CN111117946B - Nasal mucosa organoid culture medium and culture method - Google Patents
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Abstract
The invention discloses a culture medium and a culture method for nasal mucosa tissues, wherein epithelial and mesenchymal/matrix components are integrated into an organoid culture system based on a gas-liquid interface method for the first time, and an organoid which is composed of a plurality of cells including cilium cells, goblet cells, rod cells, basal cells and the like and is close to the structure and the function of an in-vivo mucosa can be obtained by inducing the proliferation and the differentiation of adult stem cells in fresh nasal mucosa epithelial tissues, so that the organoid becomes the nasal mucosa in-vitro model with the proliferation and multi-lineage differentiation capability.
Description
Technical Field
The invention relates to the field of organoid culture, further relates to the field of nasal mucosa organoid culture, and particularly relates to a nasal mucosa organoid culture medium and a culture method.
Background
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. Organoid culture is a major innovation over conventional cell biology techniques. 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.
Various nasal sinus diseases are closely related to the functions of nasal mucosa, and the research on repairing damage of mucosa, stem cells, cilium functions and the like can be developed by taking in-vitro culture of nasal mucosa epithelial cells as a disease model. The literature reports that bronchial and alveolar epithelial organoids are cultured by using a respiratory tract mucosa 3D culture system to induce differentiation and proliferation of adult stem cells in lower respiratory tract epithelium to form a 3D epithelial cell mass, but the respiratory tract mucosa organoids grow in a full-liquid environment, have a large difference with the physicochemical environment of the growth of a respiratory tract mucosa gas-liquid interface in a human body, and cannot form a complete pseudo-multilayer columnar ciliated epithelial structure. In addition, lower respiratory mucosal organoids are also difficult to apply to nasal mucosal studies due to differences in tissue characteristics. Some laboratories adopt a two-dimensional (2D) gas-liquid interface culture method to establish a nasal mucosa epithelial in-vitro model, but only can culture a single layer of nasal mucosa epithelial cells in vitro, and also lack differentiation and tissue structure and cannot simulate the tissue morphology and physiological functions of human nasal mucosa. Therefore, a brand-new gas-liquid interface nasal mucosa 3D organoid culture system is proposed.
The nasal mucosa in-vitro model has important significance for researching the cell tissue structure and function of the nasal mucosa and the nasal sinus diseases. Although various human tissues can be successfully cultured in vitro by different methods and under different culture conditions, the research and report on the culture method of nasal mucosa tissue organoid based on gas-liquid interface method are not available at present, and particularly, no attempt and report on specific test flow, operation steps, culture conditions, namely culture medium formula are available.
Disclosure of Invention
The invention aims to provide a culture medium and a culture method for nasal mucosa organoids. The method has good culture operability and repeatability, and the formula price of the culture medium is moderate.
The technical scheme adopted by the invention is as follows:
a culture medium for nasal mucosa organoid comprises the following components: b27 without Vit-A, N-acetyl cysteine, EGF, Noggin, R-spondin1, Wnt3a, A83-01, Nicotinamide, Y-27632, Glutamax, Gastrin, SB202190, penicillin streptomycin mixed solution, Wnt7a, IL-6 and HEPES.
Further, the components and contents of the culture medium are as follows: b27, 1-5X without Vit-A; n-acetyl cysteine, 0.2-5 mM; EGF, 10-250 ng/ml; noggin, 20-500 ng/ml; r-spondin1, 50-1000 ng/ml; wnt3a, 50-1000 ng/ml; a83-01, 100-2500 nM; nicotinamide, 2-50 mM; y-27632, 2-50 μ M; glutamax, 1-5X; gastrin, 1-25 nM; SB202190, 2-50. mu.M; penicillin streptomycin mixed liquor, 1-5X; wnt7a, 2-50 ng/ml; IL-6, 10-250 ng/ml; HEPES, 0.2-5 mM.
Further, the components and contents of the culture medium are as follows: vit-a free B27, 1X; n-acetyl cysteine, 1 mM; EGF, 50 ng/ml; noggin, 100 ng/ml; r-spondin1, 250 ng/ml; wnt3a, 250 ng/ml; a83-01, 500 nM; nicotinamide, 10 mM; y-27632, 10. mu.M; glutamax, 1X; gastrin, 5 nM; SB202190, 10. mu.M; penicillin streptomycin mixed solution, 1X; wnt7a, 10 ng/ml; IL-6, 50 ng/ml; HEPES, 1 mM.
The invention also discloses a nasal mucosa organoid culture method, which comprises the following specific steps: pretreating nasal mucosa tissue from different parts of turbinate, nasal septum, nasal bottom, olfactory region, paranasal sinus, nasopharynx, etc. to obtain cell precipitate, adding the cell precipitate into transwell small chamber containing mixed collagen solution, and coagulating into gel; the culture medium was added to a transwell cuvette and incubated at constant temperature.
Further, the pretreatment step includes washing, chopping, digesting, and cracking steps.
Further, the pretreatment step specifically comprises: washing nasal mucosa tissue with 2% double antibody-containing Hanks solution, and cutting the tissue; adding digestive enzyme I for digestion, centrifuging to remove supernatant, adding digestive enzyme II for digestion, centrifuging to remove supernatant, blowing heavy suspension digested tissue precipitate with Hanks liquid, filtering to obtain filtrate containing nasal mucosa single cells, centrifuging to remove supernatant, adding erythrocyte lysate for lysis, centrifuging to remove supernatant to obtain cell precipitate.
Further, digestive enzymes I include collagene type III, hyaluronidase, Insulin and EGF; digestive enzymes II include TrypLE Express and DNaseI enzymes.
Further, the preparation method of the mixed collagen solution is as follows: A. collagen matrix (rat tail type I collagen), b.1x concentrated sterile medium (DMEM), c.advanced DMEM/F12, d.sterile NaOH solution (1 mol/L); first 100. mu.l B and 1ml C were mixed, then 875. mu. lA was added, and finally 15. mu.l D was added to obtain a mixed collagen solution.
Further, the method also comprises the following culture medium replacement steps: the medium is changed every 2-3 days, the changed medium being the medium according to any of claims 1-3, preferably a conditioned medium.
Further, the method also comprises a passage step: the mixed collagen containing cells in the inner dish was transferred to a 15ml centrifuge tube and dissociated for 15-20 minutes at 37 ℃ with the addition of 100Unit/ml collagenase IV, and organoids were passaged 1:2 every 30 days.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention integrates epithelial and mesenchymal/matrix components into an organoid culture system based on a gas-liquid interface method for the first time, and can obtain an organoid which is composed of a plurality of cells including cilium cells, goblet cells, rod cells, basal cells and the like and is close to the structure and the function of the in-vivo mucosa by inducing the proliferation and the differentiation of adult stem cells in the epithelial tissue of fresh nasal mucosa so as to become the in-vitro model of the nasal mucosa with the proliferation and the multi-lineage differentiation capability.
2. The system can continuously proliferate and differentiate primary nasal mucosa epithelium for more than 30 days, which shows that the ecological niche of stem cells is successfully copied in microenvironment, and the system can be used for ideal research models of mucosa stem cells, cilium function, secretion function, nasal tumors, hereditary, allergic diseases and infectious diseases.
3. The organoid culture and identification method of nasal mucosa tissues has the advantages of simple and convenient operation process, high success rate, high reliability of identification results, stable technology and high repeatability. The experimental cost is lower than that of the prior art.
Drawings
FIG. 1 is a schematic view of a gas-liquid interface culture method
FIG. 2 nasal mucosa organoids in matrigel gel drop culture
FIG. 3 is HE staining photograph of nasal mucosa organoid and tissue section cultured by gas-liquid interface method
FIG. 4 scanning electron micrograph of cilia of nasal mucosa organoid cultured by gas-liquid interface method
Detailed Description
The invention is further described with reference to the accompanying drawings, which are not intended to be limiting in any way, and any variations based on the teachings of the invention are intended to fall within the scope of the invention.
Description of materials:
DMEM, purchased from GIBCO, was stored at 4 ℃ and was a medium containing various amino acids and glucose, which was developed on the basis of MEM culture. Compared with MEM, the dosage of each component is increased, and the components are divided into a high-sugar type (lower than 4500mg/L) and a low-sugar type (lower than 1000 mg/L). The high-sugar type is favorable for the growth of cells anchored at one position, and is suitable for tumor cells which grow fast and are difficult to attach, and the like. The culture medium is widely applied to vaccine production and cell culture and single cell culture of various primary virus host cells. Normal ovarian tissue was cryopreserved using DMEM4 for short-term transport.
Advanced DMEM/F12: purchased from GIBCO, stored at 4 ℃, modified DMEM/F-12 (Dulbecco's modified Eagle's Medium/Ham's F-12) is a widely used serum-free formulated basal medium. Compared with the classical DMEM/F-12, the serum addition amount can be reduced by 50-90%, and the growth rate or the form is not changed. Unlike other media, the following components were added to minimize serum: ethanolamine, glutathione, ascorbic acid, insulin, transferrin, for cell culture
Bovine serum albumin rich in lipids, and trace elements sodium selenite, ammonium metavanadate, copper sulfate and manganese chloride. The modified DMEM/F-12 was also supplemented with 4mM L-glutamine or GlutaMAXTM supplements.
Rat tail type I collagen: purchased from Corning, stored at 2-8 ℃ and prepared aseptically by the Birkedal-Hansen method to a purity of over 95% and soluble in 0.006mol/L acetic acid. Can be used as a thin layer on a tissue culture surface to enhance cell attachment and proliferation, or as a gel to promote expression of cell morphology and function.
NaOH: purchased from sigma, inc, for the preparation of mixed collagen solutions.
B27 (Vit-A free): supplement, available from GIBCO, B27, is a serum-free additive for growth and maintenance of short-term or long-term activity of hippocampal and other Central Nervous System (CNS) neurons. Without dimensionBiotin A type
The additives being tailored
The additive, vitamin a (retinol), can be converted to retinoic acid, which can induce differentiation of stem cells into neural cells. Formulations without vitamin a are ideal for stem cell culture.
WNT3 a: WNT signaling pathway agonists, available from Peprotech corporation.
N-acetyl cysteine, available from SIGMA, N-acetylcysteine.
EGF: epidermal growth factor from R & D.
Noggin: cell growth protein fraction purchased from Peprotech corporation.
R-mapping 1 Wnt signal pathway activator, available from R & D Systems, Inc.
A83-01: purchased from Tocris Bioscience.
Nicotinamide: from SIGMA, niacinamide.
Y-27632 dihydrochloride: ROCK specific pathway blockers purchased from Abmole Bioscience.
WNT7 a: activation of the wnt7a classical pathway, purchased from Peprotech, potentiates airway epithelial cell proliferation.
IL-6: purchased from Peprotech corporation, promotes ciliated cell differentiation.
Glutamax: glutamine, available from GIBCO.
SB 202190: small molecule inhibitors purchased from Sigma-Aldrich.
HEPES (high efficiency particulate air): purchased from invitrogen, is a weak acid that primarily serves to prevent rapid changes in the pH of the medium.
Penicillin-streptomycin 100X solution: purchased from Gibco, and primarily used to prevent bacterial contamination.
Collagen type IV Collagenase: purchased from Stem Cell corporation, for dissolving type I collagen.
Example 1
A nasal mucosa organoid culture medium comprises the following components by weight: vit-a free B27, 1X; n-acetyl cysteine, 0.2 mM; EGF, 10 ng/ml; noggin, 20 ng/ml; r-spondin1, 50 ng/ml; wnt3a, 50 ng/ml; a83-01, 100 nM; nicotinamide, 2 mM; y-27632, 2. mu.M; glutamax, 1X; gastrin, 1 nM; SB202190, 2. mu.M; penicillin streptomycin mixed solution, 1X; wnt7a, 2 ng/ml; IL-6, 10 ng/ml; HEPES, 0.2 mM.
Example 2
A nasal mucosa organoid culture medium comprises the following components by weight: vit-a free B27, 5X; n-acetylcysteine, 5 mM; EGF, 250 ng/ml; noggin, 500 ng/ml; r-spondin1, 1000 ng/ml; wnt3a, 1000 ng/ml; a83-01, 2500 nM; nicotinamide, 50 mM; y-27632, 50. mu.M; glutamax, 5X; gastin, 25 nM; SB202190, 50. mu.M; penicillin streptomycin mixed solution, 5X; wnt7a, 50 ng/ml; IL-6, 250 ng/ml; HEPES, 5 mM.
Example 3
A nasal mucosa organoid culture medium comprises the following components by weight: vit-a free B27, 1X; n-acetyl cysteine, 1 mM; EGF, 50 ng/ml; noggin, 100 ng/ml; r-spondin1, 250 ng/ml; wnt3a, 250 ng/ml; a83-01, 500 nM; nicotinamide, 10 mM; y-27632, 10. mu.M; glutamax, 1X; gastrin, 5 nM; SB202190, 10. mu.M; penicillin streptomycin mixed solution, 1X; wnt7a, 10 ng/ml; IL-6, 50 ng/ml; HEPES, 1 mM.
Example 4
A nasal mucosa organoid culture method comprises the following specific steps:
(1) preparing glue:
A. collagen matrix (rat tail type I collagen), b.1x concentrated sterile medium (DMEM), c.advanced DMEM/F12, d.sterile NaOH solution (1 mol/L);
mixing 100 mu l B with 1ml C, adding 875 mu l A, and finally adding 15 mu l D to obtain a mixed collagen solution, and storing at 4 ℃; 1ml of the mixed collagen solution was poured into a 30mm diameter inner dish in a transwell chamber and solidified in an incubator at 37 ℃ for 30 minutes for use.
(2) The nasal mucosa was stored in a prepared, serum-free Ham's F12 solution containing 5% double antibody and sent to the laboratory for pretreatment within 3 hours.
(3) Fresh nasal mucosal tissue was placed in a 15ml centrifuge tube to which 3-5ml of 2% double antibody-containing Hanks solution had been added and washed with shaking (4 washes total).
(4) The washed tissue pieces were transferred to a 6cm petri dish, the cartilage was removed with sterilized forceps, and a suitable amount of HBSS solution was added to the dish.
(5) Grasping the sterilized blade with a needle holder and cutting the tissue to a size of 0-5mm on ice3The process of mincing should not exceed 10 minutes to avoid cell damage and tissue desiccation.
(6) The minced tissue pieces in the petri dish were transferred to a 15ml centrifuge tube and the digestive enzymes I (collagenase type III), hyaluronidase, Insulin, EGF) were added to 3 ml. Digesting for about 2 hours in a constant temperature shaking table at 37 ℃, taking out the centrifuge tube every 30 minutes, and blowing the digested tissue for about 5 times by using a pipette gun in a biological safety cabinet. And after the blowing, continuously placing the mixture into a constant-temperature shaking table at 37 ℃.
(7) After the first digestion, centrifuge tube 1100r was centrifuged for 5 min.
(8) After centrifugation, the supernatant was discarded, 3ml of digestive enzyme II (TrypLE Express, DNaseI enzyme) was added, blown and resuspended, and the tube was digested in a 37 ℃ constant temperature shaker for 8 min.
(9) Digestion was stopped by adding 2ml HBSS solution.
(10) The centrifuge tube was placed in a centrifuge and centrifuged at 1100r for 5 min.
(11) After centrifugation, the supernatant was discarded, and 2ml Hanks' solution was added to blow out the resuspended digested tissue pellet.
(12) Filtering the tissue mixed solution with a 100 μm filter screen to obtain filtrate containing nasal mucosa single cells, transferring the filtrate into a 15ml centrifuge tube, and centrifuging at 1100r for 3min to obtain single cell precipitate.
(13) And after centrifugation, discarding the supernatant, adding 2ml of erythrocyte lysate, after lysis for 5min, adding 2ml of HBSS for termination, finally centrifuging for 3min at 1100r, and discarding the supernatant to obtain cell sediment.
(14) Resuspend the cells with 1ml of pre-cooled mixed collagen solution and transfer to the inner dish pre-plated with mixed collagen solution as described above in step 1.
(15) After covering the outer lid, the mixed gel in the inner dish solidified into a gel in an incubator at 37 ℃ within 30 minutes.
(16) 1.5ml of the medium of example 3 was placed in a transwell cuvette, observed under a mirror, photographed, and placed in a 37 ℃ incubator without direct contact of the cells with the medium.
(17) Replacing the culture medium every 2-3 days to culture nasal mucosa organoid; the medium to be replaced is the same as the cell culture medium in step (16), preferably a conditioned medium.
(18) Passage: the mixed collagen (fixed) containing cells in the inner dish was transferred to a 15ml centrifuge tube and dissociated for 15-20 minutes at 37 ℃ with 100Unit/ml collagenase IV and organoids were passaged 1:2 every 30 days.
Comparative example 1
Compared with the common primary culture method
The traditional tissue primary culture method based on a gas-liquid interface adopts dispersed nasal mucosa cells cultured under the 2D condition of a common culture medium (DMEM + 10% FBS) at 37 ℃ and 5% CO2And (5) culturing in a cell culture box. The culture medium is replaced every 2-3 days, and as a result, the nasal mucosa cells are found to have poor adherence effect, grow slowly and die gradually after 4 weeks. In addition, primary cells are difficult to passage, cryopreservation and recovery.
Comparative example 2
Comparison of matrigel glue drop culture method proposed by Hans Cleveler
Adopting culture medium specific to the same organoid, 37 deg.C, 5% CO2Culturing in a cell culture box under 3D condition, and replacing the culture medium every 2-3 days. The results of organoid electron microscopy cultured by the glue drop method and the ALI method are compared, and the ALI method is higher in success rate and more beneficial to cilium proliferation and differentiation.
The culture medium and the culture method of the nasal mucosa tissue are designed for the proliferation and differentiation and the physiological function characteristics of the nasal mucosa tissue source cells of different anatomical parts, and an optimal experimental operation process is designed, so that the optimal culture medium is preferably selected. The contents of the cell factors and the signal channel regulating factors in the regulated culture medium are proper, and the nasal mucosa cells can successfully and stably form the nasal mucosa organoid in the 3D environment. The main feature of the 3D culture system of the present invention is that it is capable of self-renewal of self-organization into cell clusters with morphological structure and function similar to the epithelial tissue of nasal mucosa by in vitro three-dimensional (3D) culture of adult stem cells to induce the formation of epithelial cells of appropriate polarity that interact with stromal fibroblasts. The method can be used for better researching the signal transduction function of the interaction between cells and between epithelium and mesenchyme, the interaction between ciliary dysfunction and chronic nasosinusitis, a drug transport path, an absorption mechanism and the like, and meets the needs of a plurality of scientific researches. In addition, the culture method process can also be used for culturing other epithelial tissue organoids (such as respiratory tract and digestive tract mucosa) to form a standard standardized epithelial organoid culture system. Meanwhile, the culture medium can complete subculture of nasal mucosa tissues, meet the requirement of large-scale replication of nasal mucosa organoids, and control the organoids obtained by culture to have high consistency.
The foregoing is directed to the preferred embodiment of the present invention and is not intended to limit the invention to the specific embodiment described. It will be apparent to those skilled in the art that various modifications, equivalents, improvements and the like can be made without departing from the spirit of the invention, and these are intended to be included within the scope of the invention.
Claims (8)
1. A nasal mucosa organoid culture medium is characterized in that the components and the contents of the culture medium are as follows: vit-a free B27, 1X; n-acetyl cysteine, 1 mM; EGF, 50 ng/ml; noggin, 100 ng/ml; r-spondin1, 250 ng/ml; wnt3a, 250 ng/ml; a83-01, 500 nM; nicotinamide, 10 mM; y-27632, 10. mu.M; glutamax, 1X; gastrin, 5 nM; SB202190, 10. mu.M; penicillin streptomycin mixed solution, 1X; wnt7a, 10 ng/ml; IL-6, 50 ng/ml; HEPES, 1 mM.
2. A nasal mucosa organoid culture method is characterized in that nasal mucosa tissue is pretreated to obtain cell sediment, the cell sediment is added into a transwell small-chamber dish containing mixed collagen solution, and the cell sediment is solidified into gel; adding the culture medium of claim 1 into a transwell small outdoor dish, and carrying out isothermal culture;
the mixed collagen solution is prepared from the following components: A. rat tail type I collagen, B.1X concentrated sterile medium DMEM, C.advanced DMEM/F12, D.1mol/L sterile NaOH solution.
3. The method of claim 2, wherein the cultured tissue source comprises turbinates, septum, fundus, olfactory region, sinuses, nasopharynx, and the pretreatment step comprises washing, mincing, digesting, and lysing steps.
4. The method according to claim 3, characterized in that the pre-treatment step is in particular: washing nasal mucosa tissue with 2% double antibody-containing Hanks solution, and cutting the tissue; adding digestive enzyme I for digestion, centrifuging to remove the supernatant, adding digestive enzyme II for digestion, centrifuging to remove the supernatant, blowing the resuspended and digested tissue precipitate with Hanks liquid, filtering to obtain filtrate containing nasal mucosa single cells, centrifuging to remove the supernatant, adding erythrocyte lysate for lysis, centrifuging to remove the supernatant to obtain cell precipitate; digestive enzymes I include collagenase type III, hyaluronidase, Insulin and EGF; digestive enzymes II include TrypLE Express and DNaseI enzymes.
5. The method of claim 2, wherein the mixed collagen solution is prepared by the following method: first 100. mu.l B and 1ml C were mixed, then 875. mu. lA was added, and finally 15. mu.l D was added to obtain a mixed collagen solution.
6. The method according to any one of claims 2 to 5, further comprising the step of replacing the culture medium: the medium is changed every 2 to 3 days, and the changed medium is the same as the medium of claim 1.
7. The method of claim 6, wherein the replaced medium is conditioned medium.
8. The method according to any one of claims 2 to 5, further comprising the step of passaging: the mixed collagen containing cells in the inner dish was transferred to a 15ml centrifuge tube and dissociated for 15-20 minutes at 37 ℃ with the addition of 100Unit/ml collagenase IV, and organoids were passaged 1:2 every 30 days.
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| 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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