In vitro culture method and culture fluid of nasal mucosa

Abstract

The application provides a kind of nasal mucosa in vitro culture method and culture fluid, the composition of the culture fluid includes Wnt / β-catenin signal pathway activator 0.1-20 μg / ml, fibroblast growth factor 10-150 ng / ml, TGF-β inhibitor 0.02-3 μg / ml, ROCK inhibitor 0.1-100 μmol / L, P38 MAPK inhibitor 0.1-1 μmol / L, serum-free additive 1X, N-acetyl cysteine 1-10 mmol / L, nicotinamide 1-20 mmol / L, GlutaMax additive 1X, antibiotic, DMEM / F12 culture medium 1X.The application can carry out in vitro culture to nasal mucosa, can keep at least 2 weeks of activity, and have complete epithelial barrier and immune system, can realize ex vivo nasal mucosa virus infection experiment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an in vitro culture method and culture medium for nasal mucosa. Background Technology

[0002] The nasal cavity, as the main passage for air inhalation, not only serves as a frontline respiratory defense mechanism but also as a primary site for respiratory pathogen infection and replication. Anatomically, the human nasal cavity contains a layer of pseudostratified ciliated epithelium, interspersed with goblet cells containing tubulin and secreting mucus, derived from basal cells and originating from a stem cell layer of p63+ epithelium. The human nasal mucosa epithelium is susceptible to various upper respiratory tract viruses.

[0003] In current technologies, studies on viral infection in the nasal cavity are typically conducted using animal models. This involves infecting animals such as mice and rabbits with the virus, then euthanizing them and observing the pathological changes in the nasal cavity. However, some viruses exhibit species-specific characteristics, such as the novel coronavirus, which readily infects humans. Animal experiments struggle to replicate viral infection in the human nasal mucosa. Using humans for experiments violates ethical principles. Furthermore, experiments using nasal mucosa often result in rapid cell death shortly after being removed from the body, making it impossible to accurately simulate the infection process.

[0004] Therefore, there is an urgent need for an in vitro nasal mucosa survival system that can successfully carry out nasal mucosa virus infection and complete the experimental process. Summary of the Invention

[0005] The purpose of this invention is to provide an in vitro culture method for nasal mucosa and a culture medium for in vitro culture. The culture medium and culture method provided by this invention can culture nasal mucosa in vitro, maintain its activity for at least 2 weeks, and possess a complete epithelial barrier and immune system, and can replicate the viral infection process in the human body, thereby enabling in vitro nasal mucosa viral infection experiments.

[0006] The technical solution adopted in this invention is to provide an in vitro culture medium for nasal mucosa, the components of which include:

[0007] Wnt / β-catenin signaling pathway activator 0.1-20 μg / ml

[0008] Fibroblast growth factor 10-150 ng / ml

[0009] TGF-β inhibitor 0.02-3 μg / ml

[0010] ROCK inhibitor 0.1-100 μmol / L

[0011] P38 MAPK inhibitor 0.1-1 μmol / L

[0012] Serum-free additive 1X

[0013] N-acetylcysteine ​​1-10 mmol / L

[0014] Nicotinamide 1-20 mmol / L

[0015] GlutaMax Additive 1X

[0016] Antibiotic penicillin 100 U / ml

[0017] antibiotic streptomycin 100 μg / ml

[0018] Antibiotic Primocin 50 μg / ml

[0019] DMEM / F12 medium 1X.

[0020] Preferably, the culture medium is composed of the following components:

[0021] Wnt / β-catenin signaling pathway activator 0.1-20 μg / ml

[0022] Fibroblast growth factor 10-150 ng / ml

[0023] TGF-β inhibitor 0.02-3 μg / ml

[0024] ROCK inhibitor 0.1-100 μmol / L

[0025] P38 MAPK inhibitor 0.1-1 μmol / L

[0026] Serum-free additive 1X

[0027] N-acetylcysteine ​​1-10 mmol / L

[0028] Nicotinamide 1-20 mmol / L

[0029] GlutaMax Additive 1X

[0030] Antibiotic penicillin 100 U / ml

[0031] antibiotic streptomycin 100 μg / ml

[0032] Antibiotic Primocin 50 μg / ml

[0033] DMEM / F12 medium 1X.

[0034] Preferably, the DMEM / F12 culture medium contains L-glutamine and 15 mM Hepes buffer.

[0035] Preferably, the Wnt / β-catenin signaling pathway activator is one or more of R-spondin1 protein, Wnt-3a protein, and FH535. The Wnt / β-catenin signaling pathway activator is a growth factor that promotes differentiation and induces regeneration. R-spondin1 protein is preferred.

[0036] Preferably, the fibroblast growth factor is one or two of FGF7, FGF10, and FGF-4, which can promote the growth of epithelial cells.

[0037] Preferably, the TGF-β inhibitor is one or two of Noggin, A83-01, and Compound C dihydrochloride, wherein, preferably, noggin is an endogenous inhibitor of bone morphogenetic protein, and A83-01 is a TGF-β type I receptor inhibitor that regulates cell differentiation, proliferation and apoptosis.

[0038] Preferably, the ROCK inhibitor includes one or more of Y27632, S1049Y-27632 2HCl, ZINC00881524, GSK269962A, M20594, and Y-33075, with Y27632 being the most preferred. The ROCK inhibitor can prevent apoptosis.

[0039] Preferably, the P38 MAPK inhibitor includes one or more of SB202190, SB431542, and BIRB796, with SB202190 being preferred. The P38 MAPK inhibitor induces cell differentiation.

[0040] Preferably, the serum-free additive is B27, which has antioxidant properties.

[0041] N-acetylcysteine ​​can resist oxidative damage and necrosis.

[0042] Nicotinamide / vitamin B3 is a coenzyme precursor that can regulate protein and sugar metabolism.

[0043] GlutaMax additives are dipeptide nutrients that provide nutrition for cell growth.

[0044] Antibiotics such as penicillin, streptomycin, and primoxine can kill bacteria and fungi.

[0045] Furthermore, the in vitro culture medium for the nasal mucosa preferably comprises the following components:

[0046] R-spondin1 protein 0.1-20 μg / ml

[0047] Fibroblast growth factor FGF7 5-50 ng / ml

[0048] Fibroblast growth factor FGF10 5-100 ng / ml

[0049] TGF-β inhibitor Noggin 0.01-2 μg / ml

[0050] TGF-β inhibitor A83-01 0.01-1 μmol / L

[0051] ROCK inhibitor Y-27632 0.1-100 μmol / L

[0052] P38 MAPK inhibitor SB202190 0.1-1 μmol / L

[0053] Serum-free additive B27 (50X) 1X

[0054] N-acetylcysteine ​​1-10 mmol / L

[0055] Nicotinamide 1-20 mmol / L

[0056] GlutaMax Additive 1X

[0057] Antibiotic penicillin 100 U / ml

[0058] antibiotic streptomycin 100 μg / ml

[0059] Antibiotic Primocin 50 μg / ml

[0060] DMEM / F12 medium 1X.

[0061] More preferably, the in vitro culture medium for the nasal mucosa comprises:

[0062] R-spondin1 protein 500 ng / ml

[0063] Fibroblast growth factor FGF7 25 ng / ml

[0064] Fibroblast growth factor FGF10 100 ng / ml

[0065] TGF-β inhibitor Noggin 100 ng / ml

[0066] TGF-β inhibitor A83-01 500 nmol / L

[0067] ROCK inhibitor Y-27632 5 μmol / L

[0068] P38 MAPK inhibitor SB202190 500 nmol / L

[0069] Serum-free additive B27 (50X) 1X

[0070] N-acetylcysteine ​​1.25 mmol / L

[0071] Nicotinamide 5 mmol / L

[0072] GlutaMax Additive 1X

[0073] Antibiotic penicillin 100 U / ml

[0074] antibiotic streptomycin 100 μg / ml

[0075] Antibiotic Primocin 50 μg / ml

[0076] DMEM / F12 medium 1X.

[0077] The present invention also provides a method for culturing nasal mucosa in vitro using the aforementioned in vitro culture liquid, the method comprising the following steps:

[0078] Step (1): Organize the collection;

[0079] After the nasal mucosa is removed from the body, it is preserved by immersing it in physiological saline.

[0080] Step (2): Cleaning;

[0081] The isolated nasal mucosa was washed with phosphate-buffered saline (PBS) to remove blood clots, mucus and bone fragments;

[0082] Step (3): Incubate in culture medium;

[0083] The cleaned nasal mucosa was placed in in vitro culture medium and cultured in a cell culture incubator. The cultured nasal mucosa tissue can then be used for subsequent experiments.

[0084] Preferably, in step (1), the nasal mucosa is soaked in physiological saline for less than 6 hours.

[0085] Preferably, in step (3), the nasal mucosa is placed in an in vitro culture medium for culture as soon as possible within 12 hours after being removed from the body.

[0086] Preferably, in step (3), the culture environment in the cell culture incubator is 37°C and 5% CO2.

[0087] Preferably, in step (2), the integrity of the nasal mucosa is used as the activity standard. Observing ciliary movement under a microscope is considered as the epithelial cells still being active. In the culture medium of the present invention, the nasal mucosa can be cultured in vitro for 2 weeks and remains intact and active.

[0088] Preferably, the nasal mucosa cultured in vitro provided by the present invention can be used for in vitro virus infection experiments.

[0089] The present invention also provides a method for conducting in vitro viral infection experiments using nasal mucosa cultured in vitro, wherein the nasal mucosa cultured in vitro is cultured in vitro using the aforementioned in vitro culture medium for nasal mucosa.

[0090] This invention provides an in vitro culture medium that can culture nasal mucosa in vitro, maintaining its activity for at least 2 weeks and possessing a complete epithelial barrier and immune system. It can replicate the viral infection process in the human body. Our research shows that the virus can successfully infect cultured nasal mucosa, and the expression of viral proteins can be observed through sections, thus enabling in vitro nasal mucosa virus infection experiments. Attached Figure Description

[0091] Figure 1 A schematic diagram of the process for nasal mucosal tissue culture and novel coronavirus (SARS-CoV-2) infection.

[0092] Figure 2 Photographs taken under a bright field microscope on days 1, 4, and 7 of nasal mucosal tissue culture.

[0093] Figure 3 Images of nasal mucosal tissue culture on day 7 show DAPI and HE staining, with image C showing DAPI staining and image D showing HE staining.

[0094] Figure 4 Microscopic images of nasal mucosa tissue before and 72 hours after viral infection.

[0095] Figure 5 Images of nasal mucosal tissue stained with DAPI and HE 72 hours after infection and before infection.

[0096] Figure 6 Immunofluorescence staining images of the SARS-CoV-2 nucleocapsid protein (SARS-CoV-2N protein) and the viral receptor angiotensin-converting enzyme 2 (ACE2) in nasal mucosa tissue 24 hours after infection and before infection.

[0097] Figure 7 HE staining images of nasal mucosa tissue 48 hours after infection and before infection, and immunofluorescence staining images of SARS-CoV-2N protein and viral receptor ACE2 48 hours after viral infection.

[0098] Figure 8 Immunofluorescence staining images of SARS-CoV-2N protein and microtubule protein, a ciliated cell-specific marker. Detailed Implementation

[0099] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited thereto. Example

[0100] The composition of the in vitro culture medium is shown in Table 1 below.

[0101] Table 1

[0102]

[0103] A schematic diagram of the nasal mucosal tissue culture and SARS-CoV-2 infection process is shown below. Figure 1 As shown. The specific steps are as follows:

[0104] (1) Tissue collection: After the nasal mucosa is removed from the body, it is temporarily preserved by immersing it in physiological saline for 2 hours;

[0105] (2) Cleaning and cutting: Wash the isolated nasal mucosa with phosphate-buffered saline (PBS), carefully remove blood clots, mucus and bone fragments; carefully cut into 2 mm square pieces with scissors, and place each tissue sample into a 24-well plate;

[0106] (3) Culture in culture medium: Add 2 ml of in vitro culture medium to each well and culture in a cell culture incubator at 37°C and 5% CO2. The total time from in vitro removal of nasal mucosa tissue to addition of in vitro culture medium should not exceed 12 hours. In this example, it is 6 hours.

[0107] (4) Virus infection: The novel coronavirus (Pubmed NP: MT627325) was isolated from the biosafety level 3 laboratory of Naval Medical University, expanded using African monkey kidney cells (VERO E6), and frozen at -80°C. Before use, it was brought to room temperature. The culture medium was added at a specific titer to infect the nasal mucosa. Infected nasal mucosa was collected, and RNA was extracted using the Trizol method. Viral yield and infection status were then detected by reverse transcription PCR.

[0108] In step (3), the integrity of the nasal mucosa is used as the activity standard. The observation of ciliary movement under a microscope is considered as the epithelial cells still being active.

[0109] Bright-field micrographs of nasal mucosal tissue culture on days 1, 4, and 7 are shown below. Figure 2 As shown, Figure 2 As can be seen, cilia can be clearly detected even on the 7th day.

[0110] like Figure 3 As shown, DAPI and HE staining confirmed the structural integrity of the nasal mucosal tissue culture on day 7.

[0111] Two weeks after in vitro culture, cilia movement was observed under a microscope, indicating that the nasal mucosa remained intact and active.

[0112] Images of uninfected and infected tissues 72 hours after viral infection, observed under a bright-field optical microscope, are shown below. Figure 4 As shown, the nasal mucosa tissue is generally intact.

[0113] like Figure 5 As shown, DAPI and HE staining revealed that 72 hours after viral infection, the nasal mucosa tissue showed partial defects in the submucosa compared to the control group, indicating that viral infection caused damage to the nasal mucosa.

[0114] Figure 6 The results showed that viral infection within 24 hours of infection was mainly confined to the epithelial layer and blood vessels. The viral receptor ACE2 was highly expressed in the epithelium and blood vessels of the nasal mucosa; the arrows indicate viral infection near the epithelial layer and blood vessels.

[0115] Figure 7 HE staining of the nasal mucosal epithelium 48 hours after viral infection showed signs of deterioration in the nasal mucosal structure. Immunofluorescence staining images revealed SARS-CoV-2N proteins in the fragmented epithelium and mucosa, demonstrating the destructive effects of the virus.

[0116] Figure 8 The image shows immunofluorescence co-staining of SARS-CoV-2N protein and tubulin in human nasal mucosa. Tubulin is a ciliated cell-specific marker in the nasal mucosal epithelium (pseudostratified columnar epithelium), indicating that the virus has infected the ciliated cells of the epithelium.

[0117] Figures 1-8 In the middle, the scale is 100µm. Figure 2 and Figure 4 Except (scale = 200µm).

[0118] Comparative Example 1

[0119] The components of the in vitro culture medium were changed as shown in Table 2 below.

[0120] Table 2

[0121]

[0122] The steps for culturing nasal mucosa tissue in vitro are as follows:

[0123] (1) Tissue collection: After the nasal mucosa is removed from the body, it is temporarily preserved by immersing it in physiological saline for 1 hour;

[0124] (2) Cleaning and cutting: Wash the isolated nasal mucosa with phosphate-buffered saline (PBS), carefully remove blood clots, mucus and bone fragments; carefully cut into 2 mm square pieces with scissors, and place each tissue sample into a 24-well plate;

[0125] (3) Culture in culture medium: Add 2 ml of different in vitro culture media from Table 2 to each well and culture in a cell culture incubator at 37°C and 5% CO2. The total time from in vitro removal of nasal mucosa tissue to addition of in vitro culture medium should not exceed 12 hours. In this example, it is 6 hours.

[0126] Microscopic observation was performed every 8 hours on the first day, and daily thereafter. The results showed that on the second day, the cilia in the culture medium of group A were undetectable, and the epithelial cells lost their activity. Viral infection experiments could not be performed.

[0127] In Group B culture medium, tissue cilia were undetectable on day 4, indicating loss of epithelial cell activity. Viral infection experiments could not be performed.

[0128] In Group C culture medium, tissue cilia were undetectable on day 10, indicating loss of epithelial cell activity. Viral infection experiments could not be performed.

[0129] In group D culture medium, tissue cilia were undetectable on day 9, indicating loss of epithelial cell activity. Viral infection experiments could not be performed.

[0130] In Group E culture medium, tissue cilia were undetectable on day 11, indicating loss of epithelial cell activity. Viral infection experiments could not be performed.

[0131] On day 8, tissue cilia in group F culture medium were undetectable, indicating loss of epithelial cell activity. Viral infection experiments could not be performed.

[0132] On day 10, tissue cilia in group G culture medium were undetectable, indicating loss of epithelial cell activity. Viral infection experiments could not be performed.

[0133] The above description is merely a preferred embodiment of this application and does not constitute any limitation on this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of this application, and these improvements and additions should also be considered within the scope of protection described in the embodiments of this application. Any modifications, alterations, and equivalent changes made by those skilled in the art without departing from the spirit and scope of this application based on the disclosed technical content are equivalent embodiments of this application; furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of this application still fall within the scope of the technical solution of this application.

Claims

1. A method for culturing nasal mucosa in vitro, characterized in that, In vitro culture was performed using an in vitro culture medium containing nasal mucosa, the components of which included: R-spondin1 protein 0.1-20 μg / ml Fibroblast growth factor FGF7 5-50 ng / ml Fibroblast growth factor FGF10 5-100 ng / ml TGF-β inhibitor Noggin 0.01-2 μg / ml TGF-β inhibitor A83-01 0.01-1 μmol / L ROCK inhibitor Y-27632 0.1-100 μmol / L P38 MAPK inhibitor SB202190 0.1-1 μmol / L Serum-free additive B27 (50X) 1X N-acetylcysteine ​​1-10 mmol / L Nicotinamide 1-20 mmol / L GlutaMax Additive 1X Antibiotic penicillin 100 U / ml antibiotic streptomycin 100 μg / ml Antibiotic Primocin 50 μg / ml DMEM / F12 medium 1X; The method includes the following steps: Step (1): Organize the collection; After the nasal mucosa is removed from the body, it is preserved by immersing it in physiological saline. Step (2): Cleaning; The isolated nasal mucosa was washed with phosphate buffer to remove blood clots, mucus and bone fragments; Step (3): Incubate in culture medium; After cleaning, the nasal mucosa was placed in an in vitro culture medium. The nasal mucosa tissue was carefully divided into 2mm square pieces with scissors. Each tissue piece was placed in a 24-well plate and cultured in a cell culture incubator.

2. The method for in vitro culture of nasal mucosa as described in claim 1, characterized in that, The components of the in vitro culture medium include: R-spondin1 protein 500 ng / ml Fibroblast growth factor FGF7 25 ng / ml Fibroblast growth factor FGF10 100 ng / ml TGF-β inhibitor Noggin 100 ng / ml TGF-β inhibitor A83-01 500 nmol / L ROCK inhibitor Y-27632 5 μmol / L P38 MAPK inhibitor SB202190 500 nmol / L Serum-free additive B27 (50X) 1X N-acetylcysteine ​​1.25 mmol / L Nicotinamide 5 mmol / L GlutaMax Additive 1X Antibiotic penicillin 100 U / ml antibiotic streptomycin 100 μg / ml Antibiotic Primocin 50 μg / ml DMEM / F12 medium 1X.

3. The method for in vitro culture of nasal mucosa as described in claim 1, characterized in that, In step (1), the nasal mucosa is soaked in saline solution for less than 6 hours.

4. The method for in vitro culture of nasal mucosa as described in claim 1, characterized in that, In step (3), the nasal mucosa should be placed in an in vitro culture medium for culture as soon as possible within 12 hours after being removed from the body.

5. The method for in vitro culture of nasal mucosa as described in claim 1 or 4, characterized in that, In step (3), the culture environment in the cell culture incubator is 37°C and 5% CO2. 2。 6. A method for conducting in vitro viral infection experiments using nasal mucosa cultured in vitro, characterized in that, The in vitro cultured nasal mucosa was prepared using the method for in vitro cultured nasal mucosa as described in any one of claims 1 to 5.

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