CN114149958B - Method for culturing nasal mucosa organoid by taking nasopharynx swab and application - Google Patents
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Abstract
The invention provides a method for culturing nasal mucosa organoid by human nasal swab non-invasive sampling and application, the method for culturing nasal mucosa organoid is to collect nasal mucosa epithelial cells in the nasal swab after sampling, transfer the obtained nasal mucosa epithelial cells to matrigel mixed disease and culture to obtain the nasal mucosa organoid. The invention provides a method for establishing a nasal mucosa organoid sample library, which comprises the following steps: obtaining nasal mucosa organoids by the above method; subculturing nasal mucosa organoid; and (5) performing cryopreservation on the subcultured nasal mucosa organoids to build a library. The invention firstly utilizes the human nasal swab to successfully culture and obtain the nasal mucosa organoid by noninvasive sampling, the whole preparation process is simple and easy to implement, the cost is low, and the invention has good popularization and application values. In addition, the nasal mucosa organoid can grow rapidly and can be stably cultured for a long time in the process operation of the method, and the formed nasal mucosa organoid is in a cavity shape and can well keep the tissue characteristics of epithelial cells in vitro.
Description
Technical Field
The invention relates to the technical field of organoid culture, in particular to a method for cultivating nasal mucosa organoids by taking nasopharynx swabs and application thereof.
Background
The way in which viruses invade the human body, the mechanism of infection, effective detection methods, antiviral drugs, the reaction of neutralizing antibodies and the development of vaccines, all urgently require a disease model that approximates the human tissue and microenvironment. According to the "prevention and control scheme for novel coronavirus pneumonia" (sixth edition), suspected cases and aggregated cases of novel coronavirus pneumonia must be collected from acute respiratory specimens for nucleic acid detection.
The nasal cavity is the first site of respiratory tract virus invasion into the human body, and the viral load of the nasal mucosa is often much higher than that of the oropharynx and the lower respiratory tract in the early stage of virus infection. Meinhardt et al found that the olfactory mucosa was the site of highest neocoronal load in the nasal cavity, and that virus directly damaged the sertoli and basal cells causing dysosmia in infected individuals. The Hernes H compares the nasal swab with the pharyngeal swab for sampling and detecting the influenza viruses, and the comparison result of the sampling and detecting of 32 influenza virus patients shows that the detection rate of the nasal swab sampling sample is 54 times higher than that of the pharyngeal swab. This suggests that the nasal mucosa is also the first target organ for direct damage by the new coronavirus. The nasal mucosa organoid contains ciliated cells, secretory cells, basal cells and other cell components, not only has the histological characteristics of the typical pseudo-multilayer columnar ciliated epithelium, can form a glandular cavity structure with a secretory function, but also can observe a mucociliary transmission system consisting of cilia and a mucous blanket which swing in rhythm. More importantly, ciliated cells of nasal mucosa organoid, and ACE2 and TMPRSS2 are all expressed highly, and the experiment of new coronavirus infection is successful, so that the method is an ideal model for researching new coronavirus variation, infection and transmission, immune mechanism, antiviral drug and vaccine development. However, normal human nasal mucosa can be obtained only in a small amount during operations of malformation of nasal cavity structures, such as alveolar middle turbinate and the like, the source is severely limited, and ethical risks exist.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for cultivating nasal mucosa organoid by taking nasopharynx swab materials and application thereof. The technical scheme of the invention is as follows:
in a first aspect, the invention provides a method for culturing nasal mucosa organoids by human nasal swab non-invasive sampling, which comprises the steps of collecting nasal mucosa epithelial cells after the nasal swab samples, transferring the obtained nasal mucosa epithelial cells to matrigel, uniformly mixing, and then adding a nasal mucosa organoid culture medium for culturing to obtain the nasal mucosa organoids.
Further, the method specifically comprises:
step 1, inserting a nasal swab into a nasal cavity of a detected person for sampling, and then putting the sampled nasal swab into tissue preservation solution for preservation at a certain temperature;
step 2, collecting tissues on the stored sampled nasal swab, adding a DTT solution, uniformly mixing, oscillating at a constant temperature of 37 ℃, filtering by a 100-micron filter screen to obtain cell filtrate, and centrifuging to retain cell precipitate;
step 3, adding BEGM culture solution to the cell sediment to resuspend the cells, incubating at 37 ℃, removing adherent fibroblasts by adopting a differential adherence method, and collecting nasal mucosa epithelial cells;
step 4, resuspending nasal mucosa epithelial cells by adopting a proper amount of Advanced DMEM/F12 culture medium, adding matrigel, uniformly mixing, inoculating gel drops to a culture dish, inverting at a constant temperature of 37 ℃ for a period of time after the gel drops are solidified, adding nasal mucosa organoid to culture on the basis of 5% CO at a temperature of 37 DEG C 2 Culturing for 6-12 days under the condition, and replacing the culture medium every 2-3 days.
Preferably, the nasal swab is a nylon flocked nasal swab.
Optionally, the tissue preservation solution comprises the following components in terms of final concentration: 5-10% (wt/wt) FBS, 1000U/ml streptomycin, 50-100 μ g/ml gentamicin, 2-3 μ g/ml amphotericin B, and DMEM culture medium as solvent.
Furthermore, the BEGM culture solution also contains 50-200 IU/ml penicillin and 50-200 IU/ml streptomycin.
Further, the final concentration of the matrigel in the gel drop is 5-8 mg/ml.
Preferably, the nasal mucosa organoid medium comprises, in final concentration composition: 2% -5% of FBS; BMP4, 2-20 ng/ml; FGF10, 50-200 ng/ml; FGF7, 2-20 ng/ml; bFGF, 20-50 ng/ml; SB-431542, 0.2-1. mu.M; y-276322 HCl, 1-20 μ M; CHIR99021, 1-20. mu.M; all-trans retinoic acid, 10-100 nM; Wnt-7A, 20-200 ng/ml; HEPES, 5-20 mM; TGF-beta 1, 5-20 ng/ml; penicillin, 100U/ml; streptomycin, 0.1 mg/ml; the concentrations of the above components are based on the concentration of the components in the BEGM culture solution.
Optionally, the nasal mucosa organoid culture medium further comprises: nicotinamide, 1-20 mM; a83-01, 0.2-1. mu.M.
In a second aspect, the present invention provides a method for creating a nasal mucosa organoid sample library, comprising:
obtaining nasal mucosa organoids by the above method;
subculturing nasal mucosa organoid;
and (5) performing cryopreservation on the subcultured nasal mucosa organoids to build a library.
The invention firstly utilizes the human nasal swab to successfully culture and obtain the nasal mucosa organoid by noninvasive sampling, the whole preparation process is simple and easy to implement, the cost is low, and the invention has good popularization and application values. In addition, the nasal mucosa organoid can grow rapidly and can be stably cultured for a long time in the process operation of the method, and the formed nasal mucosa organoid is in a cavity shape and can well keep the tissue characteristics of epithelial cells in vitro. The invention also provides a method for establishing the nasal mucosa organoid sample library, and provides an ideal nasal mucosa organoid model for researching new coronavirus variation, infection and transmission, an immune mechanism, antiviral drugs and vaccine development.
Drawings
FIG. 1 is a structural morphology diagram of a nasal mucosa organoid obtained in example 1 of the present invention.
FIG. 2 is a structural morphology diagram of nasal mucosa organoids obtained in example 2 of the present invention.
FIG. 3 is a structural morphology diagram of nasal mucosa organoids obtained in example 3 of the present invention.
FIG. 4 is a structural morphology diagram of nasal mucosa organoids obtained in example 4 of the present invention.
FIG. 5 is a structural morphology of nasal mucosa organoids passaged according to example 5 of the present invention.
FIG. 6 is a structural morphology of nasal mucosa organoids passaged according to example 6 of the present invention.
FIG. 7 is a structural morphology of nasal mucosa organoids passaged according to example 7 of the present invention.
FIG. 8 is a structural morphology of nasal mucosa organoids passaged according to example 8 of the present invention.
FIG. 9 is a structural configuration diagram of the organoids of example 1 of the present invention after cryopreservation and resuscitation.
FIG. 10 is a structural configuration diagram of the organoids after cryopreservation and resuscitation according to example 2 of the present invention.
FIG. 11 is a structural configuration diagram of the organoids after cryopreservation and resuscitation according to example 3 of the present invention.
FIG. 12 is a structural configuration diagram of the organoids of example 4 of the present invention after cryopreservation and resuscitation.
FIG. 13 is a structural morphology diagram of the cell of comparative example 1 of the present invention.
FIG. 14 is a structural morphology diagram of a nasal mucosa organoid of comparative example 2 of the present invention.
Detailed Description
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention will be described in further detail with reference to the following figures and specific examples, which are given by way of illustration and not by way of limitation. Example 1
The embodiment provides a method for culturing nasal mucosa organoids by noninvasive sampling of a human nasal swab, which specifically comprises the following steps:
method for collecting nasal swab
(1) Disposable nasal sampling swab: the nylon flocking sampling swab is selected, and the sample collection time is 3-5 seconds, which is far faster than the collection time of 15-120 seconds of the cotton swab. The upright nylon fibers act like a soft brush, the capillary action between the nylon fibers enhances the loading of the aqueous swatch, and the sample is more easily eluted by concentrating on the surface of the swab. Thus, the nylon flocked nasopharyngeal swab is able to adsorb and release more cell samples.
(2) The nasal sampling swab is inserted into the nasopharynx of the subject to sample.
Secondly, storage, transportation and pretreatment of the nose swab
Putting the sampled swab into a sampling tube which is added with tissue preservation solution (DMEM culture medium containing 10% FBS, 1000U/ml penicillin, 50 mu g/ml gentamicin and 2.5 mu g/ml amphotericin B) in advance, screwing a sampling tube cover, sticking a sealing film, placing the sampling tube cover into a specimen transport box which is provided with a plurality of ice bags in advance, and transporting the sampling tube cover to a laboratory. Gently wiping mucus on the surface of a tissue by using a sterile medical cotton swab in a biological safety cabinet, holding a swab handle to repeatedly extrude a swab head on the inner tube wall, repeatedly blowing a specimen to collect epithelial cells as much as possible, transferring the tissue to a centrifuge tube, adding an equivalent 10mMol/LDTT solution, uniformly mixing on an oscillator, oscillating for 10min at a constant temperature of 37 ℃ at a rotating speed of 50r/min, and removing the mucus as much as possible. Filtering with 100 μm filter screen to obtain cell filtrate, centrifuging at 1500r for 3min, and discarding supernatant. The cells were resuspended by adding prepared BEGM (100 IU/ml penicillin, 100 IU/ml streptomycin) culture medium, transferred to a petri dish, and incubated at 37 ℃ for 1 h. After removing adherent fibroblasts by adopting a differential adherence method, the nasal mucosa epithelial cells can be collected, and the total cell number is determined by utilizing a cell counter.
Culture of nasal swab sampling organoid
The cell pellet was resuspended in the appropriate amount of Advanced DMEM/F12 medium and mixed with overnight dissolved matrigel (final concentration of 5.5 mg/ml) all the way through the low temperature ice, avoiding air bubbles. The gel drops were seeded into 60mm petri dishes using a 200. mu.l pipette (200. mu.l pipette tips pre-cooled at 4 ℃) and each gel drop was approximately 35. mu.l in size. Standing the culture dish for 2min, observing whether the gel drops flow, if not, placing the culture dish into a constant temperature incubator at 37 ℃, and inverting for 45 min. Approximately 4ml of preheated nasal mucosa organoid medium was added. Observing under a mirror, taking a picture, putting into a constant-temperature incubator at 37 ℃, replacing the culture medium every 2-3 days, and culturing for 8 days.
The organoid culture medium used comprises the following components: 3% of FBS; BMP4, 10 ng/ml; FGF10, 100 ng/ml; FGF7, 12 ng/ml; bFGF, 35 ng/ml; SB-431542, 0.5. mu.M; y-276322 HCl, 10. mu.M; CHIR99021, 10 μ M; all-trans retinoic acid, 60 nM; Wnt-7A, 120 ng/ml; HEPES, 15 mM; TGF-beta 1, 10 ng/ml; penicillin, 100U/ml; streptomycin, 0.1 mg/ml; nicotinamide, 10 mM; a83-01, 0.5. mu.M; the concentrations of the above components are based on the concentration of the components in the BEGM culture solution.
The organoids obtained in this example are shown in fig. 1, and the cellular structure is clear and complete, and is in the shape of a round cavity with uniform morphology.
Example 2
The present embodiment provides a method for non-invasively sampling and culturing nasal mucosa organoids by using a human nasal swab, which is different from the specific operation method of embodiment 1 in that: the organoid culture medium comprises the following components: 5% of FBS; BMP4, 15 ng/ml; FGF10, 200 ng/ml; FGF7, 2 ng/ml; bFGF, 25 ng/ml; SB-431542, 1. mu.M; y-276322 HCl, 20. mu.M; CHIR99021, 3 μ M; all-trans retinoic acid, 20 nM; Wnt-7A, 200 ng/ml; HEPES, 5 mM; TGF-beta 1, 20 ng/ml; penicillin, 100U/ml; streptomycin, 0.1 mg/ml; the concentrations of the above components are based on the concentration of the components in the BEGM culture solution.
The organoids obtained in this example are shown in fig. 2, with clear and complete cell structure and uniform morphology.
Example 3
The present embodiment provides a method for non-invasively sampling and culturing nasal mucosa organoids by using a human nasal swab, and the specific operation method is different from that of embodiment 2 in that: the organoid culture medium comprises the following components: FBS, 2%; BMP4, 5 ng/ml; FGF10, 50 ng/ml; FGF7, 20 ng/ml; bFGF, 40 ng/ml; SB-431542, 0.2. mu.M; y-276322 HCl, 1. mu.M; CHIR99021, 20 μ M; all-trans retinoic acid, 100 nM; Wnt-7A, 20 ng/ml; HEPES, 20 mM; TGF-beta 1, 5 ng/ml; penicillin, 100U/ml; streptomycin, 0.1 mg/ml; the concentrations of the above components are based on the concentration of the components in the BEGM culture solution.
The organoids obtained in this example are shown in fig. 3, with clear and complete cell structure and uniform morphology.
Example 4
The present embodiment provides a method for non-invasively sampling and culturing nasal mucosa organoids by using a human nasal swab, and the specific operation method is different from that of embodiment 2 in that: the organoid culture medium comprises the following components: FBS, 4%; BMP4, 2 ng/ml; FGF10, 200 ng/ml; FGF7, 15 ng/ml; bFGF, 50 ng/ml; SB-431542, 0.5. mu.M; y-276322 HCl, 5. mu.M; CHIR99021, 18 μ M; all-trans retinoic acid, 80 nM; Wnt-7A, 30 ng/ml; HEPES, 10 mM; TGF-beta 1, 8 ng/ml; penicillin, 100U/ml; streptomycin, 0.1 mg/ml; the concentrations of the above components are based on the concentration of the components in the BEGM culture solution.
The organoids obtained in this example are shown in fig. 4, with clear and complete cell structure and uniform morphology.
Example 5
Organoid passaging of example 1
Sucking out the culture medium from the culture dish, adding HBSS to slightly rinse the gel drops, slightly blowing off the gel drops by using TrypLE, and placing the gel drops in CO 2 Digesting in the culture box for 3-5min, adding HBSS to terminate digestion when the organoid is digested to a uniform cell mass with a diameter of about 20-30 μm, centrifuging for 3min at 1500 rpm, discarding supernatant to obtain cell precipitate, adding 100 μ l of culture medium for resuspension, and adding 120 μ l of Matrigel to re-inoculate and culture the organoid.
The obtained organoid has better activity, most of the organoids are in a round cavity shape, the structural form diagram of a single organoid is shown in figure 5, and clear and complete cell structure and uniform form can be clearly observed.
Example 6
Organoid passaging of example 2
The operation is the same as that in example 5, the obtained organoid has better activity, most of the organoid is in a round cavity shape, and the structural morphology of a single organoid is shown in figure 6, so that clear and complete cell structure and uniform morphology can be clearly observed.
Example 7
Organoid passaging of example 3
The operation is the same as that of example 5, the obtained organoid has better activity, most of the organoid is in a round cavity shape, and the structural morphology graph of a single organoid is shown in figure 7, so that the clear and complete cell structure and uniform morphology can be clearly observed.
Example 8
Organoid passaging of example 4
The operation is the same as that in example 5, the obtained organoid has better activity, most of the organoid is in a round cavity shape, and the structural morphology graph of a single organoid is shown in figure 8, so that the clear and complete cell structure and uniform morphology can be clearly observed.
Example 9
Example 5 organoid cryopreservation and Resuscitation obtained
Digesting the subcultured organoids to a uniform cell mass with a diameter of about 20-30 μm, adding 5ml HBSS to terminate digestion, and transferring the cell mass to a 15ml centrifuge tube; 1500 rpm, 3min, centrifuging, discarding the supernatant, collecting cell sediment, resuspending the cell sediment with 1ml of a cryopreservation solution (FBS: DMSO =9: 1), placing the cell sediment in a cryopreservation tube, marking relevant information on the tube wall, performing programmed cooling, and storing in liquid nitrogen for a long time. Can establish a nasal mucosa organoid sample library, and provides an ideal nasal mucosa organoid model for researching new coronavirus variation, infection and transmission, an immune mechanism, antiviral drugs and vaccine development.
And (3) quickly dissolving the organoids to be recovered at 37 ℃, centrifuging to obtain the organoids, uniformly mixing the organoids with matrigel again, inoculating the organoids on a culture plate, and observing the growth state of the organoids.
The organoids obtained after 7 days of culture are mostly in the form of thin-walled vacuoles, and have good activity, large diameter, regular shape and large number as shown in fig. 9.
Example 10
Example 6 organoid cryopreservation and Resuscitation obtained
The same operation as in example 9 was performed to obtain organoids having better organoid activity, mostly in the form of round cavities or solid spheres, and more in number and regular in shape, as shown in fig. 10.
Example 11
Example 7 organoid cryopreservation and Resuscitation obtained
The operation was performed as in example 9, and the obtained organoids were mostly in the form of thin-walled vacuoles, with better activity, larger diameter and more regular morphology, as shown in fig. 11.
Example 12
Example 8 organoid cryopreservation and Resuscitation obtained
The same procedure as in example 9 was carried out, and the obtained organoids had better organoid activity, mostly round cavities, larger number and more regular shapes, as shown in fig. 12.
Comparative example 1
This comparative example provides a method of tissue culture of nasal mucosal organoids obtained by surgical resection, comprising the specific operations of:
first, preservation, transportation and pretreatment of surgically resected nasal polyp tissue
The nasal polyp tissue excised by the operation is preserved by DMEM, transported to a laboratory at 2-8 ℃ by using an ice bag, and then cut into pieces of about 1 cubic millimeter by using surgical scissors. The minced tissue was collected into a centrifuge tube, 5ml of collagenase type IV was added, and digestion was carried out at 37 ℃ for 30 minutes. The digestion was stopped by adding DMEM, and the cells were collected by centrifugation.
Second, tissue organoid culture
With the conventional commercially available complete medium (DMEM +10% FBS), otherwise as in example 1, nasal mucosal cells grew slowly and adhered easily to the bottom of the culture dish during the culture, and similar to the general cell culture results, no spherical-structured, multi-cellular organoid structure could be formed, as shown in fig. 13, where (a) and (b) are graphs of 5 days and 8 days of culture, respectively.
The process of comparative example 1 is the research direction generally performed by researchers at present, the nasal mucosa organoid is cultured by the tissue excised by the operation, on one hand, the operation mode can only obtain the cultured tissue when the operation is performed, the source is very limited, the ethical risk exists, on the other hand, the nasal mucosa organoid is difficult to obtain by the conventional general culture medium, and therefore, a new way for developing the nasal mucosa organoid is required to be found.
Comparative example 2
This comparative example provides another method of tissue culture of nasal mucosal organoids obtained by surgical resection, differing from comparative example 1 in that: the nasal mucosa organoid culture medium is adopted for culture. As shown in fig. 14, organoids were successfully obtained with clear and intact cellular structure and uniform morphology. Obviously, the nasal mucosa organoid culture medium of the present invention can also be applied to the culture of nasal mucosa organoids of tissues removed by nasal surgery.
In summary, on one hand, compared with the source of the surgical tissue sample, the nasal swab of the invention has relatively simple operation, can stay in the nose for a long time during sampling, and can collect the sample without anesthesia as long as the patient has good tolerance. Compared with respiratory tract samples from other surgical tissue sources, the nasal swab has the advantages that the collection is noninvasive and does not relate to ethical problems, the sample sources are rich, and the problem of human tissue sources which are the biggest bottleneck in organoid culture is solved. On the other hand, the invention firstly utilizes the noninvasive sampling of the human nasal swab to successfully culture and obtain the nasal mucosa organoid, and can stably culture for a long time, and the formed nasal mucosa organoid is in a cavity shape, and can well keep the tissue characteristics of epithelial cells in vitro. Moreover, the invention also provides a method for establishing the nasal mucosa organoid sample library, and provides an ideal nasal mucosa organoid model for researching new coronavirus variation, infection and transmission, an immune mechanism, antiviral drugs and vaccine development.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. A method for cultivating nasal mucosa organoid by taking nasopharyngeal swab material is characterized in that: the method specifically comprises the following steps:
step 1, inserting a nylon flocked nose swab into a nasal cavity of a detected person for sampling, and then putting the sampled nose swab into tissue preservation solution for preservation at a certain temperature; the tissue preservation solution comprises the following components in final concentration: 5-10% (wt/wt) FBS, 1000U/ml streptomycin, 50-100 μ g/ml gentamycin, 2-3 μ g/ml amphotericin B, and DMEM culture medium as solvent;
step 2, collecting tissues on the stored sampled nasal swab, adding a DTT solution, uniformly mixing, oscillating at a constant temperature of 37 ℃, filtering by a 100-micron filter screen to obtain cell filtrate, and centrifuging to retain cell precipitate;
step 3, adding BEGM culture solution to the cell sediment to resuspend the cells, incubating at 37 ℃, removing adherent fibroblasts by adopting a differential adherence method, and collecting nasal mucosa epithelial cells;
step 4, resuspension with appropriate amount of Advanced DMEM/F12 mediumAdding matrigel into nasal mucosa epithelial cells, mixing, inoculating gel drop into culture dish, solidifying gel drop, inverting at 37 deg.C for a period of time, adding nasal mucosa organoid, culturing at 37 deg.C and 5% CO 2 Culturing for 6-12 days under the condition, and replacing the culture medium every 2-3 days; the nasal mucosa organoid culture medium comprises the following components according to final concentration: 2% -5% of FBS; BMP4, 2-20 ng/ml; FGF10, 50-200 ng/ml; FGF7, 2-20 ng/ml; bFGF, 20-50 ng/ml; SB-431542, 0.2-1. mu.M; y-276322 HCl, 1-20 μ M; CHIR99021, 1-20. mu.M; all-trans retinoic acid, 10-100 nM; Wnt-7A, 20-200 ng/ml; HEPES, 5-20 mM; TGF-beta 1, 5-20 ng/ml; penicillin, 100U/ml; streptomycin, 0.1 mg/ml; the above components were dissolved in BEGM culture medium.
2. The method for cultivating nasal mucosa organoid by using nasopharyngeal swab as material as recited in claim 1, wherein: the BEGM culture solution in the step 3 also contains 50-200 IU/ml penicillin and 50-200 IU/ml streptomycin.
3. The method for cultivating nasal mucosa organoid by using nasopharyngeal swab as material as recited in claim 1, wherein: the final concentration of the matrigel in the gel drops is 5-8 mg/ml.
4. The method for cultivating nasal mucosa organoid by using nasopharyngeal swab as material as recited in claim 1, wherein: the nasal mucosa organoid medium was replaced with a medium consisting of the following final concentrations: 2% -5% of FBS; BMP4, 2-20 ng/ml; FGF10, 50-200 ng/ml; FGF7, 2-20 ng/ml; bFGF, 20-50 ng/ml; SB-431542, 0.2-1 μ M; y-276322 HCl, 1-20 μ M; CHIR99021, 1-20. mu.M; all-trans retinoic acid, 10-100 nM; Wnt-7A, 20-200 ng/ml; HEPES, 5-20 mM; TGF-beta 1, 5-20 ng/ml; penicillin, 100U/ml; streptomycin, 0.1 mg/ml; nicotinamide, 1-20 mM; a83-01, 0.2-1 μ M; the above components were dissolved in BEGM culture medium.
5. A method for establishing a nasal mucosa organoid sample bank is characterized by comprising the following steps: the method comprises the following steps:
obtaining a nasal mucosal organoid using the method of any one of claims 1 to 4;
subculturing nasal mucosa organoids;
and (5) performing cryopreservation on the subcultured nasal mucosa organoids to build a library.
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| CN104877964A (en) * | 2015-04-24 | 2015-09-02 | 赵振民 | In vitro construction method for salivary glands organs and acinus |
| WO2019027834A1 (en) * | 2017-07-31 | 2019-02-07 | The Regents Of The University Of Michigan | Compositions and method for establishing organoid cultures from cryogenically preserved tissue |
| CN111117946B (en) * | 2020-01-08 | 2022-03-22 | 南方医科大学南方医院 | Nasal mucosa organoid culture medium and culture method |
| CN111471643B (en) * | 2020-04-09 | 2020-12-29 | 创芯国际生物科技(广州)有限公司 | Universal culture medium and culture method for upper respiratory mucosa organoid |
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