CN111471643B - Universal culture medium and culture method for upper respiratory mucosa organoid - Google Patents
Universal culture medium and culture method for upper respiratory mucosa organoid Download PDFInfo
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Abstract
The invention provides a universal culture medium and a culture method for upper respiratory tract mucosa organoid, wherein the liquid culture medium comprises basic cell factor, specific cell factor, inhibitor, penicillin, streptomycin, sterile water and BEGM culture solution. The gel culture medium is added with Matrigel with the concentration of 3-6mg/ml on the basis of the liquid culture medium. The culture medium is used for culturing the upper respiratory tract mucosal tissue organoid, has wide application range, can culture tissues from a plurality of sample sources such as nose, mouth, throat and the like, obviously improves the culture effect, the differentiation of mucosal tissue cells and the growth speed of cell cilia, and is beneficial to the maintenance of the growth and the function of the upper respiratory tract mucosal tissue cells.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a culture medium and a culture method for general upper respiratory mucosa organoids.
Background
The upper respiratory tract mainly includes the organs of nose, pharynx, larynx, etc. The inner surface of the upper respiratory tract is distributed with secretion and cilia (except nostril, pharyngeal posterior wall and vocal cords mucosa), which can warm (or cool), moisten and purify the inhaled air and has protective effect on respiratory organs and human body. The upper respiratory tract has very important filtering and cleaning functions for human body, and the filtering and cleaning functions of the respiratory tract can block and eliminate particles, pathogens and other foreign matters entering the respiratory tract along with air, so that the air entering alveolus is almost clean and sterile, and the functions are mainly realized through the mucosa tissue of the upper respiratory tract.
The cells of the mucosal tissue of the upper respiratory tract usually have a long cylindrical compact structure, and a large number of cilia grow on the surface through which the airflow passes. The special structure can block pathogens such as bacteria, viruses and the like in the air, and a first layer of defense line is constructed for the immune system of the human body. Respiratory infections are the most common mode of infection by pathogens and are also the most common mode of infection that causes large-scale public health incidents. The chief culprit of the "new crown" epidemic is the 2019 novel coronavirus (2019-nCoV), which is transmitted mainly through respiratory tract infection. The research function and structure are helpful for people to know the pathogenesis of respiratory tract infection, thereby laying a solid foundation for the prevention, control and diagnosis and treatment of diseases. Because of the special structure of the upper respiratory tract mucosal tissue, the success rate of culture in the traditional classical in-vitro culture model is low, and the original structure and function are damaged or lost.
Organoids (Organoids) are organ-specific collections of cells derived from stem cells or precursor cells. Organoids cultured in vitro are highly similar to the corresponding organs in terms of cellular composition and tissue architecture, and possess corresponding functional characteristics. Unlike conventional cell culture in two-dimensional environment, organoid culture is a three-dimensional environment in which multiple cell populations contained in a particular tissue or organ are cultured, and the culture system is more similar to the in vivo microenvironment. Therefore, the compound has a huge application prospect in the aspects of basic research of various organ physiopathologies, precise medical treatment, drug screening and development, gene therapy, regenerative medicine and the like.
Although many other human tissues (such as liver, intestine, etc.) can be successfully cultured in vitro to form organoids under different culture conditions by using different methods, few studies and reports on the culture method of upper respiratory tract mucosa tissue organoids exist, and particularly, no report on specific experimental procedures, operation steps, culture conditions, namely culture medium formulas exists.
Disclosure of Invention
In view of the above, there is a need to provide a culture medium and a culture method for a universal upper respiratory mucosa organoid. The technical scheme of the invention is as follows:
in a first aspect, the present invention provides a universal liquid culture medium for upper respiratory mucosal organoids comprising a basal cytokine, a specific cytokine, an inhibitor, penicillin, streptomycin, a becm broth, and sterile water; wherein the basal cytokines comprise: 10-100ng/ml EGF, 20-500ng/ml Noggin, 20-500ng/ml R-spondin 1, 20-500ng/ml Wnt3 a; the specific cell factors include: 1-50ng/ml Wnt7a, 10-200ng/ml IL-6; the concentration of the penicillin is 100U/ml; the streptomycin concentration is 0.1 mg/ml; the concentrations of the components are based on the concentration of the components in the mixed solution of the BEGM culture solution and the sterile water.
Further, the preparation method of the liquid culture medium comprises the following steps: preparing the components of basic cell factor, specific cell factor, inhibitor, penicillin and streptomycin into mixed mother liquor by using sterile water, and then adding BEGM culture solution to obtain a liquid culture medium.
In a second aspect, the present invention provides a universal upper respiratory tract mucosal organoid gel medium comprising basic cytokines, specific cytokines, inhibitors, penicillin, streptomycin, Matrigel, becm broth, and sterile water, wherein the basic cytokines comprise: 10-100ng/ml EGF, 20-500ng/ml Noggin, 20-500ng/ml R-spondin 1, 20-500ng/ml Wnt3 a; the specific cytokines include: 1-50ng/ml Wnt7a, 10-200ng/ml IL-6; the concentration of the penicillin is 100U/ml; the streptomycin concentration is 0.1 mg/ml; the concentration of the Matrigel is 3-6 mg/ml; the concentrations of the above components are based on the concentrations in the mixed solution of BEGM culture solution and sterile water.
Further, the preparation method of the gel culture medium comprises the following steps: preparing the components of basic cell factors, specific cell factors, inhibitors, penicillin and streptomycin into mixed mother liquor by using sterile water according to the final concentration, adding BEGM culture solution, and mixing the mixed liquor with Matrigel at the temperature of 4 ℃ to obtain the gel culture medium.
Further, the inhibitor includes: 100-2000nM EW-7197-HCl (EW-7197Hydrochloride), 1-40 μ M SB 203580-HCl (SB 203580Hydrochloride), 2-50 μ M Y-27632dihydrochloride, the concentration of each component is based on the concentration in the mixed liquid of BEGM culture liquid and sterile water.
In a third aspect, the present invention provides a method for culturing a universal upper respiratory mucosa organoid, comprising the steps of:
1) pretreating an operation excision specimen or biopsy tissue from a fresh upper respiratory tract position to obtain a cell mass with the cell number of 3-50 cells, centrifuging, removing a supernatant, and precipitating the cell mass for later use;
2) forming a gel layer with the thickness of 1-2mm on the bottom surface of the gel culture medium in the transwell chamber;
3) resuspending the cell mass precipitate obtained in the step 1) in the gel culture medium, uniformly mixing, and adding cell liquid to a gel layer in a transwell chamber to form another cell gel layer with the thickness of 1-5 mm;
4) placing the transwell chamber of step 3) in a culture dish, adding the above liquid culture medium into the culture dish, wherein the liquid level of the liquid culture medium is not higher than the total height of the two gel layers, and then adding 5% CO at 37 deg.C2Culturing under the concentration;
5) replacing the liquid culture medium every 2-3 days, and culturing for 4-14 days to obtain upper respiratory tract tissue organoid.
The invention has the beneficial effects that: the culture medium contains the minimum components required by the culture of upper respiratory tract mucosa tissue organoid, has wide application range, and can culture tissues from a plurality of sample sources such as nose, oropharynx, larynx and the like. The culture medium of the invention has the following specific characteristics:
the component (i) does not require bovine serum albumin (FBS), the most common component in cell culture, and does not require the addition of other large protein molecules such as hormones. The components are simpler in composition, the cost is saved, and the cytotoxicity and inhibitors brought by FBS are reduced.
The basic culture medium commonly used for organoid culture is DMEM/F12, and the BEGM culture medium selected by the invention is particularly suitable for the growth of upper respiratory tract mucosal histiocyte, and the culture effect, the differentiation of mucosal histiocyte and the growth speed of cell cilia are obviously improved.
The specific cell factor added in the invention is beneficial to the growth and the function maintenance of the upper respiratory tract mucosa histiocyte.
Fourthly, all the added inhibitors are water-soluble inhibitors, and DMSO dissolution is not needed, so that the toxicity of DMSO on in-vitro cell culture is reduced while the effect is ensured.
Drawings
FIG. 1 is a schematic view showing the procedure of the culture method of the present invention.
FIG. 2 is an optical microscope photograph of pharyngeal mucosa tissue organoids cultured according to the present invention.
FIG. 3 is a histological structural view of a pharyngeal mucosa organoid in example 6 of the present invention.
FIG. 4 is a transmission electron microscope scan of nasal mucosa organoids according to example 8 of the present invention.
Detailed Description
The penicillin solid adopted in the embodiment of the invention is purchased from biological engineering (Shanghai) GmbH.
The streptomycin solid adopted in the embodiment of the invention is purchased from bio-engineering (Shanghai) GmbH.
EW-7197-HCl used in the examples of the present invention is available from MedChemexpress, Inc., USA.
SB 203580-HCl used in the examples of the present invention is available from MedChemExpress, USA.
Y-27632dihydrochloride used in the examples of the present invention is available from MedChemexpress, USA.
In the description of the present invention, it should be noted that those who do not specify specific conditions in the examples are performed according to the conventional conditions or conditions recommended by the manufacturers. The reagents or apparatus used are conventional products available from commercial sources, not indicated by the manufacturer.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The present embodiment provides a liquid culture medium for universal upper respiratory mucosal organoids, comprising basic cytokines, specific cytokines, inhibitors, penicillins, streptomycins, sterile water, and becm culture fluid, wherein the basic cytokines comprise: 50ng/ml EGF, 200ng/ml Noggin, 250ng/ml R-spondin 1, 300ng/ml Wnt3 a; the specific cytokines include: 20ng/ml Wnt7a, 100ng/ml IL-6; the inhibitor comprises: EW-7197-HCl at 500nM, SB 203580-HCl at 10. mu.M, Y-27632dihydrochloride at 20. mu.M; the concentration of the penicillin is 100U/ml; the streptomycin concentration is 0.1 mg/ml; the concentrations of the components are based on the concentration of the components in the mixed solution of the BEGM culture solution and the sterile water.
Example 2
The present embodiment provides a liquid culture medium for universal upper respiratory mucosal organoids, comprising basic cytokines, specific cytokines, inhibitors, penicillins, streptomycins, sterile water, and becm culture fluid, wherein the basic cytokines comprise: 20ng/ml EGF, 400ng/ml Noggin, 200ng/ml R-spondin 1, 100ng/ml Wnt3 a; the specific cytokines include: wnt7a at 10ng/ml, IL-6 at 50 ng/ml; the inhibitor comprises: 200nM EW-7197-HCl, 20. mu.M SB 203580-HCl, 40. mu.M Y-27632 dihydrochloride; the concentration of the penicillin is 100U/ml; the streptomycin concentration is 0.1 mg/ml; the concentrations of the above components are based on the concentrations in the mixed solution of BEGM culture solution and sterile water.
Example 3
This example provides a universal upper respiratory mucosal organoid gel medium comprising basic cytokines, specific cytokines, inhibitors, penicillins, streptomycins, Matrigel, sterile water, and becm broth, wherein the basic cytokines comprise: 50ng/ml EGF, 200ng/ml Noggin, 250ng/ml R-spondin 1, 300ng/ml Wnt3 a; the specific cytokines include: 20ng/ml Wnt7a, 100ng/ml IL-6; the inhibitor comprises: EW-7197-HCl at 500nM, SB 203580-HCl at 10. mu.M, Y-27632dihydrochloride at 20. mu.M; the concentration of the penicillin is 100U/ml; the streptomycin concentration is 0.1 mg/ml; the concentration of the Matrigel is 5 mg/ml; the concentrations of the above components are based on the concentrations in the mixed solution of BEGM culture solution and sterile water.
Example 4
This example provides a universal upper respiratory mucosal organoid gel medium comprising basic cytokines, specific cytokines, inhibitors, penicillins, streptomycins, Matrigel, sterile water, and becm broth, wherein the basic cytokines comprise: 20ng/ml EGF, 400ng/ml Noggin, 200ng/ml R-spondin 1, 100ng/ml Wnt3 a; the specific cytokines include: wnt7a at 10ng/ml, IL-6 at 50 ng/ml; the inhibitor comprises: 200nM EW-7197-HCl, 20. mu.M SB 203580-HCl, 40. mu.M Y-27632 dihydrochloride; the concentration of the penicillin is 100U/ml; the streptomycin concentration is 0.1 mg/ml; the concentration of the Matrigel is 4 mg/ml; the concentrations of the above components are based on the concentrations in the mixed solution of BEGM culture solution and sterile water.
Example 5
Pharyngeal mucosa organoid culture, the mode of culture is shown in figure 1.
The specific culture method is as follows:
(1) fresh pharyngeal tissues were stored in prepared BEGM solution containing 5% double antibody and sent to the laboratory for pretreatment within 12 hours.
(2) Sample washing: the tissue was transferred to a 15ml centrifuge tube and washed with 5ml of BEGM containing 5% double antibody for 30 seconds with shaking, the supernatant was removed and 5ml of BEGM containing 5% double antibody was added again for washing.
The impurities on the tissue surface were removed by repeated washing 3 times as described above.
(3) Sample shearing: in a biosafety cabinet, samples were transferred to 6cm petri dishes and the tissue was minced to 1-5mm in size by working on ice with sterilized surgical scissors3The shearing process should not exceed 10 minutes to avoid cell damage.
(4) First digestion of tissue: the minced tissue was transferred to a 15ml centrifuge tube, digested with shaking at 37 ℃ for 60 minutes after adding 2ml collagenase III and 1ml hyaluronidase. After the first digestion, 5ml of sterile physiological saline was added to terminate the digestion, and then the mixture was centrifuged at 1000rpm for 3min, and the supernatant was removed by retaining the precipitate.
(5) Second digestion of the tissue: adding 2ml of collagenase type III and 1ml of hyaluronidase into the precipitate in the step (4), resuspending and uniformly mixing, and digesting with shaking at 37 ℃ for 60 minutes. After the second digestion was completed, 5ml of sterile physiological saline was added to terminate the digestion.
(6) Cell filtration: and (4) filtering the digestive liquid in the step (5) by using a 100-micron filter screen to remove undigested large tissue blocks. The filtered cell fluid was centrifuged at 1000rpm for 5min, and the supernatant was carefully removed to obtain cell pellet for use.
(7) The gel medium of example 3 was allowed to form a 2mm gel layer on the bottom surface in the transwell chamber.
(8) Suspending 150. mu.l of the cell pellet obtained in step 6) in a gel medium of example 3, mixing the suspension and the pellet, and adding the cell suspension to the gel layer in the transwell chamber to form another 2mm cell gel layer;
(9) the transwell chamber of step 8) was placed in a petri dish, and then the liquid medium of example 1, which had a liquid level not higher than the total height of the two gel layers, was added to the petri dish, followed by 5% CO at 37 deg.C2Culturing under the concentration;
(10) the liquid medium of example 1 was replaced every 2 to 3 days and cultured for 8 days to obtain pharyngeal mucosa tissue organoids, and the structure under a light microscope is shown in FIG. 2.
Example 6
Morphological identification of pharyngeal mucosa organoid
The pharyngeal mucosa tissue organoids obtained in example 5 were subjected to paraffin-embedded section preparation. The embedded organoids were sectioned and then observed by HE staining.
1) Organoid collection and fixation: the mixture was put into a prepared fixing solution (4% formaldehyde fixation) and fixed for 2 hours. After completion of the fixation, the solution was centrifuged at 1200rpm for 5 minutes, and formalin-fixed solution was discarded.
2) Gradient dehydration: the fixed organoids were immersed in 85% alcohol, 95% alcohol and 100% alcohol in sequence for 30 minutes each.
3) Transparent wax dipping: adding xylene to immerse the organoids for 20 minutes, and repeating twice; paraffin wax was then added and the wax was dipped at 60 ℃ for 1.5 hours.
4) Embedding the section: the organoids were wrapped with an embedding mold and then sliced into 4-6 μm sections with a microtome and attached to anti-detachment slides.
5) Baking slices: placing the glass slide on a glass slide frame, placing in an oven, drying at 65 deg.C for 30min, and baking water and paraffin on the glass slide.
6) Dewaxing: dewaxing with xylene three times for 10 minutes each; then dipping and washing the fabric with 100 percent alcohol for three times, wherein each time lasts for 1 minute; finally, the mixture is soaked and washed for 1 minute by running water.
7) H & E staining: staining with hematoxylin for 8min, washing with water for 1min, decomposing with 1% hydrochloric acid alcohol for 1-2 s, washing with flowing water for 30min, staining with 1% eosin for 1-2min, and washing with flowing water for 1 min.
8) Fixing after dyeing: the solution was sequentially immersed in 95% alcohol and 100% alcohol, twice for each reagent, 2 minutes each time.
9) And (3) transparent and sealing: and (5) using dimethylbenzene for transparence for 2min, taking out and airing, and sealing by using neutral gum.
10) The morphological structure of the tissue observed under a normal light microscope is shown in FIG. 3.
Example 7
Nasal mucosa organoid culture in the manner shown in FIG. 1.
The specific culture method is as follows:
(1) fresh nasal tissue was stored in prepared BEGM solution containing 5% double antibody and sent to the laboratory for pretreatment within 12 hours.
(2) Sample washing: the tissue was transferred to a 15ml centrifuge tube and washed with 5ml of BEGM containing 5% double antibody for 30 seconds with shaking, the supernatant was removed and 5ml of BEGM containing 5% double antibody was added again for washing.
The impurities on the tissue surface were removed by repeated washing 3 times as described above.
(3) Sample shearing: in a biosafety cabinet, the samples were transferred to 6cm petri dishes, cartilage was removed with sterilized forceps, and tissue was minced to 1-5mm in size by operating sterilized surgical scissors on ice3The shearing process should not exceed 10 minutes to avoid cell damage.
(4) First digestion of tissue: the minced tissue was transferred to a 15ml centrifuge tube, digested with shaking at 37 ℃ for 60 minutes after adding 2ml collagenase III and 1ml hyaluronidase. After the first digestion, 5ml of sterile physiological saline was added to terminate the digestion, and then the mixture was centrifuged at 1000rpm for 3min, and the supernatant was removed by retaining the precipitate.
(5) Second digestion of the tissue: adding 2ml of collagenase type III and 1ml of hyaluronidase into the precipitate in the step (4), resuspending and uniformly mixing, and digesting with shaking at 37 ℃ for 60 minutes. After the second digestion, 5ml of sterile physiological saline was added to terminate the digestion, and then the mixture was centrifuged at 1000rpm for 5min, and the supernatant was removed by retaining the precipitate. Adding 2ml of erythrocyte lysate for resuspension, and adding 55ml of sterile normal saline to stop lysis after 5min of lysis at room temperature.
(6) Cell filtration: and (4) filtering the digestive liquid in the step (5) by using a 100-micron filter screen to remove undigested large tissue blocks. The filtered cell fluid was centrifuged at 1000rpm for 5min, and the supernatant was carefully removed to obtain cell pellet for use.
(7) The gel medium of example 4 was allowed to form a gel layer of about 1.5mm on the bottom surface in the transwell chamber.
(8) Suspending the pellet from step 6) in 150. mu.l of the gel medium of example 4, mixing the pellet and the pellet, and adding the cell suspension to the gel layer in the transwell chamber to form another 1mm cell gel layer;
(9) placing the transwell chamber of step 8) in a petri dish, then adding the liquid culture medium of example 2 into the petri dish, wherein the liquid level of the liquid culture medium is not higher than the total height of the two gel layers, and then culturing at 37 ℃ and at a concentration of 5% CO 2;
(10) the liquid medium of example 2 was changed every 2 to 3 days and cultured for 14 days to obtain a gel containing a nasal mucosal tissue organoid.
Example 8
Morphological identification of nasal mucosa organoid
The gel drops containing the nasal mucosal tissue organoids obtained in example 7 were cut to 1mm with scissors3The left and right pieces were collected in 15ml centrifuge tubes and centrifuged at 1000rpm for 5 minutes to remove the supernatant. 5ml of PBS solution was added thereto, the mixture was washed by mixing with the mixture upside down, and the supernatant was removed after centrifugation at 1000rpm for 5 minutes. Washing with PBS for 3 times according to the above method, and fixing with 2.5% glutaraldehyde solution for more than 4 hours. And scanning the fixed sample by a transmission electron microscope according to a using method of the transmission electron microscope to obtain the shape of the nasal mucosa organoid as shown in the attached figure 4. The nasal mucosa organoid tissue structure shown in the figure comprises a ciliated structure having an outer ring of 9 sets of two microtubules joined together, and an inner pair of central microtubules.
Comparative example 1
The liquid culture medium provided by the comparative example does not contain specific cytokines, and the rest is the same as the liquid culture medium provided by the example 1; the gel medium also contained no specific cytokine, as in example 3.
Pharyngeal mucosa cell organoid culture was performed according to the method of example 5 using the above-mentioned medium.
As a result, after culturing for 4 days in the step (10), growth arrest and gradual apoptosis of pharyngeal mucosa cells are found, and after 10 days, the cells are almost completely apoptotic. This suggests that these specific cytokines are beneficial for the growth and maintenance of upper respiratory mucosal tissue cell function.
Comparative example 2
The culture solution of BEGM in the liquid medium and the gel medium provided in this comparative example was changed to DMEM/F12 culture solution. The other examples were the same as example 1 and example 3, respectively.
Pharyngeal mucosa organoid culture was performed according to the method of example 5 using the above-mentioned medium.
As a result, the pharyngeal mucosa cells were found to grow slowly after culturing for 4 days in the step (10), and after collecting the cells after 8 days, the total number of the cells and the proportion of the living cells were significantly less than those in the example 5 by trypan blue staining. The organoid cells cultured in example 3 were found to be large in number and high in the proportion of viable cells, indicating that the BEGM medium is more suitable for growth of pharyngeal mucosa organoids than DMEM/F12 medium. In addition, example 5 was able to observe a significant cilia structure in the transmission electron micrograph after 14 days of culture, whereas the fibro-hair structure was not observed in comparative example 2. After the prolonged culture period, a small amount of cilia structure was observed in control example 2 after 21 days of culture. The above shows that BEGM culture medium is more beneficial to the differentiation of pharyngeal mucosa histiocyte and the growth of cell cilia than DMEM/F12 culture solution.
| Contrast item | Number of viable cells | Total number of cells | Proportion of viable cells |
| Example 5 | 3.5×107 | 3.8×107 | 92.11％ |
| Comparative example 2 | 4.2×106 | 9.7×106 | 43.30％ |
In conclusion, the culture medium has wide application range and can culture tissues from a plurality of sample sources such as the nose, the oropharynx, the throat and the like; the most common component bovine serum albumin (FBS) in cell culture does not need to be added into the components, and other large protein molecules such as hormone and the like do not need to be added, so that the cost is saved, and the cytotoxicity and inhibitors brought by the FBS are reduced. In addition, the culture medium and the culture method are suitable for the growth of the upper respiratory tract mucosal tissue cells, the culture effect, the differentiation of the mucosal tissue cells and the growth speed of cell cilia are obviously improved, and the growth and the function maintenance of the upper respiratory tract mucosal tissue cells are facilitated.
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 universal liquid culture medium for upper respiratory tract mucosa organoids is characterized in that: consists of basic cell factors, specific cell factors, inhibitors, penicillin, streptomycin, BEGM culture solution and sterile water; wherein the basic cytokine consists of: 10-100ng/ml EGF, 20-500ng/ml Noggin, 20-500ng/ml R-spondin 1, 20-500ng/ml Wnt3 a; the specific cytokine consists of: 1-50ng/ml Wnt7a, 10-200ng/ml IL-6; the concentration of the penicillin is 100U/ml; the streptomycin concentration is 0.1 mg/ml; the inhibitor comprises the following components: 100-2000nM EW-7197-HCl (EW-7197Hydrochloride), 1-40 μ M SB 203580-HCl (SB 203580Hydrochloride), 2-50 μ M Y-27632 dihydrochloride; the concentrations of the above components are based on the concentrations in the mixed solution of BEGM culture solution and sterile water.
2. The universal liquid culture medium for upper respiratory tract mucosa organoids according to claim 1, wherein: the preparation method of the liquid culture medium comprises the following steps: preparing the components of basic cell factor, specific cell factor, inhibitor, penicillin and streptomycin into mixed mother liquor by using sterile water, and then adding BEGM culture solution to obtain a liquid culture medium.
3. A gel culture medium for universal upper respiratory tract mucosa organoids is characterized in that: the composition of the basic cell factor comprises a basic cell factor, a specific cell factor, an inhibitor, penicillin, streptomycin, Matrigel, BEGM culture solution and sterile water, wherein the basic cell factor comprises the following components: 10-100ng/ml EGF, 20-500ng/ml Noggin, 20-500ng/ml R-spondin 1, 20-500ng/ml Wnt3 a; the specific cytokine consists of: 1-50ng/ml Wnt7a, 10-200ng/ml IL-6; the concentration of the penicillin is 100U/ml; the inhibitor comprises the following components: 100-2000nM EW-7197-HCl (EW-7197Hydrochloride), 1-40 μ M SB 203580-HCl (SB 203580Hydrochloride), 2-50 μ M Y-27632 dihydrochloride; the streptomycin concentration is 0.1 mg/ml; the concentration of the Matrigel is 3-6 mg/ml; the concentrations of the above components are based on the concentrations in the mixed solution of BEGM culture solution and sterile water.
4. The universal upper respiratory tract mucosa organoid gel culture medium according to claim 3, wherein: the preparation method of the gel culture medium comprises the following steps: preparing the components of basic cell factors, specific cell factors, inhibitors, penicillin and streptomycin into mixed mother liquor by using sterile water according to the final concentration, adding BEGM culture solution, and mixing the mixed liquor with Matrigel at the temperature of 4 ℃ to obtain the gel culture medium.
5. A culture method of upper respiratory mucosa organoids is characterized in that: the method comprises the following steps:
1) pretreating an operation excision specimen or biopsy tissue from a fresh upper respiratory tract position to obtain a cell mass with the cell number of 3-50 cells, centrifuging, removing a supernatant, and precipitating the cell mass for later use;
2) forming a gel layer of 1-2mm on the bottom surface of the inside of the transwell chamber by using the gel medium of claim 3 or 4;
3) resuspending the pellet of cells obtained in step 1) in the gel medium of claim 3 or 4, mixing, and adding the cell fluid to the gel layer in the transwell chamber to form another 1-5mm layer of cell gel;
4) placing the transwell chamber of step 3) in a petri dish, adding the liquid culture medium of claim 1 or 2 to the petri dish at a liquid level not higher than the total height of the two gel layers, and then adding 5% CO at 37 deg.C2Culturing under the concentration;
5) replacing the liquid medium according to claim 1 or 2 every 2 to 3 days, and culturing for 4 to 14 days to obtain the upper respiratory tissue organoid.
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| CN112430568B (en) * | 2020-11-25 | 2022-01-04 | 创芯国际生物科技(广州)有限公司 | Method for feeding epithelial source organoid by umbilical cord mesenchymal stem cells |
| CN112553142B (en) * | 2020-12-11 | 2023-02-07 | 山东大学 | 3D organ of nasal mucosa epithelial cells and culture method and application thereof |
| CN114149958B (en) * | 2021-09-28 | 2022-09-02 | 创芯国际生物科技(广州)有限公司 | Method for culturing nasal mucosa organoid by taking nasopharynx swab and application |
| CN115181730B (en) * | 2022-07-29 | 2023-05-16 | 创芯国际生物科技(广州)有限公司 | Gastric stromal tumor organoid culture medium and culture method |
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