 10 percent; on day 7 of incubation the size of the organoids reaches 200-300 μm; mechanically separating the organoids by enzymolysis and repeated blowing, wherein the size of the organoids is 100 mu m; adding an organoid culture medium, centrifuging, resuspending in the organoid culture medium containing 50-70% matrigel, adding a differentiation culture medium to induce differentiation, subculturing the organoid by using a gel embedding method, diluting, then performing resuspension inoculation in a cell culture vessel, adding the differentiation culture medium, and completely covering the solidified matrigel droplets; the differentiation medium is a human bronchial epithelial cell gas-liquid interface medium, and the culture medium is 1 to 5 x.
2. The culture method according to claim 1, wherein the centrifugation is carried out at 1500rpm for 2 minutes in the U-bottomed organoid culture method.
3. The culture method according to claim 1, wherein the incubation is specifically at 37 ℃ for 20 minutes in the gel vapor-liquid planar organoid culture method.
4. The culture method according to claim 1, wherein in the gel-vapor-liquid planar organoid culture method, the step of resuspending nasal mucosal epithelial cells in an organoid culture medium containing matrigel is performed, and the content of matrigel is controlled to be 5%.
5. 3D organoids of nasal mucosal epithelial cells obtained by the culture method according to any one of claims 1 to 4.
6. Use of the 3D organoids of nasal mucosal epithelial cells according to claim 5 for in vitro screening and/or detection of drugs.
7. The use of claim 6, wherein the in vitro drug screening is an in vitro drug high throughput screening.
8. An in vitro assay platform comprising the nasal mucosal epithelial cell 3D organoid of claim 5.
9. A method of high-throughput drug screening in vitro, comprising: applying a drug to be tested to the 3D organoid of the nasal mucosal epithelial cells according to claim 5 and/or the in vitro detection platform according to claim 8, and carrying out detection analysis on the 3D organoid of the nasal mucosal epithelial cells and/or the in vitro detection platform;
the detection and analysis method comprises fluorescent staining, RNA extraction, image capture and biomarker analysis.
10. The in vitro high throughput drug screening method of claim 9, wherein in the fluorescent staining method, the reagents used for fluorescent staining are a washing solution, a blocking solution and an antibody dilution solution;
the washing liquid comprises the following components: PBS +0.3% TritonX-100;
the confining liquid comprises the following components: PBS +10% FBS +0.3% TritonX-100;
the antibody diluent comprises the following components: PBS +1% FBS +0.3% TritonX-100.
11. The method for in vitro high throughput drug screening of claim 9 wherein said RNA extraction method comprises lysis using TRIzol.
12. The method for in vitro high throughput drug screening according to claim 9, wherein in the image capturing and biomarker analysis method, a confocal Z-step imaging method is used for imaging processing.
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