Preparation method of airway epithelial cell gas-liquid interface culture paraffin section

Abstract

A preparation method of an airway epithelial cell gas-liquid interface culture paraffin section relates to the technical field of paraffin section preparation, and comprises the following steps: amplifying basal cells from a clinical lung tissue sample, and culturing by using a differential medium to obtain a gas-liquid interface culture after a Transwell chamber is full of the cells; the preparation method comprises the following steps: fixing by using paraformaldehyde, cutting a Transwell membrane containing a gas-liquid interface culture in half by using surgical scissors, pre-embedding the gas-liquid interface culture by using agarose, dehydrating the pre-embedded gas-liquid interface culture, embedding the pre-embedded gas-liquid interface culture by using paraffin, and slicing the pre-embedded gas-liquid interface culture. According to the paraffin section of the airway epithelial cell gas-liquid interface culture, the form of the cells can be completely kept, and the growth state, polarity and the like of the cells can be reflected. Various types of epithelial cells can be identified through immunofluorescence staining, the proportion of various types of cells can be determined, and the method has important significance on in-vitro research of respiratory system diseases.

Description

Technical Field

[0001] The invention relates to the technical field of paraffin section preparation, in particular to a method for preparing paraffin sections of airway epithelial cell air-liquid interface cultures. Background Art

[0002] Air-liquid culture of human airway epithelial cells is considered to be a highly relevant culture system for respiratory physiology, and has laid a good foundation for studying the function and related diseases of human airway epithelium. The main feature of air-liquid interface culture is that the basal surface of the cells is in contact with the liquid culture medium, while the apical surface is exposed to the air. The common method of cell inoculation is to inoculate cells onto the permeable membrane of the cell culture insert. In the early stage of cell culture, both the apical and basal chambers need to be added with culture medium. When the number of cells is close to full plate, the cells need to be cultured at the air-liquid interface, that is, the culture medium in the apical chamber is blown dry and the culture medium in the basal chamber is retained. The air-liquid interface culture system simulates the situation in the human airway and induces basal cells to differentiate into secretory cells and ciliated cells. The air-liquid interface culture of airway epithelial cells contains a variety of cell types, so the expression and localization of cell markers can be detected by immunofluorescence technology to study the differences in airway epithelial function and status under different experimental conditions.

[0003] However, the overall staining of air-liquid interface culture can also reflect the expression of various cell markers. However, since the air-liquid interface is in a pseudo-stratified state and involves multiple layers of cells, the overall staining is not easy to intuitively reflect the proportion of each type of cell and the position in the air-liquid interface culture.

[0004] Making paraffin sections for immunofluorescence can reflect the location and proportion of various types of cells. However, since the air-liquid interface culture grows on a permeable membrane, the common fixation, dehydration, transparency, wax immersion, embedding and sectioning processes may cause cells to detach from the permeable membrane, which may easily lead to sample loss and changes in cell morphology, thus affecting the wide application of this model in research related to respiratory diseases. Summary of the invention

[0005] The present invention aims to solve the problems of sample loss and morphological changes that are prone to occur during the embedding process of airway epithelial cell air-liquid interface culture, and to provide a preparation method for obtaining complete air-liquid interface culture morphological structure slices to facilitate immunohistochemical research.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for preparing paraffin sections of airway epithelial cell air-liquid interface cultures, comprising the following steps:

[0008] (1) Using clinical lung tissue samples to induce differentiation to form inward-facing cilia-oriented lung airway organoids, the samples were digested into single cells and expanded and cultured using basal cell culture medium. The obtained basal cells were induced to differentiate and obtain air-liquid interface cultures;

[0009] (2) Fixing, pre-embedding, dehydrating, transparentizing, wax-immersing, embedding, and slicing the obtained air-liquid interface culture, thereby obtaining paraffin sections of the airway epithelial cell air-liquid interface culture.

[0010] As a preferred technical solution of the present invention, the method for fixing the gas-liquid interface culture is:

[0011] After culturing the air-liquid interface culture for 14 days, the medium was discarded and the cells were fixed with 4% paraformaldehyde. 200 μL of the solution was added to the upper chamber of the Transwell and 500 μL of the solution was added to the lower chamber. After fixation at room temperature for 30 min, the paraformaldehyde was discarded and the cells were washed three times with PBS for 5 min each time.

[0012] As a preferred technical solution of the present invention, the method for fixing the pre-embedded package is:

[0013] Use surgical forceps to peel off the PET film containing the air-liquid interface culture from the Transwell plug, and use surgical scissors to cut it in half; use ultrapure water to prepare a 2% agarose solution by mass volume ratio, use a Pasteur pipette to add it to a 24-well cell culture plate, and when the agarose temperature is about 60°C, put the PET film containing the cell culture vertically into the agarose, with the cell surface perpendicular to the bottom of the plate, and place the agarose-air-liquid interface culture mixture at 4°C to solidify for 10-20 minutes; take out the solidified air-liquid interface culture gel block from the 24-well plate, and trim the gel block with a surgical blade.

[0014] As a preferred technical solution of the present invention, the method of dehydration, transparency and wax dipping after pre-embedding is:

[0015] Place the trimmed air-liquid interface culture-agarose block into a disposable dehydration box and put it into a tissue dehydrator for dehydration, transparency, and wax immersion; the dehydration process is to immerse the sample in 70%, 80%, 95%, 100%, 100%, and 100% alcohol in sequence, with each step taking 1 hour; the transparency process is to immerse the sample in xylene twice in sequence, each time for 1 hour; the transparent sample is waxed 3 times with paraffin wax, the first 2 times for 1 hour each time, and the last time for 1.5 hours.

[0016] As a preferred technical solution of the present invention, the method of embedding and slicing after wax immersion is:

[0017] The air-liquid interface culture-agarose gel block was embedded in paraffin and then sliced ​​into paraffin using a microtome. The thickness of the air-liquid interface culture slices was 6 μm and the slices were stored at room temperature.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The method for preparing paraffin sections of airway epithelial cell air-liquid interface culture provided by the present invention can solve the problems of sample loss and structural morphological changes in the traditional embedding process. The paraffin sections prepared by this method can completely maintain the morphology of the cells and can intuitively reflect the growth state, polarization direction, etc. of the culture. In addition, the sections are subjected to immunofluorescence staining to observe the expression and location of various types of cell markers in air-liquid interface culture, which is suitable for evaluating the differences in the expression of various cell markers under different experimental conditions and has high practicality. At the same time, various types of epithelial cells can be identified and the proportion of each type of cells can be determined, which is of great significance for in vitro research on respiratory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the method for preparing paraffin sections of airway epithelial cell air-liquid interface cultures in an embodiment of the present invention.

[0021] Figure 2 This is the bright field image and identification image of the amplified basal cells in Example 3 of the present invention.

[0022] Figure 3 This is a bright field image of the ALI culture process in Example 4 of the present invention.

[0023] Figure 4 HE staining, AB-PAS staining and immunofluorescence identification of paraffin sections of gas-liquid interface culture in Example 6 of the present invention. DETAILED DESCRIPTION

[0024] See also Figure 1 As shown, the present invention proposes a method for preparing paraffin sections of airway epithelial cell air-liquid interface culture. First, clinical lung tissue samples are used to induce differentiation to form cilia-facing inward lung airway organoids, which are digested into single cells and expanded and cultured using basal cell culture medium. The obtained basal cells are induced to differentiate to obtain air-liquid interface cultures; then, the obtained air-liquid interface cultures are fixed, pre-embedded, dehydrated, transparentized, wax-impregnated, embedded, and sliced ​​to obtain paraffin sections of airway epithelial cell air-liquid interface cultures.

[0025] The present invention is further described in detail below in conjunction with embodiments and drawings.

[0026] Example 1 Experimental Materials

[0027] 1. Cell and organoid culture medium

[0028]

[0029]

[0030] 2. ALI culture materials

[0031]

[0032] 3. Immunofluorescence Antibodies

[0033]

[0034] Example 2 Construction of lung airway organoids with inward-facing cilia

[0035] (1) Clinical lung tissue samples were analyzed by AdDF +++ The lung tissue was washed with culture medium (DMEM / F12, 10 mM HEPES, 1×GlutaMax, 1×Penicillin-streptomycin) and centrifuged to remove blood cells; the lung tissue was transferred to a 35 mm culture dish and 500 μL of digestion solution (AdDF containing 2 mg / mL collagenase I) was added. +++ ), mince the lung tissue into 1 mm pieces with a blade 3 , transfer the chopped lung tissue into a 15 mL centrifuge tube, add 6 mL of digestion solution, and digest at 37 °C for 2 h.

[0036] (2) After digestion, the tissue was blown with a Pasteur pipette to separate the cell clusters from the tissue. The digested tissue was filtered through a 40 μm cell sieve into a 50 mL centrifuge tube. AdDF was added to the cell suspension. +++ When the volume reaches 10 mL, transfer all of it into a 15 mL centrifuge tube and centrifuge at 500 rpm for 5 min to precipitate the cell pellet. Take out the centrifuge tube after centrifugation, discard the supernatant, and add 1 mL of red blood cell lysis buffer to mix well.

[0037] (3) After centrifugation, discard the supernatant, add a small amount of cilia-facing lung airway organoid culture medium to resuspend and count. Mix the cells with 40% cell suspension and 60% matrix gel, and seed the cells in a 48-well plate at 30 μL per well, with approximately 300 cell clusters per well; place the 48-well plate in a 37°C incubator to gel for 10 minutes, then add 300 μL of cilia-facing lung airway organoid culture medium to each well and place the plate in a 37°C incubator for culture.

[0038] Example 3 Basal cell expansion culture

[0039] The cilia-oriented lung airway organoids obtained in Example 2 were isolated using the cell dissociation reagent TrypLE TM Express enzyme digestion at 37℃ for 6-8min, with AdDF +++The cells were transferred to a 15 mL centrifuge tube and centrifuged at 850 rpm for 5 min. The cells were resuspended in 1 mL of basal cell expansion medium. The single cell suspension was seeded in a six-well plate and 2 mL of basal cell expansion medium was added. The cells were cultured at 37°C and 5% CO2 for 3-4 days. Cells cultured in basal cell medium widely expressed basal cell markers ( Figure 2 shown).

[0040] Example 4 Obtaining Air-Liquid Interface (ALI) Cultures

[0041] When the basal cell confluence of Example 3 reaches 70%-80%, the cell dissociation reagent TrypLE TM The cells were digested with Express enzyme and seeded in Transwell chambers, with 3.3×10 cells per well. 4 cells, 200 μL and 500 μL of basal cell expansion medium were added to the upper and lower chambers of the Transwell chamber, respectively. When the cell confluence reached 100% (approximately 3 days), the cells were cultured in the basal cell expansion medium and then switched to air-liquid interface culture, i.e., the upper chamber medium of the Transwell was discarded, and 500 μL of ALI differentiation medium was added to the lower chamber for 14 days, with the lower chamber medium replaced every 2 days.

[0042] Figure 3 This is a bright field image of the ALI culture process. Day 0 in the figure shows basal cells cultured in a six-well plate. Day 2-14 is a bright field image of the culture process using ALI differentiation medium. The cells change from a single layer of cells to a multilayer of cells, and the cilia can be seen beating under the microscope.

[0043] Example 5 Paraffin section preparation

[0044] 1. Fixation of Air-Liquid Interface Cultures

[0045] Example 4: After culturing the air-liquid interface culture for 14 days, the culture medium was discarded, and the culture was fixed with 4% paraformaldehyde. 200 μL was added to the upper chamber of the Transwell and 500 μL was added to the lower chamber. After fixation at room temperature for 30 minutes, the paraformaldehyde was discarded and the culture was washed 3 times with PBS, each time for 5 minutes.

[0046] 2. Pre-embedding

[0047] The PET membrane containing the air-liquid interface culture was peeled off from the Transwell insert using surgical forceps and cut in half using surgical scissors.

[0048] Use ultrapure water to prepare a 2% agarose solution by mass-volume ratio, add it to a 24-well cell culture plate using a Pasteur pipette, and when the agarose temperature is about 60°C, place the PET film containing the cell culture vertically into the agarose with the cell surface perpendicular to the bottom of the plate, and place the agarose-air-liquid interface culture mixture at 4°C to solidify for 10-20 minutes.

[0049] The solidified air-liquid interface culture block was taken out from the 24-well plate and trimmed with a surgical blade to reduce the volume of the block as much as possible without removing the culture.

[0050] 3. Dehydration, transparency, wax dipping

[0051] The trimmed air-liquid interface culture-agarose gel block was placed in a disposable dehydration box and placed in a tissue dehydrator for dehydration, transparency, wax dipping and other processes.

[0052] The dehydration process was to immerse the samples in 70%, 80%, 95%, 100%, 100%, and 100% alcohol in sequence, with each step lasting 1 h.

[0053] The transparent process was to immerse the sample in xylene twice, each time for 1 h;

[0054] The transparent sample was immersed in paraffin wax for 3 times, with the first 2 times for 1 h each time and the last time for 1.5 h.

[0055] 4. Paraffin Embedded Sections

[0056] The air-liquid interface culture-agarose gel block was embedded in paraffin and then sliced ​​into paraffin using a microtome. The thickness of the air-liquid interface culture slices was 6 μm and the slices were stored at room temperature.

[0057] Example 6 HE, AB-PAS staining and immunofluorescence identification

[0058] 1. HE staining

[0059] (1) Dewaxing sections into water (xylene I, xylene II, 100% ethanol I, 100% ethanol II, 90% ethanol, 80% ethanol, ddH2O, 5 min each);

[0060] (2) Hematoxylin staining for 30 seconds, followed by rinsing with tap water;

[0061] (3) Differentiate with 0.2% HCl in ethanol solution for 3-5 seconds and rinse with tap water;

[0062] (4) Immerse the sections in 80% ethanol for 5 min;

[0063] (5) Eosin staining for 15 seconds;

[0064] (6) The sections were quickly immersed in 100% ethanol three times;

[0065] (7) After being transparentized with xylene, the slides were sealed with neutral gum.

[0066] like Figure 4 The HE staining result in the figure shows that the slices obtained by using the air-liquid interface culture prepared in Example 4 have complete morphological structure.

[0067] 2. AB-PAS staining

[0068] (1) Dewax the sections into water (xylene I, xylene II, 100% ethanol I, 100% ethanol II, 90% ethanol, 80% ethanol, ddH2O for 5 min each), and wash with ddH2O for 2 min;

[0069] (2) Stain with Alcian blue solution for 20 min and wash with ddH2O three times, 2 min each time;

[0070] (3) Add oxidant and oxidize for 8 min, then wash twice with ddH2O, each time for 2 min;

[0071] (4) Add Schiff staining solution to cover the sections and stain for 20 min, then rinse twice with ddH2O, 5 min each time;

[0072] (5) Stain the nucleus with hematoxylin solution for 1 min and wash with ddH2O;

[0073] (6) Differentiate with acidic differentiation solution for 2-5 seconds and wash with ddH2O;

[0074] (7) Use Scott bluing solution to reverse blue for 3 min, and wash with ddH2O for 3 min;

[0075] (8) Stepwise ethanol dehydration (80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, 5 min each);

[0076] (9) After being transparentized with xylene, the slides were sealed with neutral gum.

[0077] like Figure 4 The AB-PAS staining result diagram in the figure shows that the air-liquid interface culture prepared in Example 4 has the function of mucin secretion, blue represents acidic mucin, and red represents neutral mucin.

[0078] 3. Immunofluorescence on Paraffin Sections

[0079] (1) The obtained paraffin sections were first baked at 60°C for 30 min;

[0080] (2) hydration, specifically: 100% xylene for 5 min, 100% xylene for 5 min, 100% ethanol for 5 min, 100% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, ddH2O for 5 min;

[0081] (3) Antigen retrieval: Antigen retrieval was performed using 0.1 M, pH 6.2 citrate buffer followed by immunofluorescence staining;

[0082] (4) Use 3% BSA and 0.5% TritonX-100 to block the membrane at room temperature for 1 h;

[0083] (5) Primary antibody incubation: P63, MUC5AC, FOXJ1, Acetylated αTubulin, E-Cadherin, and CC10 antibodies were diluted with 3% BSA according to the corresponding ratio according to the required identification indicators;

[0084] (6) Wash away the primary antibody with PBS for 3 times, 15 min each time.

[0085] (7) Add fluorescently labeled secondary antibody working solution and incubate at room temperature for 2 h;

[0086] (8) After washing with PBS three times, add 1 μg / mL Hoechst 33342 dye solution and incubate for 10 min;

[0087] (9) After sealing with an anti-fluorescence quencher, take photos using an inverted fluorescence microscope or a high-content imaging system.

[0088] like Figure 4 The immunofluorescence identification result graph shows that the air-liquid interface culture obtained in Example 4 expresses the epithelial cell marker E-Cadherin and contains basal cells (P63 + ), ciliated cells (FOXJ1 + ), Club Cell (CC10 + ), goblet cells (MUC5AC + ).

[0089] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing paraffin sections of airway epithelial cell air-liquid interface cultures, characterized in that: Here are the steps: (1) Using clinical lung tissue samples to induce differentiation to form inward-facing cilia-oriented lung airway organoids, the samples were digested into single cells and expanded and cultured using basal cell culture medium. The obtained basal cells were induced to differentiate and obtain air-liquid interface cultures; (2) Fixing, pre-embedding, dehydrating, transparentizing, wax-immersing, embedding, and slicing the obtained air-liquid interface culture, thereby obtaining paraffin sections of the airway epithelial cell air-liquid interface culture.

2. The preparation method according to claim 1, characterized in that The method for fixing the air-liquid interface culture is: After culturing the air-liquid interface culture for 14 days, the medium was discarded and the cells were fixed with 4% paraformaldehyde. 200 μL of the solution was added to the upper chamber of the Transwell and 500 μL of the solution was added to the lower chamber. After fixation at room temperature for 30 min, the paraformaldehyde was discarded and the cells were washed three times with PBS for 5 min each time.

3. The preparation method according to claim 2, characterized in that: The method of fixing pre-embedding is: Use surgical forceps to peel off the PET film containing the air-liquid interface culture from the Transwell plug, and use surgical scissors to cut it in half; use ultrapure water to prepare a 2% agarose solution by mass volume ratio, use a Pasteur pipette to add it to a 24-well cell culture plate, and when the agarose temperature reaches 60°C, place the PET film containing the cell culture vertically into the agarose, with the cell surface perpendicular to the bottom of the plate, and place the agarose-air-liquid interface culture mixture at 4°C to solidify for 10-20 minutes; remove the solidified air-liquid interface culture gel block from the 24-well plate and trim the gel block with a surgical blade.

4. The preparation method according to claim 3, characterized in that: The method for dehydration, transparency and wax dipping after pre-embedding is: Place the trimmed air-liquid interface culture-agarose block into a disposable dehydration box and put it into a tissue dehydrator for dehydration, transparency, and wax immersion; the dehydration process is to immerse the sample in 70%, 80%, 95%, 100%, 100%, and 100% alcohol in sequence, with each step taking 1 hour; the transparency process is to immerse the sample in xylene twice in sequence, each time for 1 hour; the transparent sample is waxed 3 times with paraffin wax, the first 2 times for 1 hour each time, and the last time for 1.5 hours.

5. The preparation method according to claim 4, characterized in that: The method of embedding and sectioning after wax immersion is: The air-liquid interface culture-agarose gel block was embedded in paraffin and then sliced ​​into paraffin using a microtome. The thickness of the air-liquid interface culture slices was 6 μm and the slices were stored at room temperature.

6. Use of the method according to any one of claims 1 to 5 for preparing paraffin sections of airway epithelial cell air-liquid interface cultures, characterized in that: Immunofluorescence staining was used to observe the expression and localization of cell markers in cell culture and to evaluate the differences in the expression of various cell markers under different experimental conditions.

Images