Method for establishing an in vitro three-dimensional lung model based on acellular matrix

Abstract

The present invention belongs to the field of biotechnology and materials science, and specifically relates to a method for establishing an in vitro three-dimensional lung model based on a decellularized matrix. Specifically, the present invention provides a method for establishing an in vitro three-dimensional lung model based on a decellularized matrix, the method comprising freeze-drying and sterilizing the decellularized matrix, and adding cells and cell culture medium to the decellularized matrix and performing cell culture. The three-dimensional lung model prepared by the method of the present invention can be used as an in vitro three-dimensional virus infection model.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and materials science, and specifically relates to a method for establishing an in vitro three-dimensional lung model based on a decellularized matrix. Background Art

[0002] The goal of developing in vitro 3D models is to construct biomimetic tissue or organ structures that have similar structural and functional characteristics to natural tissues or organs and can promote interactions between cells and between cells and the extracellular matrix (ECM). However, to date, no natural or synthetic material can fully replicate all the characteristics of the natural extracellular matrix.

[0003] The extracellular matrix is ​​a complex network composed of biomacromolecules secreted by cells, which provides a suitable place for cell survival and activity. It not only plays a physical role such as support, connection, and protection, but also can dynamically have an all-round impact on cells. The key to the decellularization strategy is to remove all cellular components and genetic material in natural tissues and organs, and to retain the three-dimensional structure and components of the extracellular matrix to the greatest extent. Decellularized matrix materials retain the specific structure and components of natural tissues and have mechanical properties similar to those of natural tissues. They can provide tissue-specific microenvironments to support cell adhesion, proliferation and differentiation, and thus have great potential in the study of in vitro three-dimensional models of various diseases. Therefore, decellularized matrix materials have been widely used in various fields of tissue engineering and regenerative medicine, and have made significant progress. Using tissue engineering strategies to inoculate seed cells on biological scaffold materials to construct a three-dimensional tissue model is a relatively mature in vitro model establishment method.

[0004] Unlike other solid tissues, the structure of lung tissue is loose and complex, containing a large number of bronchial and alveolar structures, and two sets of pipeline systems, airway and blood vessels. These complex structures pose new challenges to the preparation of lung decellularized matrix materials. Summary of the invention

[0005] In terms of establishing an in vitro three-dimensional model of the lung, the structure of the lung tissue is loose and complex, and contains a large number of bronchial and alveolar structures. These complex structures make the preparation of an in vitro three-dimensional model of the lung a difficult point in the field of tissue engineering research. The present invention provides a method for constructing an in vitro three-dimensional model based on a decellularized lung matrix, and the three-dimensional model prepared by the method is infected with a SARS-CoV-2 pseudovirus to establish an in vitro three-dimensional virus infection model.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for establishing an in vitro three-dimensional lung model based on a decellularized matrix, the method comprising the steps of freeze-drying and sterilizing the decellularized matrix, adding cells and cell culture medium to the decellularized matrix, and conducting cell culture.

[0008] More preferably, in the method, the lung decellularized matrix is ​​first cut into small pieces with a thickness of 3 mm and / or a diameter of 1 cm and then freeze-dried and sterilized.

[0009] Preferably, the method for preparing the decellularized matrix comprises the following steps:

[0010] 1) Take heart and lung tissues and perfuse the lungs with PBS through the retention needle;

[0011] 2) PBS is injected into the lungs along the trachea to inflate both lungs;

[0012] 3) The artery clamp is used to close the trachea, so that the PBS solution remains in the lungs;

[0013] 4) Continue to perfuse with PBS after deflation;

[0014] 5) Use Triton X-100, SDS, and Chaps to perfuse in sequence, and then use PBS to elute.

[0015] Preferably, the Triton X-100 is 1% Triton X-100.

[0016] Preferably, the SDS is 0.1% SDS.

[0017] Preferably, the CHAPS is 8 mM CHAPS.

[0018] Preferably, in step 5), 1% Triton X-100 is perfused for 1-4 h (including 1, 1.5, 2, 2.5, 3, 3.5, 4 h); preferably, 2-3 h; more preferably, 2.5 h (hours).

[0019] Preferably, in step 5), 0.1% SDS is perfused for 0.5-3 h (including 0.5, 1, 1.5, 2, 2.5, 3 h); preferably, 1-2 h; more preferably, 1.5 h.

[0020] Preferably, in step 5), 8 mM CHAPS is perfused for 0.5-3 h (including 0.5, 1, 1.5, 2, 2.5, 3 h); preferably, 1-2 h; more preferably, 1.5 h.

[0021] Preferably, the PBS elution in step 5) lasts for at least 8 hours (including 8, 9, 10, 11, 12, 13, 14 or more hours); preferably, at least 10 hours; more preferably, at least 12 hours; more preferably, 12 hours.

[0022] Preferably, in step 5), 1% Triton X-100 is perfused for 2.5 h, 0.1% SDS is perfused for 1.5 h, 8 mM CHAPS is perfused for 1.5 h, and PBS is eluted for 12 h.

[0023] More preferably, in step 5), 1% Triton X-100 is perfused for 2.5 h (first 1 h: 3000 μL / min, last 1.5 h: 5000 μL / min), 0.1% SDS is perfused for 1.5 h (5000 μL / min), 8 mM CHAPS is perfused for 1.5 h (5000 μL / min), and PBS is eluted for 12 h (5000 μL / min).

[0024] Preferably, the cardiopulmonary tissue is obtained from a model organism.

[0025] Preferably, the model organism comprises a mammal or a non-mammal.

[0026] Preferably, the model organism includes rats, mice, sheep, cattle, horses, pigs, alpacas, rabbits, fish or monkeys.

[0027] Most preferably, in the specific embodiment of the present invention, rat lung decellularized matrix is ​​prepared as an example.

[0028] Preferably, the cardiopulmonary tissue is obtained by conventional sampling methods in the art, as specifically shown in General Method 1 of the present invention.

[0029] More preferably, the preparation method provided by the present invention is carried out in any type of decellularization device. Specifically, the decellularization device used in the present invention is Figure 1 Indicates.

[0030] Preferably, the PBS perfusion in step 1) is performed by a peristaltic pump.

[0031] More preferably, the flow rate controlled by the peristaltic pump is 1 mL / min.

[0032] The purpose of PBS perfusion in step 1) is to remove air bubbles.

[0033] Preferably, in the above step 2), a sufficient amount of PBS is perfused to expand both lungs, and residual blood can be irrigated and air in the lungs can be expelled; in the present invention, 40 mL of PBS is perfused.

[0034] Preferably, in step 3), PBS remains in the lungs for at least 20 min, specifically including 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more min.

[0035] Specifically, the present invention is prepared by taking a stay of 30 minutes as an example.

[0036] Preferably, in step 4), the PBS perfusion lasts for 0-30 min; preferably, 10-20 min; more preferably, 15 min. The 0-30 specifically includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30.

[0037] Preferably, the specific freeze-drying operation steps in the model establishment method are: placing the lung acellular matrix pieces into a -80°C refrigerator overnight, taking them out the next day and placing them into a freeze dryer for freeze drying.

[0038] More preferably, the freeze drying process in the freeze dryer lasts for about 5 hours.

[0039] Preferably, the sterilization step in the model building method includes sterilization methods well known in the art, including chemical reagent sterilization, radiation sterilization, dry heat sterilization, and moist heat sterilization.

[0040] Preferably, the lung decellularized matrix is ​​sterilized by radiation sterilization.

[0041] Preferably, the radiation used in the radiation sterilization includes Co 60 Radiation (Cobalt-60), Cesium 137 Rays (Caesium-137, Cs-137), ultraviolet rays (UV), X-rays and gamma rays, etc.

[0042] Preferably, the sterilization step in the model establishment method is sterilized using cobalt 60, and more specifically, the freeze-dried lung decellularized matrix is ​​irradiated and sterilized using cobalt 60.

[0043] Preferably, the radiation dose used in the radiation sterilization is 10-15 kGy.

[0044] Preferably, the lung decellularized matrix in the model establishment method is placed in a suitable container, and the cell culture is carried out in the container, and the cell density when inoculating cells is 2-3×10 5 cells / mL.

[0045] Preferably, the cell density when inoculating cells is 2.5×10 5 cells / mL.

[0046] Preferably, the cell culture step comprises adding cells and culture medium, culturing for 1-2 hours, and then replenishing the culture medium to continue culturing; preferably, replenishing the medium after 1.5 hours.

[0047] The cell culture medium described in the present invention includes any conventional cell culture medium in the art, and exemplary ones include TeSR-E8, mTESR1, E8, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, α-Minimal Essential Medium (α-MEM), G-Minimal Essential Medium (Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Dulbecco's Medium), AmnioMax, Amino MaxⅡcomplete Medium (Gibco, Newyork, USA), Chang's medium, Mesem Cult-XF medium (STEMCELL Technologies, Vancouver, Canada), RPMI1640, Ham's F12, DMEM / F12, Ham's F-12KMedium, Hepato ZYME-SFM, William's EMedium, Waymouth's Medium or HepatocyteCulture Medium, etc.

[0048] Most preferably, the experiments were conducted in DMEM medium in the specific embodiments of the present invention.

[0049] That is, most preferably, the preparation method includes the steps of preparing a lung decellularized matrix and constructing a three-dimensional model based on the lung decellularized matrix. The step of preparing the lung decellularized matrix is ​​as described in Specific Embodiment 1 of the present invention, and the step of constructing the three-dimensional model is as described in Specific Embodiment 8 of the present invention.

[0050] Preferably, the cells comprise lung-associated cells.

[0051] Preferably, the lung-related cells include: pulmonary vascular pericytes, microvascular smooth muscle cells, pulmonary artery fibroblasts, lung cancer cells, and pulmonary vascular endothelial cells.

[0052] Preferably, the lung cancer cells include any type of lung cancer cells, including commercial cell lines or cells isolated from the lungs of lung cancer patients.

[0053] Specifically, the cells inoculated in the present invention are human lung adenocarcinoma cells A549 or Calu-3.

[0054] As used in the present invention, "freeze drying" utilizes the principle of ice crystal sublimation. In a highly vacuum environment, the moisture in frozen food materials is sublimated directly from ice solid to steam without melting the ice. Freeze drying is also called freeze sublimation drying. The dried material maintains its original chemical composition and physical properties (such as porous structure, colloidal properties, etc.).

[0055] On the other hand, the present invention provides an in vitro three-dimensional lung model prepared by the above preparation method.

[0056] More specifically, the in vitro three-dimensional lung model can be infected with viruses and used as a model for studying viral infection of the lungs.

[0057] On the other hand, the present invention provides the use of the in vitro three-dimensional lung model in simulating viral infection in vitro.

[0058] Preferably, the virus includes viruses that cause human disease, exemplarily including Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), H5 subtype avian influenza virus, canine coronavirus (CCoV-HuPn-2018), Ebola virus, and Zika virus.

[0059] Preferably, the virus includes SARS-CoV-2 and its D614G, Alpha, Delta, Omicron variants, and other variants of SARS-CoV-2, such as XBB, BA.5, BQ1 and other series of variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a decellularization device used in the present invention.

[0061] Figure 2 These are the gross observation results of rat decellularized lung tissue.

[0062] Figure 3 These are the results of scanning electron microscopy observations of natural lungs and decellularized lungs.

[0063] Figure 4 These are the HE staining results of native lung and decellularized lung.

[0064] Figure 2-4 Middle, A: native lung tissue; B: Triton+SDS treatment group; C: Triton+Chaps treatment group; D: Triton+SDS+Chaps combined treatment group.

[0065] Figure 5 It is the result of DNA quantitative analysis.

[0066] Figure 6 These are the results of Alcian blue staining of native lung and decellularized lung.

[0067] Figure 7 It is the result of GAG quantitative analysis.

[0068] Figure 8 These are the results of immunohistochemical staining of collagen in native lung and decellularized lung. A: Immunohistochemical staining of type I collagen in native lung and decellularized lung; B: Immunohistochemical staining of type III collagen in native lung and decellularized lung; C: Immunohistochemical staining of type IV collagen in native lung and decellularized lung.

[0069] Fig. 9 The histological staining results of functional proteins in native lung and decellularized lung.

[0070] Fig.10 The results of proteomic analysis of lung decellularized matrix, A: Venn diagram and protein quantity bar graph of intracellular (IN) and extracellular (EX) proteins in D-Lung and Matrigel; B: percentage of protein quantity in different subcellular localizations in D-Lung and Matrigel; bar graph of protein quantity percentage in five subcellular localizations in D-Lung and Matrigel; C: protein GO analysis of D-Lung and Matrigel. Data are shown as mean ± standard deviation. n(D-Lung) = 3, n(Matrigel) = 3. Fig.11 yes Figure 3-8 The differential expression results of Matrisome subclass proteins in D-Lung and Matrigel, A: differential analysis of the number of proteins matched to the Matrisome database in D-Lung and Matrigel; B: differential analysis of the expression levels of Matrisome subclass proteins in D-Lung and Matrigel; C: volcano plot of differentially expressed proteins of Matrisome subclasses in D-Lung and Matrigel; D: plot of differentially expressed proteins of Matrisome subclasses in D-Lung and Matrigel.

[0071] Fig.12 The results of the growth status test of Calu-3 cells in the lung decellularized matrix material are shown in Figure 1. A: HE staining of the lung decellularized matrix material after inoculation of Calu-3 cells for 1 day; B: HE staining of the lung decellularized matrix material after inoculation of Calu-3 cells for 3 days; C: HE staining of the lung decellularized matrix material after inoculation of Calu-3 cells for 5 days. Scale bar = 20 μm. Fig.13 This is the growth curve of Calu-3 cells in three-dimensional culture and two-dimensional culture in vitro. ** indicates p<0.01.

[0072] Fig.14 The immunofluorescence staining results of lung function proteins in the in vitro three-dimensional lung tissue model are shown in Figure 1. The immunofluorescence staining results of CDH1 protein (A), TJP1 protein (B), and SFTPC protein (C) in the in vitro three-dimensional lung tissue model are shown in Figure 2. Scale bar = 100 μm.

[0073] Fig.15 These are the results of mRNA expression of lung function genes in an in vitro three-dimensional lung tissue model, A: mRNA expression of CDH1 gene; B: mRNA expression of TJP1 gene; C: mRNA expression of SFTPC gene. ** indicates p<0.01.

[0074] Fig.16 The results of ACE2 expression identification in an in vitro three-dimensional lung tissue model, A: immunofluorescence staining of ACE2 protein; B: mRNA expression of ACE2 gene; CD: WB analysis of ACE2 protein expression. Scale bar = 100 μm, * indicates p < 0.05, ** indicates p < 0.01.

[0075] Fig.17 This is the result of immunofluorescence staining of the in vitro three-dimensional model of SARS-CoV-2D614G pseudovirus infection.

[0076] Fig.18 This is the result of immunofluorescence staining of the in vitro three-dimensional model of SARS-CoV-2Alpha variant pseudovirus infection.

[0077] Fig.19 This is the result of immunofluorescence staining of the in vitro three-dimensional model of SARS-CoV-2Delta variant pseudovirus infection.

[0078] Fig. 20 This is the result of immunofluorescence staining of the in vitro three-dimensional model of SARS-CoV-2Omicron variant pseudovirus infection.

[0079] Fig.21The results are the infection rates of SARS-CoV-2 pseudoviruses in the three-dimensional model of lung decellularized matrix, and the infection rates of SARS-CoV-2D614G (A), Alpha (B), Delta (C), and Omicron (D) pseudoviruses in the three-dimensional model of lung decellularized matrix. * indicates p < 0.05.

[0080] Figure 17-21 In the figure, A: Immunofluorescence staining of an in vitro three-dimensional model of lung decellularized matrix inoculated with A549 cells; B: Immunofluorescence staining of an in vitro three-dimensional model of lung decellularized matrix inoculated with Calu-3 cells; C: Immunofluorescence staining of an in vitro three-dimensional model of Matrigel inoculated with Calu-3 cells. Scale bar = 20 μm. DETAILED DESCRIPTION

[0081] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0082] The experimental materials involved in this invention are:

[0083] 1. Experimental Animals

[0084] Sprague-Dawley (SD) rats (200±20 g, either sex, clean grade, Vital River).

[0085] 2. Cell lines

[0086] (1) Human lung adenocarcinoma cells; A549

[0087] (2) Human lung adenocarcinoma cells; Calu-3

[0088] The above cell lines were provided by Professor Wang Xuejun from the Academy of Military Medical Sciences.

[0089] 3. Main reagents

[0090] Table 1. Main reagents

[0091]

[0092] 4. Preparation of main solutions

[0093] (1) 1× PBS: Dissolve 1.44 g Na2HPO4, 8 g NaCl, 0.24 g KH2PO4, and 0.2 g KCl in 500 mL distilled water and adjust the volume to 1 L. Sterilize by autoclaving.

[0094] (2) 0.1% SDS solution: Dissolve 1 g of SDS in 500 mL of distilled water and adjust the volume to 1 L. Sterilize by high pressure.

[0095] (3) 1% Triton solution: Dissolve 10 mL Triton in 500 mL 1× PBS and adjust the volume to 1 L. Sterilize by high pressure.

[0096] (4) 8 mM Chaps solution: Dissolve 4.92 g Chaps in 500 mL distilled water and adjust the volume to 1 L. Autoclave.

[0097] (5) DMEM culture medium: Dissolve DMEM dry powder, 3.7 g NaHCO3, and 2.383 g HEPES in 1 L ultrapure water, adjust the pH to 7.2-7.4, filter and sterilize in a clean bench, and store at 4°C. When used, aliquot and add 10% FBS and 1% penicillin-streptomycin.

[0098] The general method of the present invention:

[0099] Method 1: Cardiopulmonary sampling

[0100] SD rats were fasted for 12 hours before surgery and anesthetized with 7% chloral hydrate at 0.5 mL / 100 g according to the rat's weight. The anesthesia period lasted about 2 minutes. The rats were fixed in a supine position, and the neck, chest, and abdomen were disinfected with 75% ethanol in turn. The skin and muscles of the neck were separated layer by layer to expose the trachea, and the trachea was intubated between the 3rd and 4th tracheal cartilage rings. The small animal ventilator was connected for mechanical ventilation, with a tidal volume of 6 mL and a respiratory rate of 80 times / min. The operation was started after the rats were observed to breathe steadily and the respiratory rate was consistent with the ventilator.

[0101] Open the abdominal cavity through an incision in the middle of the chest and abdomen, cut the diaphragm to enter the chest cavity, and cut the ribs on both sides vertically until the heart and lungs are completely exposed. Open the right atrium to bleed, insert the syringe needle into the bottom of the right ventricle, and inject 15mL of sodium heparin solution (50U / mL) to prevent blood clotting. After the color of the lungs becomes lighter, separate the trachea from the esophagus and remove the heart and lungs as a whole.

[0102] Method 2: SEM detection

[0103] Fresh lung tissue and decellularized lung tissue obtained by the above three methods were freeze-dried in a vacuum freeze dryer for 2 hours. A small amount of freeze-dried sample was directly adhered to the conductive glue and gold was sprayed for 45 seconds using an Oxford Quorum SC7620 sputtering coater at 10 mA. The sample morphology was then photographed using a scanning electron microscope at an accelerating voltage of 3 kV.

[0104] Method 3: DNA quantitative detection

[0105] (1) Drying: Fresh lung tissue and three types of lung decellularized tissues were taken out of the refrigerator and placed in 1.5 mL EP tubes, labeled, sealed with sealing film, and placed in a vacuum freeze dryer for lyophilization for 2 h.

[0106] (2) Quantification: Each tissue was weighed, ground, and homogenized at a mass ratio of 10 mg / mL.

[0107] (3) Centrifuge at 10,000 rpm (~11,200 × g) for 1 min, pour off the supernatant, add 200 μL of buffer GA, and shake until completely suspended.

[0108] (4) Add 20 μL of Proteinase K solution and mix well. Then place at 56°C until the tissue is dissolved and centrifuge to remove water droplets on the inner wall of the tube cap.

[0109] (5) Add 200 μL of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 min. The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0110] (6) Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0111] (7) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0112] (8) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 × g) for 30 sec, discard the waste liquid, and return the adsorption column CB3 to the collection tube.

[0113] (9) Add 700 μL of buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 × g) for 30 sec, discard the waste liquid, and return the adsorption column CB3 to the collection tube.

[0114] (10) Repeat step (9).

[0115] (11) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400 × g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0116] (12) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μl of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400 × g) for 2 min, and collect the solution in a centrifuge tube.

[0117] (13) The DNA content in the tissue was measured using an ultra-micro spectrophotometer.

[0118] Method 4: GAG quantitative detection

[0119] (1) Sample pretreatment

[0120] 1) Take out fresh lung tissue and three types of lung decellularized tissues, weigh and take 200 mg, 2) put them into a pre-cooled 15mL conical centrifuge tube, 3) add 3mL GENMED cleaning solution to wash once, 4) transfer them into a liquid nitrogen cryotube, 5) immediately put them into a liquid nitrogen tank overnight, 6) take them out of the liquid nitrogen tank the next day, and immediately (at the fastest speed) grind the tissue into powder with a grinding rod, 7) put them into a 1.5mL centrifuge tube, 8) add 500μLGENMED extraction solution, 9) vortex vigorously for 1min to mix thoroughly, 10) put them into a 56℃ constant temperature metal bath and incubate for 16h, 11) put them into a 90℃ constant temperature metal bath and incubate for 10min, 12) put them into a micro desktop centrifuge and centrifuge for 10min at a speed of 13000rpm, 13) carefully transfer the supernatant to a new 1.5mL centrifuge tube, 14) put them in an ice box for later use

[0121] (2) Preparation of standard samples

[0122] 1) Prepare 5 1.5mL centrifuge tubes, label them as tubes 1 to 5, 2) Add 50μL GENMED cleaning solution to each tube, 3) Pipette 50μL GENMED standard solution into tube 1 and mix well, 4) Carefully transfer 50μL of the diluted GENMED standard solution from tube 1 to tube 2 and mix well, 5) Carefully transfer 50μL of the diluted GENMED standard solution from tube 2 to tube 3 and mix well, 6) Carefully transfer 50μL of the diluted GENMED standard solution from tube 3 to tube 4 and mix well, 7) Put tubes 1 to 5 in an ice box for later use; the concentrations of the standard tubes are shown in the table below

[0123] Table 2, Standard tube concentration

[0124]

[0125] (3) Standard curve determination

[0126] 1) Pipette 50 μL of the prepared GENMED standard solution into a 1.5 mL centrifuge tube; 2) Add 1 mL of GENMED staining solution; 3) Vortex for 15 seconds; 4) Incubate at room temperature for 30 minutes, avoid light exposure, and vortex for 15 seconds every 5 minutes; 5) Immediately centrifuge in a micro-tabletop centrifuge for 10 minutes at 13,000 rpm; 6) Carefully remove the supernatant to ensure that no water droplets remain. Purple or pink deposits can be seen on the wall or bottom of the tube; 7) Add 1 mL of LGENMED dissociation solution, 8) vortex for 15 seconds, 9) incubate at room temperature for 5 minutes, avoid light, to ensure full dissolution, 10) transfer to a new cuvette, 11) immediately put into the spectrophotometer for detection (wavelength 656nm): obtain the standard sample absorbance reading, the reference reading is about 0.2 to 1.0, 12) repeat the experimental steps 1 to 11 four times, 13) construct a standard curve: the vertical axis (Y axis) is the absorbance reading; the horizontal axis (X axis) is the standard glycosaminoglycan content (μg)

[0127] (4) Sample measurement

[0128] 1) Pipette 50 μL of the prepared sample into a 1.5 mL centrifuge tube; 2) Add 1 mL of GEMNED staining solution; 3) Vortex for 15 seconds; 4) Incubate at room temperature for 30 minutes, avoid light, and vortex for 15 seconds every 5 minutes; 5) Immediately centrifuge in a micro-tabletop centrifuge for 10 minutes at a speed of 13,000 rpm; 6) Carefully remove the supernatant to ensure that no water droplets remain. Purple or pink deposits can be seen on the wall or bottom of the tube; 7) Add 1 mL of GEMNED dissociation solution; 8) Vortex for 15 seconds; 9) Incubate at room temperature for 5 minutes, avoid light, and ensure full dissolution; 10) Transfer to a new cuvette; 11) Immediately place in a spectrophotometer for detection (wavelength 656 nm): obtain the sample absorbance reading; 12) Obtain the corresponding glycosaminoglycan content (μg) of the sample according to the above standard curve

[0129] (5) Concentration calculation

[0130] According to the above standard curve, the corresponding glycosaminoglycan content of the sample (μg) / 0.050 (sample volume; mL) = μg glycosaminoglycan / mL Method 5. Histological Detection 1. Preparation of Paraffin Sections

[0131] (1) Fresh lung tissue and three types of decellularized lung tissue were fixed in 4% paraformaldehyde for 24 hours, or three-dimensional models cultured for 1, 5, and 7 days were fixed in 4% paraformaldehyde for 24 hours. (2) Gradient dehydration: Take out the tissue block and place it in 70%, 80%, 90%, 95% and 100% ethanol solutions for 15 minutes each. (3) Transparent: Take out the tissue block and soak it in xylene twice, 10 minutes each time. (4) Wax dipping: Take out the tissue block and soak it in paraffin solution twice, 1 hour each time. (5) Embedding: Take out the tissue block and place it in the middle of the embedding box for embedding, and place it on the freezing table to solidify. (6) Sectioning: After the wax block is completely solidified, section it with a thickness of 4μm. (7) Spreading: Place the cut wax slices in a slide floater for spreading. (8) Retrieving: Use a glass slide to retrieve the slices and mark them. (9) Baking: Place the glass slide on a slide baker for 2 to 3 hours. (10) Place the slide in a 37°C incubator overnight. (11) Place the slide in a baking oven for 2 to 3 hours and place in a slide box for later use.

[0132] 2. HE staining

[0133] (1) Dewaxing: immerse the sections in xylene twice for 10 minutes each time, then immerse in 100%, 95%, 70%, and 60% ethanol for 5 minutes each, and finally dewax in distilled water for 5 minutes. (2) Hematoxylin staining for 5 minutes. (3) Differentiation with 1% hydrochloric acid for 5 seconds, rinse with tap water for 5 minutes. (4) Reblue with 1% ammonia water, rinse with tap water for 5 minutes. (5) Eosin staining for 5 minutes, rinse with tap water for 5 minutes. (6) Dehydration: immerse in 60%, 75%, 95%, and 100% ethanol for 2 seconds each. (7) Immerse in xylene twice, 5 minutes each time. (8) Seal the sections with neutral gum.

[0134] 3. EVG staining

[0135] (1) Dewaxing: immerse the sections in xylene twice for 10 minutes each time, then immerse in 100%, 95%, 70%, and 60% ethanol for 5 minutes each, and finally dewax in distilled water for 5 minutes. (2) Verhoeffs hematoxylin staining for 30 minutes, rinse with tap water for 5 minutes. (3) Differentiation with 2% ferric chloride solution until black fibers and gray background are observed under the microscope, and then rinse with distilled water for 5 seconds. (4) 5% sodium thiosulfate to remove iodine for 1 minute, and then rinse with distilled water for 5 seconds. (5) Eosin staining for 5 minutes, and then rinse with tap water for 5 minutes. (6) Van Gieson's solution counterstaining for 5 minutes. (7) Dehydration: immerse in 60%, 75%, 95%, and 100% ethanol for 2 seconds each. (8) Immerse in xylene twice, 5 minutes each time. (9) Seal the sections with neutral resin.

[0136] 4. Alcian Blue staining

[0137] (1) Dewaxing: immerse the sections in xylene twice for 10 minutes each time, then immerse in 100%, 95%, 70%, and 60% ethanol for 5 minutes each, and finally dewax in distilled water for 5 minutes. (2) Soak in Alcian acidification solution for 3 minutes. (3) Stain in Alcian staining solution for 30 minutes. (4) Rinse in running water. (5) Restain in Nuclear Fast Red staining solution for 5 minutes. (6) Rinse in running water for 1 minute. (7) Dehydrate: immerse in 60%, 75%, 95%, and 100% ethanol for 2 seconds each. (8) Immerse in xylene twice, 5 minutes each time.

[0138] 5. Immunohistochemical staining

[0139] (1) Dewaxing: Immerse the sections in xylene twice for 10 minutes each time, then immerse in 100%, 95%, 70%, and 60% ethanol for 5 minutes each, and finally dewax in distilled water for 5 minutes. (2) Incubate in 3% hydrogen peroxide at room temperature for 15 minutes, then rinse in tap water and distilled water for 5 seconds. (3) Immerse in pH 8.0 EDTA and fix at 140℃ for 3 minutes. (4) Wash with PBS 3 times, 5 minutes each time. (5) Add goat serum / rabbit serum and place in a 37℃ incubator for blocking for 20 minutes. (6) Add primary antibodies Rabbit Anti-Collagen TypeⅠ, Rabbit Anti-Collagen TypeⅢ, Rabbit Anti-Collagen TypeⅣ, Rabbit Anti-Elastin, Rabbit Anti-Fibronectin and Rabbit Anti-LAMININ respectively, and place in a 4℃ refrigerator overnight. (7) Wash with PBS 3 times, 5 minutes each time. (8) Add the secondary antibody corresponding to the above primary antibody (see main reagents) respectively, and incubate in a 37℃ constant temperature incubator for 20 minutes. (9) Wash with PBS 3 times, 5 minutes each time. (10) DAB color development, observe under the microscope and terminate in time. (11) Stain the nucleus with hematoxylin for 3 minutes and rinse with running water. (12) Differentiate with 1% hydrochloric acid for 5 seconds and rinse with running water. (13) Reblue with 1% ammonia water and rinse with running water. (14) Stain with eosin for 5 minutes and rinse with running water. (15) Dehydrate, immerse in 60%, 75%, 95% and 100% ethanol for 2 seconds each. (16) Immerse in xylene twice, 5 minutes each time. (17) Seal the slide with neutral resin.

[0140] 6. Proteomic analysis

[0141] 1) Draw materials

[0142] After decellularization, 30 mg×3 of lung decellularized matrix material and 30 mg×3 of commercial Matrigel were taken from the control group and stored at low temperature (-80°C).

[0143] 2) Protein extraction

[0144] Take out the cryopreserved samples, weigh an appropriate amount into a pre-cooled mortar, add liquid nitrogen and grind thoroughly into powder. Add an appropriate amount of lysis buffer (8M urea, 1% protease inhibitor) to each group of samples, and ultrasonically lyse. Centrifuge at 4°C, 12000g for 10 minutes to remove cell debris, transfer the supernatant to a new centrifuge tube, and use the BCA kit to determine the protein concentration.

[0145] 3) Protein concentration determination

[0146] Take 5 μL of protein sample, add 0 μL, 5 μL, 10 μL, 15 μL, and 20 μL of standard to the sample wells of the enzyme label strip, add sample diluent to make up to 20 μL, and test 3 replicate wells for each;

[0147] Add 5 μL of the protein sample to be tested to the sample well of the enzyme label strip, add sample diluent to make up to 20 μL, and test 3 replicate wells each;

[0148] Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min;

[0149] A570 was measured using an ELISA reader (the optimal absorption wavelength is 562 nm, and other wavelengths between 540 and 595 nm can also be used);

[0150] Calculate the protein concentration of the samples based on the standard curve and the sample volume used.

[0151] 4) Enzymatic hydrolysis with pancreatin

[0152] Take an equal amount of each sample protein for enzymatic hydrolysis, and adjust the final volume to a uniform volume with lysis buffer. Slowly add 20% TCA, mix thoroughly, and let it stand at 4°C for 2 hours. Centrifuge at 4500g for 5 minutes, discard the supernatant, and wash the precipitate with pre-cooled acetone 2 to 3 times. Dry the precipitate, add 200mM TEAB and ultrasonically break up the precipitate, and add trypsin at a ratio of 1:50 (protease: protein) for overnight enzymatic hydrolysis. Add dithiothreitol (DTT) to a final concentration of 5mM and reduce at 56°C for 30 minutes. Add iodoacetamide (IAA) to a final concentration of 11mM and incubate at room temperature for 15 minutes (protect from light).

[0153] Take 5 μL of protein sample, add 0 μL, 5 μL, 10 μL, 15 μL, and 20 μL of standard to the sample wells of the enzyme label strip, add sample diluent to make up to 20 μL, and test 3 replicate wells for each;

[0154] Add 5 μL of the protein sample to be tested to the sample well of the enzyme label strip, add sample diluent to make up to 20 μL, and test 3 replicate wells each;

[0155] Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min;

[0156] A570 was measured using an ELISA reader (the optimal absorption wavelength is 562 nm, and other wavelengths between 540 and 595 nm can also be used);

[0157] Calculate the protein concentration of the samples based on the standard curve and the sample volume used.

[0158] 5) Liquid chromatography-mass spectrometry analysis

[0159] The peptides after enzymatic hydrolysis were dissolved in liquid chromatography mobile phase A and separated using EASY-nLC 1200 ultra-high performance liquid phase system with a stable flow rate of 550.00 nL / min. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase parameters are shown in Table 2-2.

[0160] Table 3. Liquid chromatography parameters

[0161]

[0162] The separated peptides were injected into the NSI ion source (2.3 kV) for ionization. Exploris480 was used for mass spectrometry analysis. The detection and analysis of peptide parent ions and their secondary fragments were performed using a high-resolution Orbitrap. The primary mass spectrometry scanning range was set to 400-1200 m / z, and the scanning resolution was 60000.00; the secondary mass spectrometry scanning range fixed starting point was set to 100 m / z, and the scanning resolution was 15000.00. The data acquisition mode used a data-dependent scanning (DDA) program. In order to improve the effective utilization of the mass spectrometer, the parameters were set as follows: automatic gain control (AGC) was 100%, the signal threshold was 5E4ions / s, and the maximum injection time was 50ms; the dynamic exclusion time of the tandem mass spectrometry scan was set to 20s to avoid repeated scanning of the parent ion.

[0163] 6) Database search

[0164] Secondary mass spectrometry data were searched using PD 2.4. The database was Rattus_norvegicus_10116 (29951 sequences), and a reverse library was added to the database to calculate the false positive rate (FDR) caused by random matching. At the same time, a common contamination library was added to the database to eliminate the influence of contaminating proteins in the identification results; the search parameters were set as follows: the enzyme cleavage method was Trypsin / P; the number of missed cleavage sites was 2; the minimum length of the peptide was 7 amino acid residues; the maximum number of peptide modifications was 5; the primary parent ion mass error tolerance of Firstsearch was 20.0ppm, the primary parent ion mass error tolerance of Main search was 5ppm, and the mass error tolerance of secondary fragment ions was 20.0ppm. Cysteine ​​alkylation Carbamidomethyl (C) was set as a fixed modification, and ['Acetyl (Protein N-term)', 'Oxidation (M)', 'Deamidation (NQ)'] was set as a variable modification. The quantitative method was set to LFQ, and the FDR of protein identification and PSM identification was set to 1%.

[0165] 7) Bioinformatics analysis

[0166] Subcellular localization: The cells of eukaryotic organisms are subdivided into membrane-bound regions with different functions. Some of the major components of eukaryotic cells are: extracellular space, cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum (ER), peroxisomes, vacuoles, cytoskeleton, nucleoplasm, nucleosomes, nuclear matrix, and ribosomes. We used Wolfpsort subcellular localization prediction software to analyze subcellular localization. Wolfpsort software is an updated version of PSORT / PSORT II for predicting eukaryotic sequences.

[0167] Secondary mass spectrometry data were searched using PD 2.4. The database was Rattus_norvegicus_10116 (29951 sequences), and a reverse library was added to the database to calculate the false positive rate (FDR) caused by random matching. At the same time, a common contamination library was added to the database to eliminate the influence of contaminating proteins in the identification results; the search parameters were set as follows: the enzyme cleavage method was Trypsin / P; the number of missed cleavage sites was 2; the minimum length of the peptide was 7 amino acid residues; the maximum number of peptide modifications was 5; the primary parent ion mass error tolerance of Firstsearch was 20.0ppm, the primary parent ion mass error tolerance of Main search was 5ppm, and the mass error tolerance of secondary fragment ions was 20.0ppm. Cysteine ​​alkylation Carbamidomethyl (C) was set as a fixed modification, and ['Acetyl (Protein N-term)', 'Oxidation (M)', 'Deamidation (NQ)'] was set as a variable modification. The quantitative method was set to LFQ, and the FDR of protein identification and PSM identification was set to 1%.

[0168] Method 6. Statistical analysis

[0169] SPSS 17.0 and GraphPad Prism8 software were used for data processing and analysis. The quantitative data of each group were expressed as "mean ± standard deviation". The statistical difference between the two groups was analyzed by t test; the comparison between more than two groups was analyzed by One-Way ANOVA combined with the least significant difference (LSD) test. p < 0.05 was considered significant.

[0170] Method 7. Cell culture

[0171] 7.1 Cell recovery

[0172] (1) Take out the cryopreserved tubes of Calu-3 and A549 cells from the liquid nitrogen tank and quickly thaw them in a 37°C constant temperature water bath.

[0173] (2) Transfer the cell suspension to a 15 mL centrifuge tube, add 1 mL of complete culture medium, mix thoroughly, centrifuge, and discard the supernatant.

[0174] (3) Add 1× PBS to resuspend the cells, centrifuge, and discard the supernatant.

[0175] (4) Add complete culture medium to resuspend the cells, inoculate them into the culture dish, shake well, and place them in a cell culture incubator for culture.

[0176] 7.2 Cell passaging

[0177] (1) Wash and replace the medium in a timely manner according to the cell growth situation, and subculture when the cell density reaches about 80%.

[0178] (2) Discard the culture medium and wash the cells with 1× PBS. Add an appropriate amount of 0.25% trypsin to digest the adherent cells. For semi-adherent cells, just blow them off.

[0179] (3) Observe under a microscope. When the cells become round, add complete medium to terminate digestion. Vacuum the cells thoroughly to make them completely detach. Transfer the cells to a 15 mL centrifuge tube and centrifuge. Discard the supernatant.

[0180] (4) Add 1× PBS to resuspend the cells, centrifuge, and discard the supernatant.

[0181] (5) Add complete culture medium to resuspend the cells, select an appropriate ratio based on the cell quantity and inoculate them into a new culture dish, shake well, and place in a cell culture incubator for culture.

[0182] 7.3 Cell cryopreservation

[0183] (1) Once the cells have grown to the logarithmic growth phase, they can be frozen.

[0184] (2) Discard the culture medium and wash the cells with 1× PBS. Add an appropriate amount of 0.25% trypsin to digest the adherent cells. For semi-adherent cells, just blow them off.

[0185] (3) Observe under a microscope. When the cells become round, add complete medium to terminate digestion. Vacuum the cells thoroughly to make them completely detach. Transfer the cells to a 15 mL centrifuge tube and centrifuge. Discard the supernatant.

[0186] (4) Add 1× PBS to resuspend the cells, centrifuge, and discard the supernatant.

[0187] (5) Add 1.5 mL of 4°C cell freezing solution, mix well and transfer to a cryotube. Mark the tube wall and freeze the cells using the gradient freezing method.

[0188] Method 8: Immunofluorescence

[0189] (1) Dewaxing, immerse the sections in xylene twice, 10 minutes each time, then immerse in 100%, 95%, 70%, and 60% ethanol for 5 minutes each, and finally dewax in distilled water for 5 minutes. (2) Incubate in 3% hydrogen peroxide at room temperature for 15 minutes, and then rinse in tap water and distilled water for 5 seconds. (3) Immerse in pH 8.0 EDTA and repair at 140℃ for 3 minutes. (4) Wash 5 times with PBS, 5 minutes each time. (5) Add goat serum / rabbit serum and place in a 37℃ incubator for blocking for 10 minutes. (6) Add primary antibodies Anti-CDH1 / E-cadherinAntibody, Anti-TJP1 Antibody, Anti-Prosurfactant Protein C Antibody and Anti-ACE2Antibody respectively, and place in a 4℃ refrigerator overnight. (7) Wash 5 times with PBS, 5 minutes each time. (8) Add Alex Flour 594 Affini Pure Donkey Anti-Rabbit IgG and incubate at 37°C for 20 min in a dark room. (9) Wash with PBS 3 times, 5 min each time in a dark room. (10) Seal the slides with glycerol and store in a 4°C refrigerator in a dark room.

[0190] Method 9: RT-qPCR detection of gene expression

[0191] (1) RNA extraction

[0192] 1) Extraction of total RNA from Calu-3 cells: Wash the cells to be extracted with pre-cooled PBS, add 1 mL of Trizol to each 10 cm dish, shake well to allow the lysate to fully contact the cells to lyse the cells, and use a pipette to aspirate the lysate after about 1 minute and transfer it to a 15 mL centrifuge tube. Mix well by pipetting and let stand at room temperature for 5 minutes.

[0193] 2) Extraction of total RNA from Calu-3 cells in a three-dimensional model based on Matrigel: The Matrigel matrix balls to be extracted were washed with pre-cooled PBS, and the Matrigel matrix balls were directly resuspended with Trizol. After about 1 minute, the lysate was aspirated using a pipette and transferred to a 15 mL centrifuge tube. After pipetting and mixing, the tube was allowed to stand at room temperature for 5 minutes.

[0194] 3) Extraction of total RNA from Calu-3 cells in an in vitro three-dimensional lung model based on acellular matrix: The decellularized matrix to be extracted was washed with pre-cooled PBS and taken out and placed in a 1.5 mL EP tube. Trizol was added and the matrix was crushed in a small ball mill. After sufficient crushing, the matrix was allowed to stand at room temperature for 5 minutes.

[0195] 4) Transfer the lysate to a new 1.5 mL EP tube, add 1 / 5 of its volume of chloroform, shake vigorously for 30 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4°C, 12,000 rpm for 15 minutes.

[0196] 5) Carefully aspirate the colorless liquid on the upper layer and transfer it to a new 1.5 mL EP tube. Add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 12,000 rpm for 10 minutes to see a small amount of white RNA precipitate.

[0197] 6) Discard the supernatant, add 1 mL of 75% pre-cooled ethanol (prepared with DEPC water), vortex, and centrifuge at 7500 rpm at 4°C for 5 min.

[0198] 7) Discard the supernatant and place the EP tube in a clean bench to dry until the white precipitate becomes transparent and then dissolve it in an appropriate amount of DEPC water. The obtained RNA can be used for subsequent experiments or stored in a -80°C refrigerator for future use.

[0199] (2) Reverse transcription of cDNA

[0200] 1) Determine RNA concentration and quantify 500 ng.

[0201] 2) Thaw the reverse transcription reagent at room temperature, and quickly place it on ice after thawing. Prepare a mixed solution to remove genomic DNA, mix thoroughly, centrifuge briefly, and incubate in a 42°C constant temperature water bath for 3 minutes. Then place it on ice.

[0202] 3) Prepare the reverse transcription reaction system 4) Add the Mix in the reverse transcription reaction to the reaction solution in the gDNA removal step and mix thoroughly.

[0203] 5) Incubate at 42°C for 15 min, then at 95°C for 3 min and place on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperature.

[0204] (3) Real-time fluorescence quantitative PCR (4) Statistical analysis: calculate the relative expression levels of relevant gene mRNA in cells based on the CT values ​​of different groups.

[0205] Method 10: Western Blotting

[0206] (1) Protein sample preparation

[0207] 1) Prepare RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors 2 minutes before use.

[0208] 2) Extraction of total protein from Calu-3 cells: digest the cells to be extracted, centrifuge, remove the supernatant, wash with 1× PBS, centrifuge, resuspend with RIPA, lyse on ice for 15-20 min (flick or vortex for 10 s every 5 min), centrifuge at 12000 rpm, 4°C for 20 min, and aspirate the supernatant into a new tube.

[0209] 3) Extraction of cell proteins in Matrigel-based three-dimensional models: discard the culture medium, wash three times with 1× PBS, resuspend the Matrigel matrix balls directly with RIPA, lyse on ice for 15-20 min (flick or vote for 10 s every 5 min), centrifuge at 12000 rpm, 4°C for 20 min, and aspirate the supernatant into a new tube.

[0210] 4) Extraction of cellular proteins in the in vitro three-dimensional lung model based on decellularized matrix: discard the culture medium, wash three times with 1×PBS, take out the lung decellularized matrix and place it in a 1.5 mL EP tube, add RIPA, place it in a small ball mill to crush the matrix, and after it is fully crushed, place it on ice for 15-20 minutes (flick or votex for 10 s every 5 minutes), centrifuge at 12000 rpm, 4°C for 20 minutes, and aspirate the supernatant into a new tube.

[0211] (2) Determination of protein concentration by BCA method

[0212] A. Dilute BSA standard B. Prepare BCA working solution to detect protein sample concentration

[0213] 1) Mix reagents A and B in a ratio of 50:1 to prepare the working solution. Add the mixed working solution into a 96-well plate, 200 μL of working solution per well, and set three wells for each sample as a control.

[0214] 2) Add the diluted BSA standard and the sample to be tested to a 96-well plate, 10 μL per well, mix thoroughly with the working solution, and place in a 37°C incubator in the dark for 40 minutes.

[0215] 3) Take out the 96-well plate, use a microplate reader to measure the OD value at 562 nm, and draw a standard curve of protein concentration. Calculate the protein concentration based on the measured OD value of the protein sample.

[0216] 4) The total sample volume per lane was 20 μL. 5× Loading buffer was added to make it 1× concentration according to the total sample volume. The required protein sample volume was calculated according to 30 μg protein sample / lane. When the final total sample volume was less than 20 μL, RIPA was used to make up the volume. The sample was mixed thoroughly and placed in a 95°C constant temperature metal bath for denaturation for 5 min. The sample was then placed in a -20°C refrigerator for storage.

[0217] (3) Preparation of SDS-PAGE stacking gel and separation gel

[0218] 1) Insert the prepared glass plate into the gel casting apparatus and check to ensure there is no leakage.

[0219] 2) Prepare the concentrated gel and separation gel. 3) Use a 1 mL pipette to add the prepared separation gel between the glass plates of the gel casting machine, add anhydrous ethanol to 1 / 4 of the glass plate to flatten it, let it stand at room temperature for about 40 minutes to solidify, then pour out the anhydrous ethanol and absorb it with absorbent paper.

[0220] 4) Use a 1 mL pipette to add the prepared concentrated gel to the upper 1 / 4 between the glass plates of the gel casting apparatus, be careful to avoid the formation of bubbles, then quickly insert the vertical sample comb and let it stand at room temperature for about 40 minutes to allow it to solidify.

[0221] (4) SDS-PAGE electrophoresis

[0222] 1) Install the electrophoresis tank: Install the solidified gel plate into the electrophoresis instrument, add 1× Running Buffer inside and outside the tank, ensure that the electrophoresis tank does not leak, and pull out the sample comb vertically.

[0223] 2) Start loading samples. First, add 2 μL of Prestained Protein Ladder to each lane on both sides, and then add the prepared protein samples in sequence to the middle lane, 20 μL per lane. Fill the unused comb holes with Loading buffer.

[0224] 3) Set the electrophoresis instrument to 120V and start electrophoresis. When the sample reaches the bottom of the separation gel, the electrophoresis is complete and the power is disconnected.

[0225] (5) Wet transfer

[0226] 1) Cut the PVDF membrane according to the size of the gel and soak it in anhydrous methanol for 20 seconds. Then soak the PVDF membrane, filter paper and sponge in 1× Running buffer for later use.

[0227] 2) Cut the gel. Take out the gel plate from the electrophoresis tank and carefully separate the two glass plates with the matching slicer. Cut off the excess gel and mark the corners according to the marker position. Rinse the gel surface with running water.

[0228] 3) Take out the soaked sponge and spread it on the black side of the transfer clamp, take out the soaked filter paper and spread it on the glue surface, turn the glue plate upside down, remove the glue plate, put the filter paper and glue on the sponge on the black side of the transfer clamp, spread the PVDF membrane on the glue surface (mark the front side of the contact surface between the membrane and the glue before spreading the membrane), spread it flat, spread the filter paper on the PVDF membrane, and spread the sponge on the filter paper. There should be no bubbles in the three-layer system. Finally, clamp it with a clamp.

[0229] 4) Start the transfer process. Place the black side of the transfer head toward the cathode in the transfer tank in the ice bath. Pour in pre-cooled Transbuffer so that the liquid covers the clamping plate. Cover the electrotransfer tank with the lid, set the voltage to 60V, and transfer for 3 hours.

[0230] (6) Closed

[0231] 1) After the transfer is completed, take out the PVDF membrane and soak it in TBST.

[0232] 2) Block with TBST containing 5% skimmed milk powder at room temperature for 1 h.

[0233] (7) Primary antibody incubation

[0234] 1) Dilute the antibody with antibody diluent.

[0235] 2) Soak the PVDF membrane in TBST, mark the target band area with a pen, put the PVDF membrane in plastic wrap, cut the membrane with a steel ruler and a knife, and soak it in TBST after cutting.

[0236] 3) After drying the PVDF membrane (to prevent excessive dilution), place it in an antibody incubation bag, add an appropriate amount of Anti-ACE2 Antibody, and incubate it in a 4°C refrigerator overnight.

[0237] (8) Mouse Anti-β-Actin Incubation

[0238] 1) Dilute the antibody in TBST containing 5% skim milk powder.

[0239] 2) Place the PVDF membrane in the antibody diluent and incubate on a shaker at room temperature for 1 hour.

[0240] 3) After incubation, wash the membrane with TBST for 3×15 min.

[0241] (9) ECL development

[0242] 1) Mix solution A and solution B in the chemiluminescence kit in a ratio of 1:1.

[0243] 2) Use filter paper to absorb the liquid on the PVDF membrane, place it in the imager tray, carefully drop the ECL mixed solution on the front of the PVDF membrane, adjust the exposure, and take a picture.

[0244] Method 11. Establishment of in vitro three-dimensional model based on Matrigel

[0245] (1) The two-dimensionally cultured Calu-3 cells were washed, digested, centrifuged, the supernatant was discarded, complete culture medium was added, the cells were resuspended into a cell suspension, and the cells were counted.

[0246] (2) After counting the cells, centrifuge and discard the supernatant. Resuspend the cells in Matrigel and inoculate them in a 24-well plate (inoculation density: 2.5×10 5 cells / mL), 30 μL per well.

[0247] (3) Place the 24-well plate in a cell culture incubator for 20 min. After the matrix gel turns into a gel, add 2 mL of complete culture medium for three-dimensional culture. Change the medium every 24 h.

[0248] Example 1. Preparation of Lung Decellularized Matrix

[0249] Preparation method:

[0250] Place the removed heart and lung tissue in a 10 cm cell culture dish, insert an indwelling needle (No. 22) into the pulmonary artery along the cardiac catheter, fix the indwelling needle with an artery clamp, and use a peristaltic pump to perfuse PBS into the lungs through the indwelling needle at a rate of 1 mL / min to expel bubbles. Then use a syringe to inject 40 mL of PBS into the lungs along the trachea to expand both lungs, lavage out residual blood and expel air from the lungs. After the lavage is completed, close the trachea with an artery clamp and allow the PBS solution to remain in the lungs for 30 minutes. After 30 minutes, remove the tracheal artery clamp to further deflate the lungs and continue to perfuse PBS for 15 minutes. After 15 minutes, the lungs were decellularized using the following three methods. See the decellularization device for details. Figure 1 .

[0251] Method 1, Triton X-100+SDS treatment group: 1% Triton X-100+ perfusion for 2.5 h (first 1 h: 3000 μL / min, last 1.5 h: 5000 μL / min), 0.1% SDS perfusion for 3 h (5000 μL / min), PBS elution for 12 h (5000 μL / min).

[0252] Method 2, Triton X-100+Chaps treatment group: 1% Triton X-100 perfusion for 2.5 h (first 1 h: 3000 μL / min, last 1.5 h: 5000 μL / min), 8 mM Chaps perfusion for 3 h (5000 μL / min), PBS elution for 12 h (5000 μL / min).

[0253] Method three, Triton X-100+SDS+Chaps combined treatment group: 1% Triton X-100 perfusion for 2.5 h (first 1 h: 3000 μL / min, last 1.5 h: 5000 μL / min), 0.1% SDS perfusion for 1.5 h (5000 μL / min), 8 mM CHAPS perfusion for 1.5 h (5000 μL / min), PBS elution for 12 h (5000 μL / min).

[0254] Morphological observation:

[0255] After decellularization by the three methods, the overall morphology of the lung tissue did not change significantly. As the elution process progressed, the three lung decellularization methods were able to remove the blood and cell components in the natural lung tissue, and the lung tissue gradually became transparent. The decellularized lungs prepared by the Triton+SDS+Chaps combined treatment group had the best transparency, and the dense vascular structures such as bronchi and blood vessels in the lung lobes could be clearly observed, indicating that this method can better preserve the tissue structure of the lung. However, only a small amount of vascular structure could be observed in the Triton+Chaps group and the Triton+SDS group ( Figure 2 , Scale bar = 10 mm).

[0256] Example 2, SEM test results

[0257] The cross-sectional SEM results of lung tissue and decellularized lung tissue showed that the microtissue structure of natural lung was well organized, and lung cells were distributed in the network structure composed of extracellular matrix. The microtissue structure of decellularized lung in the Triton+SDS treatment group was severely damaged, and no cellular components were visible; the microtissue structure of decellularized lung in the Triton+Chaps treatment group was well preserved, but some cells were visible in the extracellular matrix network structure; the Triton+SDS+Chaps combined treatment group not only effectively preserved the microtissue structure of natural lung, but also had no obvious cellular components ( Figure 3 , Scalebar=50μm).

[0258] The above results show that the Triton+SDS+Chaps combined elution method has the best effect on preserving the three-dimensional structure of natural lung extracellular matrix.

[0259] Example 3: HE staining and DNA quantitative analysis

[0260] HE staining results showed that compared with the natural lung, there were no visible cell residues in the Triton+SDS treatment group, but the lung tissue structure was severely damaged, with almost no intact alveolar and vascular structures. The lung tissue structure was better preserved in the Triton+Chaps treatment group, but there were more cell residues. The Triton+SDS+Chaps combined treatment group can better preserve the tissue structure of the natural lung and effectively remove the cellular components ( Figure 4 , Scale bar = 200 μm).

[0261] DNA quantification results show that compared with natural lungs, the three decellularization treatment groups can remove more than 95% of the DNA components in natural lungs (p<0.01). Triton+Chaps group 42.73±1.54ng / mg, Triton+SDS+Chaps group 22.51±1.20ng / mg, Triton+SDS group 11.36±0.62ng / mg. After comparison, the DNA content of the three decellularization treatment groups was Triton+Chaps group>Triton+SDS+Chaps group>Triton+SDS, and the differences among the groups were significant (p<0.01)( Figure 5 , ** indicates p<0.01).

[0262] Example 4: Alcian Blue staining and GAG quantitative analysis

[0263] Alcian blue staining can dye the polysaccharide components in the extracellular matrix blue and the cell components red. The staining results showed that compared with natural lung tissue, the tissue structure of the polysaccharide in the decellularized matrix of the Triton+SDS treatment group was more severely damaged, and the polysaccharide components were lost more, with no obvious visible cell components. The tissue structure of the polysaccharide in the decellularized matrix of the Triton+Chaps treatment group was well preserved, but some cells remained. The tissue structure of the polysaccharide in the decellularized matrix of the Triton+SDS+Chaps combined treatment group was well preserved, the polysaccharide content was relatively rich, and no visible cells remained ( Figure 6 , Scale bar = 100 μm).

[0264] The results of GAG quantification showed that the GAG ​​content of the three decellularized groups was significantly reduced compared with the natural lung tissue (p<0.01). The GAG ​​content of the Triton+Chaps group was 37.91±1.52μg / mg, the GAG ​​content of the Triton+SDS+Chaps group was 23.48±0.93μg / mg, and the GAG ​​content of the Triton+SDS group was 11.11±0.78μg / mg. After comparison, the GAG ​​content of the three decellularized treatment groups was Triton+Chaps group>Triton+SDS+Chaps group>Triton+SDS, and the differences among the groups were significant (p<0.01)( Figure 7 , ** indicates p<0.01).

[0265] Example 5: Immunohistochemical staining of structural proteins

[0266] We performed immunohistochemical staining on the collagen component of the main structural protein of the extracellular matrix in rat lungs and decellularized lungs. The results showed that the decellularized matrices obtained from the three decellularized treatment groups all expressed type I collagen, type III collagen, and type IV collagen. Among them, the network structure composed of collagen fibers in the lung decellularized matrix of the Triton+SDS treatment group was more severely damaged; the lung decellularized matrix of the Triton+Chaps treatment group and the Triton+SDS+Chaps combined treatment group could better retain the collagen fiber network structure, but there were more cell residues in the Triton+Chaps treatment group ( Figure 8 , Scale bar = 100 μm). The above results show that the lung acellular matrix obtained by the Triton+SDS+Chaps combined treatment group has a good collagen fiber network structure.

[0267] Example 6: Histological staining of functional proteins

[0268] EVG staining can make collagen fibers appear red and elastic fibers appear blue-black. The staining results showed that the lung decellularized matrix in the Triton+SDS treatment group was mainly composed of elastic fibers, with less collagen fibers, and the fiber structure was severely damaged. The lung decellularized matrix in the Triton+Chaps treatment group was mainly composed of collagen fibers, with less elastic fibers, and the fiber structure was well preserved. The lung decellularized matrix in the Triton+SDS+Chaps combined treatment group had rich collagen and elastic fibers, and the fiber structure was well preserved ( Fig. 9 A, Scale bar = 100 μm). The results of Elastin immunohistochemical staining were consistent with those of elastic fibers in EVG staining, and also proved that the composition and structure of elastic fibers in the lung acellular matrix of the Triton+SDS+Chaps combined treatment group were relatively intact ( Fig. 9 B). The immunohistochemical staining results of Fibronectin and Laminin showed that the decellularized matrices obtained from the three decellularized treatment groups all expressed fibronectin and laminin. Similarly, the composition and structure of fibronectin and laminin in the lung decellularized matrix of the Triton+SDS+Chaps combined treatment group were relatively intact ( Fig. 9 CD).

[0269] Example 7: Proteomic Analysis

[0270] Proteomic analysis of lung decellularized matrix

[0271] The differences in protein composition between lung decellularized matrix and Matrigel were further analyzed by proteomic studies. According to the analysis method reported by Huanjing Bi et al., all identified proteins can be classified as intracellular proteins and extracellular proteins. The analysis results showed that the total number of proteins in D-Lung was more than that in Matrigel. A total of 3333 proteins were identified from D-Lung samples, including 742 extracellular proteins; 3127 proteins were identified from Matrigel samples, including 588 extracellular proteins. The Venn diagram showed that the common proteins of D-Lung and Matrigel included 2234 intracellular proteins and 533 extracellular proteins ( Fig.10 A). We further analyzed the subcellular localization of the proteins and found that among all the identified proteins, the percentage differences in the number of plasma membrane proteins, extracellular matrix proteins, nuclear proteins, cytoplasmic proteins and other proteins in D-Lung and Matrigel were small. The number of extracellular matrix proteins in D-Lung and Matrigel accounted for 11.7% and 10.5% of the total protein in each of them, respectively ( Fig.10 B). GO analysis showed that the percentage of related proteins in D-Lung was higher than that in Matrigel ( Fig.10 C).

[0272] Differential expression of Matrisome subtype proteins in D-Lung and Matrigel

[0273] In order to analyze the composition differences between D-Lung and Matrigel in more detail, we used the Matrisome database to further annotate the extracellular matrix proteins in the identified proteins. The Matrisome database can divide extracellular matrix proteins into core ECM proteins and ECM-related proteins. The core ECM proteins include collagen, ECM glycoproteins and proteoglycans, and the ECM-related proteins include ECM accessory proteins, ECM regulatory factors and secretory factors.

[0274] We matched 225 proteins in the Matrisome database, including 115 core ECM proteins, including 22 collagens, 83 ECM glycoproteins, and 10 proteoglycans; and 107 ECM-related proteins, including 29 ECM accessory proteins, 52 ECM regulatory factors, and 26 secretory factors.

[0275] The number of proteins in D-Lung that matched the Matrisome database was higher than that in Matrigel. Except for ECM regulatory factors and ECM glycoproteins, the number of other extracellular matrix subclass proteins in D-Lung was higher than that in Matrigel ( Fig.11A). We further analyzed the differential expression of proteins and identified 1,170 differentially expressed D-Lung proteins, including 964 upregulated proteins, compared with Matrigel. After matching with Matrisome, the average expression levels of collagen, ECM accessory proteins, ECM glycoproteins, and proteoglycans in D-Lung proteins were significantly higher than those in Matrigel, the expression level of ECM regulatory factors was lower than that in Matrigel, and there was no significant difference in the expression level of secretory factors ( Fig.11 B). The volcano plot showed that there were significant differences in the expression levels of Matrisome subclass proteins between D-Lung and Matrigel, especially the collagen proteins were significantly up-regulated, such as Col1a1, Col4a4, Col5a2, Col6a6 and Col12a1, while a few proteins were down-regulated, such as Col8a1 ( Fig.11 C). The heat map shows that the expression levels of different subclasses of Matrisome proteins in D-Lung are higher than those in Matrigel, among which the differences in collagen, ECM accessory protein, ECM glycoprotein, proteoglycan and secretory factor subclasses are more obvious ( Fig.11 D).

[0276] Example 8: Establishment of an in vitro lung tissue model based on lung decellularized matrix and detection of cell status

[0277] Method for establishing in vitro lung tissue model

[0278] (1) Plating: Cut the prepared lung decellularized matrix into small discs with a diameter of 1 cm and a thickness of 3 mm, and spread them flat on a 24-well plate, 1 disc per well.

[0279] (2) Freeze-drying: Place the prepared 24-well plate in a -80°C refrigerator overnight. Take it out the next day and place it in a freeze dryer for freeze drying. The freeze-drying time is about 5 hours.

[0280] (3) Irradiation sterilization: The freeze-dried matrix is ​​irradiated and sterilized using cobalt 60 (irradiation dose 10-15 kGy). After sterilization, it can be stored for a long time for future use.

[0281] (4) The two-dimensionally cultured Calu-3 cells were washed, digested, centrifuged, the supernatant was discarded, complete culture medium (DMEM culture medium) was added, the cells were resuspended into a cell suspension, and the cells were counted.

[0282] (5) After counting the cells, centrifuge and discard the supernatant. Resuspend the cells in complete medium and inoculate them into irradiated 24-well plates (inoculation density: 2.5×10 5 cells / mL), 50 μL per well.

[0283] (6) Place the 24-well plate in a cell culture incubator for 1.5 h to allow the cell suspension to fully combine with the decellularized matrix, then add 2 mL of complete culture medium for three-dimensional culture, and change the medium every 24 h.

[0284] Cell state detection

[0285] After human lung epithelial cells Calu-3 were inoculated into rat lung decellularized matrix materials, samples were collected at different time points and HE staining was performed to observe the growth status of Calu-3 in rat lung decellularized matrix materials. The results showed that after 1 day of culture of Calu-3 cells inoculated, the cells were able to adhere to the surface of the lung decellularized matrix material and grow along the edge of the lung matrix. After 3 days of culture, the overall growth state of Calu-3 cells was good, and they often grew on the alveolar-like scaffold structure in the decellularized matrix material. After 5 days of culture, the number of Calu-3 cells in the lung decellularized matrix material increased significantly, and they were able to grow on the decellularized matrix alveolar structure and form alveolar-like structures. In addition, Calu-3 cells in a state of division and proliferation can be observed, as shown by the arrows ( Fig.12 ). The results showed that the rat lung decellularized matrix material had good biocompatibility and could support the three-dimensional culture of Calu-3 cells. Calu-3 cells were inoculated in the rat lung decellularized matrix material and could form alveolar-like biomimetic structures after 5 days of culture, indicating that the lung decellularized matrix material can be used to establish an in vitro human three-dimensional lung tissue model.

[0286] Example 9: Proliferation of Calu-3 cells in an in vitro three-dimensional lung tissue model

[0287] Detection Methods

[0288] (1) Calu-3 cells were cultured at 4×10 4 The cells were inoculated at a density of 100 / mL in two-dimensional culture and two three-dimensional models using 96-well plates.

[0289] (2) Cell viability was tested on the second day after inoculation (marked as Day 0) for cells cultured in two-dimensional culture; cell viability was tested on the third day after inoculation (marked as Day 0) for cells cultured in three-dimensional model.

[0290] (3) Discard the culture medium and add 100 μL of complete culture medium containing 10% CellTiter-Blue to each well.

[0291] (4) The 96-well plate was placed back into the incubator to react for 3 h, and then the 96-well plate was placed in an ELISA reader for detection.

[0292] (5) Cell viability was tested from day 1 to day 8 after inoculation.

[0293] Test results

[0294] After human lung epithelial cells Calu-3 were inoculated into rat lung decellularized matrix materials and Matrigel, the CellTiter-Blue kit was used to detect the proliferation of Calu-3 cells in the two materials, and a growth curve was drawn. The results showed that the growth rate of Calu-3 cells in lung decellularized matrix materials was significantly higher than that in Matrigel and in two-dimensional culture (p<0.01). The above results further indicate that lung decellularized matrix materials have good biocompatibility and can better support the growth and proliferation of human lung epithelial cells Calu-3 compared with Matrigel ( Fig.13 ).

[0295] Example 10: Expression of lung function proteins in an in vitro three-dimensional lung tissue model

[0296] Human lung epithelial cells Calu-3 were inoculated into rat lung decellularized matrix materials and cultured for 5 days to establish an in vitro three-dimensional lung tissue model. Immunofluorescence staining was used to detect the expression of CDH1, TJP1 and SFTPC proteins in the in vitro three-dimensional lung tissue model. The results showed that Calu-3 cells showed positive expression of epithelial function-related proteins such as CDH1, TJP1 and SFTPC on the alveolar-like structures in the lung decellularized matrix materials, indicating that rat lung decellularized matrix can support the function of human lung epithelial cells Calu-3. The three-dimensional lung tissue model based on rat lung decellularized matrix is ​​expected to simulate some functions of human lung epithelial tissue in vitro ( Fig.14 ).

[0297] Example 11. mRNA expression of lung function genes in an in vitro three-dimensional lung tissue model

[0298] In order to compare the differences in gene expression levels of Calu-3 cell functional proteins CDH1, TJP1 and SFTPC in the in vitro three-dimensional lung tissue model with the three-dimensional model based on Matrigel, we used RT-qPCR to detect the mRNA expression levels of CDH1, TJP1 and SFTPC genes in Calu-3 cells in the above two in vitro three-dimensional models and two-dimensional culture. The results showed that the mRNA expression levels of CDH1, TJP1, and SFTPC genes in the in vitro three-dimensional lung tissue model based on lung decellularized matrix were significantly higher than those in the two-dimensional culture group and the three-dimensional model group based on Matrigel (p<0.01). The results showed that compared with Matrigel, lung decellularized matrix can better maintain the function of human lung epithelial cells Calu-3 ( Fig.15 ).

[0299] Example 12: Identification of ACE2 expression in an in vitro three-dimensional lung tissue model

[0300] Studies have shown that ACE2 on the surface of host cells is the main receptor protein that binds to the S protein of SARS-CoV-2 and mediates the entry of the virus into host cells. In order to explore whether the three-dimensional lung tissue model based on lung decellularized matrix can be used to establish an in vitro three-dimensional infection model of SARS-CoV-2. We detected the expression of ACE2 protein in the in vitro three-dimensional lung tissue model by immunofluorescence staining. The results showed that Calu-3 cells showed positive expression of ACE2 protein on the alveolar-like structure in the lung decellularized matrix material, indicating that the rat lung decellularized matrix can support the expression of ACE2 in human lung epithelial cells Calu-3. Therefore, the three-dimensional lung tissue model based on lung decellularized matrix has the potential to establish an in vitro three-dimensional infection model of SARS-CoV-2 ( Fig.16 A).

[0301] In order to compare the difference in ACE2 gene expression levels of Calu-3 cells in the in vitro three-dimensional lung tissue model with the three-dimensional model based on Matrigel, we used RT-qPCR to detect the mRNA expression level of ACE2 gene in Calu-3 cells in the in vitro three-dimensional lung tissue model based on lung decellularized matrix. The results showed that the mRNA expression level of ACE2 gene in the in vitro three-dimensional lung tissue model based on lung decellularized matrix was significantly higher than that in the two-dimensional culture group and the three-dimensional model group based on Matrigel (p<0.01). It shows that compared with Matrigel, lung decellularized matrix can better maintain the mRNA expression of ACE2 gene in human lung epithelial cells Calu-3. ( Fig.16 B). WB results were consistent with RT-qPCR results, further indicating that the ACE2 protein expression level of Calu-3 cells in the lung acellular matrix material was significantly higher than that in the two-dimensional culture group and the three-dimensional model group of Matrigel (p<0.01, Fig.16 CD). The above results indicate that Calu-3 cells in the in vitro three-dimensional lung tissue model based on lung decellularized matrix may be more susceptible to SARS-CoV-2 infection compared with the two-dimensional model and Matrigel three-dimensional model.

[0302] Example 13. Establishment of SARS-CoV-2 pseudovirus infection model

[0303] Experimental methods:

[0304] 1. SARS-CoV-2 pseudovirus infection

[0305] (1) Cultivation of the three-dimensional model to be infected: The in vitro three-dimensional model inoculated with A549 and Calu-3 cells was cultured until the fifth day and then infected with pseudovirus.

[0306] (2) Virus infection: Aspirate the complete medium in the model to be infected, infect the cells at an MOI of 1, and incubate at 37°C for 12 h (slowly shake the culture dish during this period to promote viral infection). After 12 h of infection, change the medium and add complete medium to continue culturing for three days.

[0307] (3) SARS-CoV-2D614G, Alpha variant, Delta variant and Omicron variant pseudoviruses were provided by the laboratory of Professor Wang Xuejun of the Academy of Military Medical Sciences. The above pseudoviruses were produced by co-transfecting 293T cells with the lentiviral dual reporter gene plasmid pLDR, the lentiviral packaging plasmid psPAX2 (12260) and the SARS-CoV-2S plasmid. The lentiviral reporter gene plasmids of the D614G, Alpha and Omicron variant pseudoviruses carry the dual reporter genes eGFP and luc2 luciferase, and the lentiviral reporter gene plasmids of the Delta variant pseudovirus carry the dual reporter genes mcherry and luc2 luciferase.

[0308] 2. Histological identification of SARS-CoV-2 pseudovirus infection 3D model

[0309] (1) The infected in vitro three-dimensional model was paraffin-embedded and sectioned (the method is the same as 2.5.1) and prepared for immunofluorescence staining.

[0310] (2) Dewaxing: Immerse the sections in xylene twice for 10 min each time, then immerse them in 100%, 95%, 70%, and 60% ethanol for 5 min each, and finally dewax them in distilled water for 5 min.

[0311] (3) Incubate in 3% hydrogen peroxide at room temperature for 15 min, then rinse in tap water and distilled water for 5 s respectively.

[0312] (4) Immerse in pH 8.0 EDTA and perform high pressure repair at 140°C for 3 min.

[0313] (5) Wash with PBS 5 times, 5 min each time.

[0314] (6) Add goat serum or rabbit serum and place in a 37°C incubator for 10 min.

[0315] (7) Add Anti-ACE2 Antibody and place in a 4°C refrigerator overnight.

[0316] (8) Wash with PBS 5 times, 5 min each time.

[0317] (9) Depending on the fluorescence carried by the pseudovirus, add fluorescent secondary antibody Alex Flour594 AffiniPureDonkey Anti-Rabbit IgG or Alex Flour488 AffiniPure Donkey Anti-Rabbit IgG differentially, and incubate in a 37°C incubator for 20 min in the dark.

[0318] (10) Wash with PBS three times, 5 min each time, and protect from light.

[0319] (11) Seal the slides with glycerol and store in a refrigerator at 4°C away from light.

[0320] 3. Analysis of SARS-CoV-2 pseudovirus infection rate

[0321] Three-dimensional model specimens were obtained from the DL-A549 group, DL-Calu3 group and M-Calu3 group after infection with different pseudoviruses. Five sections were selected from each specimen for observation under a fluorescence microscope. Five fields of view were randomly selected from each section. The number of eGFP-positive cells (eGFP + cells) or the number of eRFP-positive cells (eRFP + The infection efficiency of SARS-CoV-2D614G, Alpha and Omicron variant pseudoviruses was measured using eGFP + The percentage of green fluorescent labeled cells to DAPI labeled cells (eGFP + cells / Total cells); infection efficiency of SARS-CoV-2Delta variant using mCherry + The percentage of red fluorescent labeled cells to DAPI labeled cells (mCherry + cells / Total cells).

[0322] Experimental results:

[0323] In order to study the feasibility of the in vitro three-dimensional lung tissue model based on lung decellularized matrix as an in vitro SARS-CoV-2 infection model, we used four SARS-CoV-2 pseudoviruses (D614G, Alpha, Delta, and Omicron) to in vitro infect the lung decellularized matrix three-dimensional model and Matrigel three-dimensional model (MOI = 1.0), and compared the infection efficiency of each pseudovirus in the above two three-dimensional models. Given that A549 cells do not express ACE2 (a key receptor for SARS-CoV-2 to infect cells), this experiment set up a three-dimensional lung tissue model inoculated with A549 cells in the lung decellularized matrix as a negative control.

[0324] Immunofluorescence staining showed that Calu-3 cells in the in vitro three-dimensional model based on lung decellularized matrix tended to form alveolar-like structures, while Calu-3 cells in the Matrigel three-dimensional model grew in aggregates to form cell spheres, lacking the biomimetic structure of lung tissue. In addition, the ACE2 expression of Calu-3 cells in the in vitro three-dimensional model based on lung decellularized matrix was higher than that in the Matrigel three-dimensional model, further confirming the previous RT-qPCR and WB results. Since cells infected with SARS-CoV-2 pseudoviruses can express the fluorescent reporter gene carried by the pseudovirus and thus have fluorescent signals, we observed and compared the infection efficiency of SARS-CoV-2 pseudoviruses of D614G, Alpha, Delta, and Omicron in the two three-dimensional models.

[0325] The results showed that SARS-CoV-2 pseudovirus could not infect A549 cells in the lung decellularized matrix, but could infect Calu-3 cells in the lung decellularized matrix and Calu-3 cells in the Matrigel three-dimensional model ( Figure 17-Figure 20 ). Quantitative analysis results showed that the infection rates of the above four SARS-CoV-2 pseudoviruses in the lung acellular matrix three-dimensional model were significantly higher than those in the Matrigel group (p<0.05, Fig.21 ), indicating that compared with the Matrigel three-dimensional model, the in vitro three-dimensional model based on lung decellularized matrix has a higher SARS-CoV-2 pseudovirus infection rate and can be used as an in vitro three-dimensional infection model of SARS-CoV-2.

Claims

1. A method for establishing an in vitro three-dimensional lung model based on a decellularized matrix, the method comprising the steps of freeze-drying and sterilizing the decellularized matrix, adding Calu-3 cells and cell culture medium to the decellularized matrix, and performing cell culture; The method for preparing the decellularized matrix comprises the following steps: 1) Take heart and lung tissues and perfuse the lungs with PBS through the retention needle; 2) PBS is injected into the lungs along the trachea to inflate both lungs; 3) The artery clamp is used to close the trachea, so that the PBS solution remains in the lungs; 4) Continue to perfuse with PBS after deflation; 5) Perfuse in the following order: 1% Triton X-100 for 2.5 h, 0.1% SDS for 1.5 h, 8 mM Chaps for 1.5 h, and then elute with PBS for 12 h.

2. The method as set forth in claim 1, wherein in step 5), 1% Triton X-100 is perfused for 2.5 h, with a flow rate of 3000 μL / min for the first h and 5000 μL / min for the last 1.5 h; 0.1% SDS is perfused for 1.5 h at a flow rate of 5000 μL / min); 8 mM CHAPS is perfused for 1.5 h at a flow rate of 5000 μL / min; and PBS elution is performed at a flow rate of 5000 μL / min.

3. The method as described in claim 1, wherein the lung decellularized matrix is ​​first cut into small pieces with a thickness of 3 mm and a diameter of 1 cm and then freeze-dried and sterilized.

4. The method as set forth in claim 1, wherein the cardiopulmonary tissue is obtained from a model organism.

5. The method of claim 4, wherein the model organism comprises rats, mice, sheep, cattle, horses, pigs, alpacas, rabbits, fish or monkeys.

6. The method according to claim 4, wherein the model organism is a rat.

7. The method as set forth in claim 1, wherein the PBS perfusion in step 1) is performed by a peristaltic pump, and the flow rate controlled by the peristaltic pump is 1 mL / min.

8. The method as set forth in claim 1, wherein in step 3) the PBS is left in the lungs for 30 minutes.

9. The method as set forth in claim 1, wherein the PBS perfusion in step 4) lasts for 15 min.

10. The method as claimed in claim 1, wherein the freeze-drying operation step is: placing the lung acellular matrix pieces in a -80°C refrigerator overnight, taking them out the next day and placing them in a freeze dryer for freeze drying.

11. The method of claim 10, wherein the freeze drying process in the freeze dryer lasts for about 5 hours.

12. The method as set forth in claim 1, wherein the sterilization method comprises chemical sterilization, radiation sterilization, dry heat sterilization, and moist heat sterilization.

13. The method of establishing as claimed in claim 12, wherein the sterilization method is radiation sterilization, and the radiation in the radiation sterilization comprises Co 60 Radiation, cesium 137 Rays, ultraviolet rays, X-rays and gamma rays.

14. The method of claim 1, wherein the sterilization method is to use Co 60 Sterilize.

15. The method of claim 14, wherein the Co 60 The irradiation dose is 10-15 kGy.

16. The method of claim 1, wherein the cell density when the cells are added is 2-3×10 5 cells / mL.

17. The method of claim 1, wherein the cell density when the cells are added is 2.5×10 5 cells / mL.

18. The method as claimed in claim 1, wherein the cell culture step comprises adding cells and culture medium, culturing for 1-2 hours, and then supplementing the culture medium to continue culturing.

19. The method as claimed in claim 18, wherein the cell culture step comprises adding cells and culture medium, culturing for 1.5 hours, and then supplementing the culture medium to continue culturing.

20. The method of establishing as claimed in claim 1, wherein the cell culture medium comprises TeSR-E8, mTESR1, E8, Dulbecco's modified Eagle's medium, minimum essential medium, Eagle's minimal essential medium, F-10, F-12, α-minimum essential medium, G-minimum essential medium, IMPM, AmnioMax, new second-generation amniotic fluid medium, Chang's medium, Mesem Cult-XF medium, RPMI1640, Ham's F12, DMEM / F12, Ham's F-12K Medium, Hepato ZYME-SFM, William'sEMedium, Waymouth's Medium or Hepatocyte Culture Medium.

21. The in vitro three-dimensional lung model prepared by the method of claim 1.

22. Use of the in vitro three-dimensional lung model prepared by the establishment method of claim 1 in simulating virus infection in vitro, wherein the virus includes severe acute respiratory syndrome coronavirus 2 and its D614G, Alpha, Delta, Omicron, XBB, and BA.5 variants.

Images