Culture medium system for inducing airway stem cells to differentiate into airway epithelial cells based on ALI and application of culture medium system

Abstract

The invention discloses a culture medium system for inducing airway stem cells to differentiate into airway epithelial cells based on ALI and application of the culture medium system. Belongs to the technical field of ALI culture. By means of the culture medium system, the airway stem cells can be efficiently and stably induced to be differentiated into multiple types of airway epithelial cells in the ALI culture process, and particularly, the generated cilia cells are close to the physiological state in vivo. Compared with an imported culture medium, the culture medium has the advantages of definite components, high batch stability, low cost and simple preparation method; the generated airway epithelial cells have a plurality of cell lineages, can well simulate airway epithelium, and show forms and functional characteristics similar to those of an in-vivo airway. According to the system, a respiratory tract epithelium-induced ALI culture system is remarkably improved, the research cost is reduced, a preferable scheme is provided for scholars engaged in studying physiological and pathological characteristics of respiratory tract epithelial cells based on ALI, and researchers are helped to establish a stable repeatable airway model.

Description

Technical Field

[0001] The present invention relates to the field of ALI culture technology, and more specifically to a culture medium system for inducing airway stem cells to differentiate into airway epithelial cells based on ALI and its application. Background Art

[0002] With the aggravation of environmental pollution and population aging, as well as the emergence and spread of new respiratory viruses, respiratory diseases seriously threaten human health and life. In the respiratory system, airway epithelial cells are the first physiological barrier to protect airway-lung tissue from invasion by exogenous substances. Airway tissue contains various types of airway epithelial cells, such as basal cells, ciliated cells, multiple types of mucus-secreting cells, neuroendocrine cells and other cell lineages. Among them, the tops of ciliated cells and mucus-secreting cells can directly contact the air interface, and basal cells are located in the lower layer of ciliated cells and mucus-secreting cells, that is, the basal layer. The distribution of these cells indicates that airway epithelial cells have polarity.

[0003] An air-liquid interface (ALI) cell culture system that can simulate the polarity of airway epithelial cells has been established internationally. This system uses a Transwell chamber to inoculate airway epithelial stem cells on the porous membrane of the Transwell chamber and culture them in culture medium until they are fully confluent. Afterwards, an "air-lifted" differentiation treatment is performed to expose the top of the cells to the air and the bottom surface to the basal differentiation culture medium, which promotes the differentiation of stem cells into airway epithelia with oscillating cilia and secretory cells in a pseudostratified morphology, which can simulate the in vivo characteristics of airway epithelial cells. At present, the airway epithelial cell ALI culture system is of great significance in the in vitro study of the physiological functions of the respiratory tract, the pathogenic mechanisms of respiratory diseases, and the diagnosis and treatment evaluation.

[0004] At present, the ALI differentiation medium used in the study is imported from abroad, with unclear specific ingredients and relatively expensive purchase price. It is of great significance to develop domestic ALI differentiation medium with independent intellectual property rights, clear composition, reliable quality and low price, and then promote scientific research based on airway epithelial cells on the pathogenesis of airway epithelial cell lesions, clinical drug screening for airway respiratory diseases, and evaluation of drug pathological toxicity.

[0005] In summary, how to provide a culture medium system based on ALI to induce airway stem cells to differentiate into airway epithelial cells is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a culture medium system and its application for inducing airway stem cells to differentiate into airway epithelial cells based on ALI. Compared with the ALI airway differentiation culture medium imported from abroad, the culture medium system has clear ingredients, high batch stability, and low price cost. At the same time, it can more efficiently induce the generation of various lineage cells of airway epithelium within the same culture time, can well simulate the airway epithelium, and show morphological and functional characteristics similar to those of the airway in vivo, especially the morphological characteristics and movement of cilia, which provides a more preferred solution for domestic research based on the ALI system to study the physiological functions of the respiratory tract, the pathogenic mechanisms of respiratory diseases, and the diagnosis and treatment evaluation. This culture medium system will greatly reduce the research cost and greatly improve the research efficiency, which is of great significance to the promotion of related research.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A culture medium system for inducing airway stem cells to differentiate into airway epithelial cells based on ALI, including airway epithelial cell differentiation medium;

[0009] The airway epithelial cell differentiation medium uses DMEM / F12 as a solvent and includes the following components at final concentrations: 0.3-1×N2 supplement, 0.3-1×B27 supplement, 0.05-0.5% fetal bovine albumin, 0.2-2.5 mM N-acetylcysteine, 1-2 mM glutamine, 10-100 μg / mL ascorbic acid, 25-200 ng / mL FGF, 75-200 ng / mL FGF, 105-200 ng / mL FGF, 5-200 ng / mL BMP4, 10.5-5 μM A830, 10-200 nM retinoic acid, 1-20 μM SJ000291942, 1-20 μM SRI-01138, 1-20 μM IBMX, 10-200 μM 8-Br-cAMP 10~200μM and dexamethasone 0.05~3μM.

[0010] Further, it also includes airway stem cell expansion medium;

[0011] The airway stem cell expansion medium is Pneumacult ExPlus supplemented with 1 μM A8301, 1 μM DMH1 and 10 μM Y-27632.

[0012] Further, including airway epithelial cell differentiation medium and airway stem cell expansion medium;

[0013] The airway epithelial cell differentiation medium uses DMEM / F12 as a solvent and includes the following components at final concentrations: 1×N2 supplement, 1×B27 supplement, 0.5% fetal bovine albumin, 2.5 mM N-acetylcysteine, 2 mM glutamine, 100 μg / mL ascorbic acid, 200 ng / mL FGF2, 200 ng / mL FGF7, 200 ng / mL FGF10, 200 ng / mL BMP4, 200 ng / mL A830, 15 μM A830, 200 nM retinoic acid, 220 μM SJ00029194, 20 μM SRI-01138, 120 μM IBMX, 200 μM 8-Br-cAMP, and 3 μM dexamethasone;

[0014] The airway stem cell expansion medium is Pneumacult ExPlus supplemented with 1 μM A8301, 1 μM DMH1 and 10 μM Y-27632.

[0015] A method for inducing airway epithelial cells, using the above-mentioned culture medium system.

[0016] Further, the specific steps are as follows:

[0017] (1) resuspending airway stem cells in airway stem cell expansion medium to obtain a cell suspension;

[0018] (2) Add the cell suspension to the upper layer of the Transwell chamber and add the airway stem cell expansion medium to the lower layer, and culture continuously for 3 to 7 days;

[0019] (3) The culture medium in the upper and lower layers of the Transwell chamber was replaced with airway epithelial cell differentiation medium. After 24 hours, the culture medium in the upper layer of the chamber was removed by aspiration;

[0020] (4) Continue to culture for 14 to 21 days.

[0021] Application of the above culture medium system in preparing airway epithelial cells.

[0022] An airway epithelial cell is a cell obtained by culturing using the above culture medium system or the above method.

[0023] The above-mentioned application of airway epithelial cells for non-diagnostic and therapeutic purposes is any one of the following:

[0024] A: Physiological functions, pathological characteristics and pathogenic mechanisms of respiratory diseases in vitro;

[0025] B: Drug development, screening, and pharmacological efficacy evaluation for respiratory diseases related to airway epithelial pathology;

[0026] C: Establish respiratory disease model;

[0027] D: Study the infection of respiratory viruses on respiratory epithelial cells.

[0028] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0029] The present invention provides a culture medium system for inducing the differentiation of airway stem cells into airway epithelial cells based on ALI. Compared with the ALI airway differentiation culture medium imported from abroad, the culture medium system has clear ingredients and low cost. At the same time, it can efficiently induce the generation of various lineage cells of the airway epithelium within the same culture time, providing a preferred solution for domestic research on the differentiation of airway stem cells into airway epithelial cells based on ALI. The culture medium system will greatly reduce the research cost and greatly improve the research efficiency, which is of great significance for promoting the research on the physiological function of the respiratory tract, the pathogenic mechanism of respiratory diseases, diagnosis and treatment evaluation, clinical drug screening, and drug toxicity evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0031] Figure 1 1 is a scanning electron microscope image of the cilia morphology of ciliated cells of mouse airway stem cells on the 21st day of ALI induction culture in Experiment 1 of the present invention, wherein a is a schematic diagram of the experiment based on Transwell-induced differentiation of airway stem cells into airway epithelial cells; b is a cilia morphology image of ciliated cells obtained using the culture medium of Example 2 of the present invention; c is a cilia morphology image of ciliated cells obtained using the culture medium of Example 3 of the present invention; d is a cilia morphology image of ciliated cells obtained using the culture medium of Example 4 of the present invention; e is a cilia morphology image of ciliated cells of mouse airway tissue;

[0032] Figure 2 The ciliary morphology of ciliated cells observed under a microscope on the 21st day of ALI-induced culture of mouse airway stem cells in Experiment 1 of the present invention, wherein a is a ciliary morphology of ciliated cells obtained using the culture medium of Example 2 of the present invention; b is a ciliary morphology of ciliated cells obtained using the culture medium of Example 3 of the present invention; c is a ciliary morphology of ciliated cells obtained using the culture medium of Example 4 of the present invention;

[0033] Figure 3The mouse airway stem cells of Example 5 in Experiment 1 of the present invention were induced and cultured in the airway epithelial cell differentiation medium of Example 3. The cross-sections were histochemically identified on the 21st day to identify airway epithelial ciliated cells (ACT), Club endocrine cells (CC10), Goblet cells (MUC5AC), and MUC5B + Representative diagram of secretory cells (MUC5B);

[0034] Figure 4 The mouse airway stem cells of Example 5 in Experiment 1 of the present invention were induced and cultured in the airway epithelial cell differentiation medium of Example 3. The longitudinal sections were histochemically identified on the 21st day for airway epithelial basal cells (KRT5), ciliated cells (ACT), Club endocrine cells (CC10), Goblet cells (MUC5AC), MUC5B + Representative diagram of secretory cells (MUC5B);

[0035] Figure 5 These are the expression results of genes of cells of various lineages of the airway epithelium after qPCR identification of the airway epithelial cells of the mouse airway stem cells of Example 5 in Experiment 2 of the present invention after induction culture with the airway epithelial cell differentiation medium of Example 3 on the 21st day, wherein Airway-SC represents airway stem cells, Lung represents lung airway tissue, and ALI represents cells induced by the culture system of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0038] The airway stem cells used in the following examples are commercially available mouse airway stem cells.

[0039] Example 1

[0040] Preparation of airway stem cell expansion medium

[0041] The culture medium Pneumacult ExPlus (StemCell Technologies, Cat. #05040) was purchased and supplemented with 1 μM A8301 (ThermoFisher Scientific, Cat. #293910), 1 μM DMH1 (ThermoFisherScientific, Cat. #412610) and 10 μM Y-27632 (Tocris, Cat. #1254).

[0042] Example 2

[0043] Preparation of airway epithelial cell differentiation medium

[0044] Using DMEM / F12 as the solvent, a culture medium containing the following final concentrations was prepared: 0.3×N2 supplement, 0.3×B27 supplement, 0.05% fetal bovine albumin, 0.2 mM N-acetylcysteine, 1 mM glutamine, 10 μg / mL ascorbic acid, 25 ng / mL FGF, 75 ng / mL FGF, 105 ng / mL FGF, 5 ng / mL BMP4, 5 ng / mL A830, 10.5 μM, 10 nM retinoic acid, 21 μM SJ000291942, 1 μM SRI-01138, 11 μM IBMX, 10 μM 8-Br-cAMP, and 0.05 μM dexamethasone.

[0045] Example 3

[0046] Preparation of airway epithelial cell differentiation medium

[0047] Using DMEM / F12 as the solvent, a culture medium containing the following final concentrations was prepared: 1×N2 supplement, 1×B27 supplement, 0.5% fetal bovine albumin, 2.5 mM N-acetylcysteine, 2 mM glutamine, 100 μg / mL ascorbic acid, 200 ng / mL FGF2, 200 ng / mL FGF7, 200 ng / mL FGF10, 200 ng / mL BMP4, 200 ng / mL A830, 15 μM, retinoic acid, 200 nM, SJ00029194, 220 μM, SRI-01138, 120 μM, IBMX, 200 μM, 8-Br-cAMP, and 3 μM dexamethasone.

[0048] Example 4

[0049] Purchased airway epithelial cell differentiation medium as a control

[0050] Mix the purchased PneumaCult ALI Medium (StemCell Technologies Cat.#05001) according to the instructions.

[0051] Example 5

[0052] A Transwell-based culture method for inducing airway stem cells to differentiate into airway epithelial cells

[0053] (1) A polyester film Transwell chamber with a diameter of 6 mm and a pore size of 0.4 μm was coated with 50 μg / mL type I rat tail collagen and air-dried for subsequent cell seeding.

[0054] (2) Airway stem cells with a cell confluence of 80% to 90% were digested with 0.25% trypsin to make them fall off the bottom of the cell culture dish, and the digestion was terminated with DMEM containing 10% FBS.

[0055] (3) Centrifugation to collect airway stem cells, and resuspend the cells in the airway stem cell expansion medium of Example 1 to a cell density of 2×10 7 / mL to obtain cell suspension.

[0056] (4) 200 μL of the cell suspension was added to the upper layer of the Transwell chamber, and 600 μL of the airway stem cell expansion medium of Example 1 was added to the lower layer. The medium was changed every other day, and the culture was continued for 3 to 7 days until the cells in the upper layer of the chamber completely covered the entire chamber interface.

[0057] (5) The upper and lower layers of the chamber are replaced with the airway epithelial cell differentiation medium of Example 2, Example 3 or Example 4. After 24 hours, the upper layer of the chamber medium is removed by aspiration, and the lower layer medium is retained to start inducing airway epithelial cell differentiation.

[0058] (6) The airway epithelial cell differentiation medium in the lower layer of the chamber was replaced every other day. The culture was continued for 14 to 21 days, and video sampling was performed.

[0059] Experiment 1

[0060] Identification of lineage cells of airway stem cells induced to differentiate into airway epithelial cells based on Transwell

[0061] In order to verify that the airway epithelial cell differentiation culture medium of the present invention can stably and efficiently induce the production of airway epithelial cells of various lineages, the cells cultured continuously for 21 days were videotaped, sampled and fixed.

[0062] (1) Video recording of ciliary cell beating:

[0063] The Transwell membrane with cells was peeled off from the chamber and placed on a glass slide with the cells facing upward. Then 50 μL of the airway stem cell expansion medium of Example 1 was added to the glass slide and the slide was inverted on the microscope stage to start the video recording mode and observe the movement of the cilia. The morphology and distribution of the cilia were then observed under an electron microscope.

[0064] The results are as follows Figure 1 As shown in the figure, it can be seen that compared with the purchased culture medium ( Figure 1 d), the culture medium system of the present invention ( Figure 1 b. Figure 1 c) The generated cilia are more mature and closer to the cilia of airway ciliated cells in animals ( Figure 1 e).

[0065] Figure 2 The morphology of ciliary cells of mouse airway stem cells was observed under a microscope on the 21st day of ALI induction culture. Compared with the purchased culture medium ( Figure 2 c), the culture medium system of the present invention ( Figure 2 a. Figure 2 b) The cilia density of ciliated cells tends to increase.

[0066] (2) Histochemical fixation experimental operation:

[0067] The well plate culture medium was discarded, and the Transwell membrane cells were fixed with 4% paraformaldehyde solution at room temperature for 30 minutes, and dehydrated with 30% sucrose for 48 hours. The Transwell membrane was embedded with OCT and longitudinal frozen sections were made with a thickness of 10 μm. The frozen sections were immunofluorescently stained, and the corresponding cell lineage was identified with airway epithelial cell-specific antibodies. The tissue sections were incubated with the corresponding primary antibody at 4°C overnight, washed 3 times with PBS, and then the fluorescent secondary antibody corresponding to the corresponding antibody was added, incubated at room temperature for 1 hour, washed 3 times with PBS, and then incubated with DAPI staining solution for 5 minutes, washed 3 times with PBS, and observed and photographed under a fluorescence microscope.

[0068] The results are as follows Figure 3 , Figure 4 As shown, KRT5 marks basal cells, ACT marks ciliated cells, CC10 marks endocrine cells (Club cells), MUCAC marks Goblet cells, and MUC5B marks MUC5B + Secretory cells. It can be seen that the airway epithelial cell differentiation medium of the present invention can induce airway stem cells to differentiate into multiple types of airway epithelial cells.

[0069] Experiment 2

[0070] Real-time fluorescence quantitative PCR verifies that airway stem cells differentiate into airway epithelial lineage cells after ALI culture

[0071] The airway epithelial cells differentiated and cultured in the medium ALI of Example 3 for 21 days in Example 5 were dissociated from the Transwell membrane, fully lysed with Trizol, and chloroform with 1 / 2 volume of Trizol was added, and the mixture was centrifuged at 4°C, 12000 rpm for 15 minutes. The upper aqueous phase was taken and an equal volume of isopropanol was added and fully mixed, and the mixture was allowed to stand at room temperature for 10 minutes, and centrifuged at 4°C, 12000 rpm for 15 minutes. After that, the precipitate was fully washed with 75% ethanol, dried at room temperature until it was translucent, and RNase-free ddH 2 O to dissolve RNA.

[0072] RNA was reverse transcribed using a reverse transcription kit (Promega, A5001). First, 500 ng of RNA was added with 1 μL of primer dT and ddH 2 O to 5 μL, heat at 70°C for 5 minutes to open the helical structure in the RNA, and immediately put it on ice for more than 5 minutes. Then, add 4 μL 5× Reaction buffer, 4 μL MgCl 2 , 1μL PCR dNTP, 0.5μL RNase inhibitors, 1μL Reverse Transcriptase and 4.5μL ddH 2 O, after fully mixing, 25°C, renature for 5 minutes, 42°C, extend for 1 hour, and 70°C, react for 15 minutes to inactivate the reverse transcriptase. The expression level of the corresponding gene was detected by real-time fluorescence quantitative PCR. The reaction system of real-time fluorescence quantitative PCR is shown in Table 1, the reaction conditions are shown in Table 2, and the primer sequences are shown in Table 3.

[0073] Table 1 Reaction system of real-time fluorescence quantitative PCR

[0074] Reagents Volume (μL) 2×SYBGreenTaq 10.0 Forwardprimer 0.6 Reverseprimer 0.6 cDNA 0.6 <![CDATA[ddH 2 The]]> 8.2 Total volume 20

[0075] Table 2 Reaction conditions of real-time fluorescence quantitative PCR

[0076]

[0077] Table 3 Real-time fluorescence quantitative PCR primer sequence list

[0078]

[0079]

[0080] like Figure 5 As shown, after 21 days of ALI differentiation culture of airway stem cells, the differentiated airway epithelial cells expressed genes of epithelial cells in airway tissue, indicating that lung epithelial cells of the corresponding cell lineage were generated.

[0081] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0082] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium system for inducing airway stem cells to differentiate into airway epithelial cells based on ALI, characterized in that: Includes Airway Epithelial Cell Differentiation Medium; The airway epithelial cell differentiation medium uses DMEM / F12 as a solvent and includes the following components at final concentrations: 0.3-1×N2 supplement, 0.3-1×B27 supplement, 0.05-0.5% fetal bovine albumin, 0.2-2.5 mM N-acetylcysteine, 1-2 mM glutamine, 10-100 μg / mL ascorbic acid, 25-200 ng / mL FGF, 75-200 ng / mL FGF, 105-200 ng / mL FGF, 5-200 ng / mL BMP4, 10.5-5 μM A830, 10-200 nM retinoic acid, 1-20 μM SJ000291942, 1-20 μM SRI-01138, 1-20 μM IBMX, 10-200 μM 8-Br-cAMP 10~200μM and dexamethasone 0.05~3μM.

2. The culture medium system according to claim 1, characterized in that Also included is an airway stem cell expansion medium; The airway stem cell expansion medium is Pneumacult ExPlus supplemented with 1 μM A8301, 1 μM DMH1 and 10 μM Y-27632.

3. The culture medium system according to claim 1, characterized in that Includes Airway Epithelial Cell Differentiation Medium and Airway Stem Cell Expansion Medium; The airway epithelial cell differentiation medium uses DMEM / F12 as a solvent and includes the following components at final concentrations: 1×N2 supplement, 1×B27 supplement, 0.5% fetal bovine albumin, 2.5 mM N-acetylcysteine, 2 mM glutamine, 100 μg / mL ascorbic acid, 200 ng / mL FGF2, 200 ng / mL FGF7, 200 ng / mL FGF10, 200 ng / mL BMP4, 200 ng / mL A830, 15 μM A830, 200 nM retinoic acid, 220 μM SJ00029194, 20 μM SRI-01138, 120 μM IBMX, 200 μM 8-Br-cAMP, and 3 μM dexamethasone; The airway stem cell expansion medium is Pneumacult ExPlus supplemented with 1 μM A8301, 1 μM DMH1 and 10 μM Y-27632.

4. A method for inducing airway epithelial cells, characterized in that: Use the culture medium system according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The specific steps are as follows: (1) resuspending airway stem cells in airway stem cell expansion medium to obtain a cell suspension; (2) Add the cell suspension to the upper layer of the Transwell chamber and add the airway stem cell expansion medium to the lower layer, and culture continuously for 3 to 7 days; (3) The culture medium in the upper and lower layers of the Transwell chamber was replaced with airway epithelial cell differentiation medium. After 24 hours, the culture medium in the upper layer of the chamber was removed by aspiration; (4) Continue to culture for 14 to 21 days.

6. Use of the culture medium system according to any one of claims 1 to 3 in preparing airway epithelial cells.

7. An airway epithelial cell, characterized in that: Cells obtained by culturing using the culture medium system described in any one of claims 1 to 3 or the method described in any one of claims 4 to 5.

8. The use of airway epithelial cells for non-diagnostic and therapeutic purposes according to claim 7, characterized in that: The application is any of the following: A: Physiological functions, pathological characteristics and pathogenic mechanisms of respiratory diseases in vitro; B: Drug development, screening, and pharmacological efficacy evaluation for respiratory diseases related to airway epithelial pathology; C: Establish respiratory disease model; D: Study the infection of respiratory viruses on respiratory epithelial cells.

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