CN116656596A

CN116656596A - Method for establishing in-vitro lung three-dimensional model based on acellular matrix

Info

Publication number: CN116656596A
Application number: CN202310673956.7A
Authority: CN
Inventors: 魏化伟; 张凯慧; 王海滨
Original assignee: Beijing Kexin Hengye Biotechnology Co ltd
Current assignee: Beijing Kexin Hengye Biotechnology Co ltd
Priority date: 2023-06-08
Filing date: 2023-06-08
Publication date: 2023-08-29

Abstract

The invention belongs to the technical field of biology and the field of materials, and particularly relates to an in-vitro lung three-dimensional model establishment method based on a decellularized matrix. Specifically, the invention provides a method for establishing an in-vitro lung three-dimensional model based on acellular matrix, which comprises the steps of freeze-drying and sterilizing the acellular matrix, adding cells and a cell culture medium like the acellular matrix, and culturing the cells. The lung three-dimensional model prepared by the method can be used as a virus in-vitro three-dimensional infection model.

Description

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

Technical Field

The invention belongs to the technical field of biology and the field of materials, and particularly relates to an in-vitro lung three-dimensional model establishment method based on a decellularized matrix.

Background

The goal of developing in vitro three-dimensional models is to construct biomimetic tissue or organ structures that have similar structural and functional characteristics as the native tissue or organ and are capable of promoting cell-to-cell and cell-to-extracellular matrix (Extra Cellular Matrix, ECM) interactions. However, to date, no natural or synthetic material can fully replicate all of the features of the natural extracellular matrix.

The extracellular matrix is a complex network of biological macromolecules secreted by cells, providing a suitable place for cell survival and activity. The cell-free protective device not only plays physical roles of supporting, connecting, protecting and the like, but also can dynamically generate omnibearing influence on cells. The key of the decellularization strategy is to remove all cellular components and genetic material in natural tissues and organs and to preserve the three-dimensional structure and composition of extracellular matrix to the greatest extent. The acellular matrix material has the advantages that the specific structure and components of the natural tissue are reserved, and the acellular matrix material has similar mechanical properties to the natural tissue, so that a tissue-specific microenvironment can be provided for supporting the adhesion, proliferation and differentiation of cells, and the acellular matrix material has great potential in the aspect of in-vitro three-dimensional model research of various diseases. Therefore, decellularized matrix materials have been widely used in various fields of tissue engineering and regenerative medicine, and have made remarkable progress. The method for constructing the three-dimensional tissue model by inoculating seed cells on the biological scaffold material by utilizing a tissue engineering strategy is a mature in-vitro model establishment method.

Unlike other solid tissues, the structure of the lung tissue is loose and complex, contains a large number of bronchi and alveoli structures, and contains both airway and vascular double-lumen tubing, and these complex structures present new challenges for the preparation of the lung decellularized matrix material.

Disclosure of Invention

In the aspect of establishing an in-vitro three-dimensional model of the lung, the structure of the lung tissue is loose and complex, and a large number of bronchus and alveolus structures are contained, and the complex structures cause the preparation of the in-vitro three-dimensional model of the lung to be a difficult point in the field of tissue engineering research. The invention provides a method for constructing an in-vitro three-dimensional model based on lung acellular matrix, which utilizes SARS-CoV-2 pseudovirus to infect the three-dimensional model prepared by the method and can establish a virus in-vitro three-dimensional infection model.

Specifically, the invention provides the following technical scheme:

in a first aspect, the present invention provides a method for establishing a three-dimensional model of an in vitro lung based on acellular matrix, said method comprising the steps of lyophilizing, sterilizing the acellular matrix, and adding cells and cell culture medium like the acellular matrix and culturing the cells.

More preferably, the method comprises cutting lung acellular matrix into small pieces with thickness of 3mm and/or diameter of 1cm, and lyophilizing and sterilizing.

Preferably, the preparation method of the acellular matrix comprises the following steps:

1) Taking cardiopulmonary tissue, and performing PBS perfusion on the lungs through detention;

2) PBS was infused into the lungs along the trachea, expanding both lungs;

3) The arterial clamp seals the trachea, leaving the PBS solution in the lungs;

4) Continuing to perfuse with PBS after deflation;

5) The cells were sequentially perfused with Triton X-100, SDS, chaps and eluted with PBS.

Preferably, the Triton X-100 is 1% Triton X-100.

Preferably, the SDS is 0.1% SDS.

Preferably, the CHAPS is 8mM CHAPS.

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

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

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

Preferably, the PBS elution in step 5) lasts 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, 12h.

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

More preferably, 1% Triton X-100 was perfused for 2.5h (first 1h: 3000. Mu.L/min, later 1.5h: 5000. Mu.L/min), 0.1% SDS was perfused for 1.5h (5000. Mu.L/min), 8mM CHAPS was perfused for 1.5h (5000. Mu.L/min), and PBS was eluted for 12h (5000. Mu.L/min) in step 5).

Preferably, the cardiopulmonary tissue is taken from a model organism.

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

Preferably, the model organism comprises a rat, mouse, sheep, cow, horse, pig, alpaca, rabbit, fish or monkey, etc.

Most preferably, the lung acellular matrix is prepared in the example of a rat in the specific embodiment of the invention.

Preferably, the cardiopulmonary tissue is obtained by means of materials conventional in the art, as specifically indicated in general method 1 of the present invention.

More preferably, the preparation method provided by the present invention is carried out in any type of decellularization device, in particular, the decellularization device used in the present invention is schematically shown in fig. 1.

Preferably, the above step 1) is performed by peristaltic pump in PBS.

More preferably, the peristaltic pump controls a flow rate of 1mL/min.

The purpose of the PBS infusion in step 1) is to expel the air bubbles.

Preferably, in the step 2), a sufficient amount of PBS is infused to expand the two lungs, and residual blood can be irrigated and air in the lungs can be discharged; in the present invention, 40mL of PBS was perfused.

Preferably, the PBS is left in the lung in step 3) above for at least 20min, including specifically 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more min (min).

Specifically, the preparation is carried out in the present invention by taking 30min of residence as an example.

Preferably, the PBS infusion in step 4) above lasts for 0-30min; preferably, 10-20min; more preferably, 15min. The 0-30 specifically comprises 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.

Preferably, the specific operation steps of lyophilization in the model building method are: the lung acellular matrix tablet is placed in a refrigerator at-80 ℃ for overnight, taken out the next day and placed in a freeze dryer for freeze drying.

More preferably, the lyophilization process of the lyophilizer lasts about 5 hours.

Preferably, the sterilization step in the modeling method includes sterilization means well known in the art, including chemical sterilization, radiation sterilization, dry heat sterilization, wet heat sterilization.

Preferably, the lung acellular matrix is sterilized by radiation sterilization.

Preferably, the radiation in the radiation sterilization comprises Co ⁶⁰ Radiation (cobalt-60), cesium ¹³⁷ Radiation (Caesium-137, cs-137), ultraviolet (UV), X-rays, gamma rays, and the like.

Preferably, the sterilization step in the model building method is to sterilize with cobalt 60, more specifically, to sterilize the lung acellular matrix after the freeze-drying treatment by irradiation with cobalt 60.

Preferably, the irradiation dose used in the irradiation sterilization is 10-15kGy.

Preferably, the lung acellular matrix in the model building method is contained in a suitable container, and then the cell culture is carried out in the container, and the cell density is 2-3×10 when the cells are inoculated ⁵ Individual cells/mL.

Preferably, the cell density at the time of cell seeding is 2.5X10 ⁵ Individual cells/mL.

Preferably, the step of culturing the cells comprises culturing for 1-2 hours after adding the cells and the culture medium, and then supplementing the culture medium for continuous culture; preferably, the fluid is fed after 1.5 h.

Cell culture media according to the present invention include any cell culture media conventional in the art, and exemplary include TeSR-E8, mTER 1, E8, dulbecco's Modified Eagle's Medium (DMEM, minimal Essential Medium), minimum essential Medium (BME, basal Medium Eagle), F-10, F-12, alpha-minimum essential Medium (alpha-MEM, alpha-Minimal Essential Medium), G-minimum essential Medium (G-MEM, glasgo's Minimal Essential Medium), IMM (Iscove's Modified Dulbecco's Medium), amnioMax, novel second generation amniotic fluid Medium (Dulbecco's Medium, gibco, new York, USA), chang's Medium, mesem Curt-XF Medium (STEMCELL Technologies, vancouver, canawa), RPMI 540, haasgo's ' 83, haasgo's, haas well as described herein, and the like, and may include any of the Modified Eagle's Medium, dulbecco's Medium, change's Modified Eagle's Medium, change's Medium, and the like.

Most preferably, experiments were performed in DMEM medium in the specific examples of the present invention.

That is, most preferably, the preparation method includes the steps of preparing a lung acellular matrix and constructing a three-dimensional model on the basis of the lung acellular matrix. The procedure for preparing the lung acellular matrix is described in embodiment 1 of the present invention, and the procedure for constructing the three-dimensional model is described in embodiment 8 of the present invention.

Preferably, the cells comprise lung-related cells.

Preferably, the lung-related cells comprise: perivascular cells of the lung, smooth muscle cells of the microvasculature, fibroblasts of the pulmonary artery, cells of the lung cancer and endothelial cells of the pulmonary vein.

Preferably, the lung cancer cells include any type of lung cancer cells, including commercial cell lines or cells isolated from the lung of a lung cancer patient.

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

As used herein, "freeze-drying" refers to the process of using ice crystal sublimation to sublimate water from frozen food material directly from ice solids to steam without melting ice under high vacuum conditions, and freeze-drying is also known as freeze-sublimation drying, wherein the dried material retains its original chemical composition and physical properties (e.g., porous structure, colloidal properties, etc.).

On the other hand, the invention provides the in-vitro lung three-dimensional model prepared by the preparation method.

More specifically, the in vitro three-dimensional model of lung may be infected with a virus, which is used as a model for studying the lungs infected with the virus.

In another aspect, the invention provides the use of the in vitro lung three-dimensional model in simulating in vitro a viral infection situation.

Preferably, the virus comprises a human-caused disease virus, illustratively including middle east respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), subtype H5 avian influenza virus, canine coronavirus (CCoV-HuPn-2018), ebola virus, zika virus.

Preferably, the virus includes SARS-CoV-2 and its D614G, alpha, delta, omicron variant, as well as other variants of SARS-CoV-2, such as XBB, BA.5, BQ1 and the like.

Drawings

FIG. 1 shows a decellularization device used in the present invention.

FIG. 2 is a general observation of rat decellularized lung tissue.

FIG. 3 shows the result of scanning electron microscopy of natural lung and decellularized lung.

FIG. 4 shows the HE staining results of natural lungs and decellularized lungs.

In fig. 2-4, a: natural lung tissue; b: triton+sds treatment group; c: triton+chaps treatment group; d: triton+sds+chaps combined treatment group.

FIG. 5 shows the results of DNA quantitative analysis.

FIG. 6 shows Alcian blue staining results of natural and decellularized lungs.

Fig. 7 is the GAG quantitative analysis results.

FIG. 8 shows 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.

FIG. 9 shows the histological staining results of functional proteins in natural lungs and decellularized lungs.

Fig. 10 is the result of proteomic analysis of lung acellular matrix, a: venn diagrams and protein number bar diagrams of Intracellular (IN) and extracellular Egg (EX) proteins IN D-Lung and Matrigel; b: percentage of protein number at different subcellular localization in D-Lung and Matrigel; protein quantity percent bar graph under five subcellular localization 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 is the result of differential expression of Matrisome subclass protein in FIGS. 3-8D-Lung and Matrigel, A: D-Lung and Matrigel are matched with the difference analysis of the protein quantity in the matriname database; b: differential analysis of the expression levels of Matrisome subclass proteins in D-Lung and Matrigel; c: volcanic plot of D-Lung differentially expressed proteins from Matrisome subclass in Matrigel; d: D-Lung and Matrisome subclasses in Matrigel.

FIG. 12 is a graph showing the results of detecting the growth state of Calu-3 cells in the lung decellularized matrix material, and HE staining of A lung decellularized matrix material after 1 day of culture with Calu-3 cells inoculated; b: HE staining after 3 days of culture of the lung decellularized matrix material inoculated with Calu-3 cells; c: HE staining after 5 days of culture with Calu-3 cells inoculated with lung decellularized matrix material. Scale bar = 20 μm. FIG. 13 is a graph showing growth curves of Calu-3 cells in three-dimensional and two-dimensional culture in vitro, representing p <0.01.

FIG. 14 shows immunofluorescence staining of a lung function protein in an in vitro three-dimensional lung tissue model, CDH1 protein (A), TJP1 protein (B), SFTPC protein (C). Scale bar = 100 μm.

FIG. 15 shows the mRNA expression results of the lung function gene 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. * Represents p <0.01.

Fig. 16 is an identification of ACE2 expression in an in vitro three-dimensional lung tissue model, a: immunofluorescent staining of ACE2 protein; b: mRNA expression of ACE2 gene; C-D: WB analysis of ACE2 protein expression. Scale bar=100 μm, representing p <0.05, representing p <0.01.

FIG. 17 shows the result of immunofluorescence staining of an in vitro three-dimensional model of SARS-CoV-2D614G pseudovirus infection.

FIG. 18 shows the result of immunofluorescence staining of a pseudo-virus infection in vitro three-dimensional model of SARS-CoV-2Alpha variant.

FIG. 19 shows the result of immunofluorescent staining of a pseudovirus infection in vitro three-dimensional model of SARS-CoV-2Delta variant.

FIG. 20 shows the result of immunofluorescence staining of an in vitro three-dimensional model of pseudovirus infection with SARS-CoV-2Omicron variant.

FIG. 21 is a graph showing the results of infection rate of SARS-CoV-2 pseudovirus in a three-dimensional model of lung acellular matrix, SARS-CoV-2D614G (A), alpha (B), delta (C), omicron (D) pseudovirus infection rate in a three-dimensional model of lung acellular matrix. * Represents p <0.05.

In fig. 17-21, a: in vitro three-dimensional model immunofluorescence staining of lung acellular matrix inoculated A549 cells; b: in vitro three-dimensional model immunofluorescence staining of lung acellular matrix inoculated Calu-3 cells; c: immunofluorescent staining of Matrigel-seeded Calu-3 cells in vitro three-dimensional model. Scale bar = 20 μm.

Detailed Description

The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.

The invention relates to experimental materials:

1. experimental animal

Sprague-Dawley (SD) rats (200+ -20 g, male and female unlimited, clean grade, vetong Lihua).

2. Cell lines

(1) Human lung adenocarcinoma cells; a549

(2) Human lung adenocarcinoma cells; calu-3

The cell lines described above were supplied by the military medical institute Wang Xuejun teacher.

3. Main reagent

TABLE 1 major reagents

4. Preparation of the Main solution

(1) 1 XPBS: 1.44g of Na ₂ HPO ₄ 8g NaCl,0.24g KH2PO4,0.2g KCl dissolved in 500mL distilled water to a volume of 1L, and autoclaved.

(2) 0.1% sds solution: 1g of SDS was dissolved in 500mL of distilled water, and the volume was set to 1L, followed by autoclaving.

(3) 1% triton solution: 10mL of Triton was dissolved in 500mL of 1 XPBS, the volume was set to 1L, and the solution was autoclaved.

(4) 8mM Chaps solution: 4.92g of Chaps was dissolved in 500mL of distilled water and the volume was set to 1L, and autoclaved.

(5) DMEM medium: DMEM dry powder, 3.7g NaHCO ₃ 2.383g HEPES was dissolved in 1L of ultra pure water, pH was adjusted to 7.2 to 7.4, sterilized by filtration in an ultra clean bench, stored at 4℃and dispensed and added with 10% FBS and 1% penicillin when used.

The general method comprises the following steps:

method 1, cardiopulmonary sampling

SD rats were fasted for 12 hours prior to surgery and anesthetized with 7% chloral hydrate at 0.5mL/100g based on their body weight for approximately 2 minutes into the anesthetic phase. The rats were fixed supine and the neck, chest and abdomen were sterilized sequentially with 75% ethanol. Separating neck skin and muscle layer by layer and exposing trachea, performing tracheal intubation between 3/4 th tracheal cartilage rings, communicating with a small animal breathing machine for mechanical ventilation, and observing that the rat breathes steadily and the breathing frequency is consistent with that of the breathing machine after the breathing frequency is 80 times/min, wherein the tidal volume is 6 mL.

The abdominal cavity is opened by the incision in the middle of the chest and abdomen, the diaphragm is cut off to enter the chest cavity, and the two ribs are cut off vertically until the heart and lung are completely exposed. The right atrium was exsanguinated, and a syringe needle was inserted into the bottom of the right ventricle, and 15mL of heparin sodium solution (50U/mL) was injected to prevent clotting. After the color of the lung is lightened, the trachea is separated from the esophagus, and the whole heart and lung is taken out.

Method 2, SEM examination

Fresh lung tissue and the lung decellularized tissue obtained by the three methods are freeze-dried for 2 hours by a vacuum freeze dryer. A small amount of freeze-dried sample is directly adhered to the conductive adhesive, and sprayed with metal for 45 seconds by using an Oxford Quorum SC7620 sputtering film plating instrument, wherein the sprayed metal is 10mA; and then a scanning electron microscope is used for shooting the appearance of the sample, and the accelerating voltage is 3kV during appearance shooting.

Method 3, DNA quantitative detection

(1) And (3) drying: fresh lung tissue and three lung decellularized tissues are taken out of the refrigerator and then put into an EP tube with 1.5mL, marked, sealed by a sealing film and put into a vacuum freeze dryer for freeze drying for 2 hours.

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

(3) Centrifuge at 10,000rpm (11,200Xg) for 1min, pour out the supernatant, add 200. Mu.L buffer GA, shake until thoroughly suspended.

(4) Adding 20 mu LProteinase K solution, mixing, standing at 56 deg.C until tissue is dissolved, and removing water drop on the inner wall of tube cover.

(5) 200 mu L of buffer GB is added, the mixture is fully and reversely mixed, the mixture is placed at 70 ℃ for 10min, the solution is clear in strain, and water drops on the inner wall of the tube cover are removed by instantaneous separation.

(6) 200 mu L of absolute ethyl alcohol is added, and the mixture is fully and uniformly shaken for 15sec, so that flocculent precipitation can occur at the moment, and water drops on the inner wall of the pipe cover can be removed immediately.

(7) The solution obtained in the previous step and the flocculent precipitate were both added to an adsorption column CB3 (the adsorption column was placed in a collection tube), centrifuged at 12,000rpm (13,400Xg) for 30sec, the waste liquid was poured off, and the adsorption column CB3 was placed back in the collection tube.

(8) To the adsorption column CB3, 500. Mu.L of the buffer solution GD was added, and the mixture was centrifuged at 12,000rpm (13,400Xg) for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was returned to the collection tube.

(9) To the adsorption column CB3, 700. Mu.L of the buffer PW was added, and the mixture was centrifuged at 12,000rpm (13,400Xg) for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was returned to the collection tube.

(10) Repeating the step (9).

(11) The adsorption column CB3 was put back into the collection tube and centrifuged at 12,000rpm (13,400Xg) for 2min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material.

(12) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 μl of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5min, centrifuging at 12,000rpm (13,400×g) for 2min, and collecting the solution into the centrifuge tube.

(13) The DNA content in the tissue was determined using an ultra-micro spectrophotometer.

Method 4, GAG quantitative detection

(1) Sample pretreatment

1) fresh lung tissue and three lung decellularized tissues were removed, 200mg weighed, 2) placed in a pre-chilled 15mL conical centrifuge tube, 3) 3mL of ENMED clear solution was added and washed 1 time, 4) transferred to a liquid nitrogen cryopreservation tube, 5) immediately placed in a liquid nitrogen tank overnight, 6) immediately (fastest) ground into powder with a grinding rod after the next day, 7) placed in a 1.5mL centrifuge tube, 8) added with 500. Mu.L ENMED extract, 9) vigorously vortexed for 1min, thoroughly mixed, 10) placed in a 56℃constant temperature metal bath for 16h, 11) placed in a 90℃constant temperature metal bath for 10min, 12) placed in a mini-table centrifuge for 10min, at 13000rpm, 13) carefully transferred supernatant to a new 1.5mL centrifuge tube, 14) placed in an ice box for later use

(2) Standard sample preparation

1) preparing 5 centrifuge tubes with 1 to 5mL, marking the centrifuge tubes as 1 to 5 # tubes, 2) respectively adding 50 mu L of GENMED cleaning solution, 3) transferring 50 mu L of GENMED standard solution to the 1 # tube, mixing, 4) carefully transferring 50 mu L of GENMED standard solution diluted by the 1 # tube to the 2 # tube, mixing, 5) carefully transferring 50 mu L of GENMED standard solution diluted by the 2 # tube to the 3 # tube, mixing, 6) carefully transferring 50 mu L of GENMED standard solution diluted by the 3 # tube to the 4 # tube, mixing, and 7) putting the 1 to 5 # tubes into an ice box for standby; standard tube concentrations are shown in the table below

TABLE 2 Standard tube concentration

(3) Standard curve determination

1) 50. Mu.L of the GENMED standard solution prepared above was removed to a 1.5mL centrifuge tube, 2) 1mL of the GENMED staining solution was added, 3) vortexing 15s, 4) incubation was performed for 30min at room temperature, avoiding light exposure, during which 15s were vortexing 15s every 5min, 5) immediately placed in a microcentrifuge for centrifugation for 10min at 13000rpm, 6) carefully removing supernatant to ensure no water droplet residue, visible wall or underviolet or pink deposition, 7) 1mL of the GENMED dissociation solution was added, 8) vortexing 15s, 9) incubation was performed at room temperature for 5min, avoiding light exposure, ensuring adequate dissolution, 10) transfer to a new cuvette, 11) immediately placed in a spectrophotometer for detection (wavelength 656 nm): obtaining absorbance readings for standard samples, reference readings at around 0.2 to 1.0, 12) repeating experimental steps 1 to 11 four times, 13) constructing a standard curve: the ordinate (Y-axis) is on absorbance reading; the abscissa (X-axis) is the standard glycosaminoglycan content (μg)

(4) Sample measurement

1) taking 50 μl of the sample to be tested prepared above into a 1.5mL centrifuge tube, 2) adding 1mL of the end staining solution, 3) vortexing for 15s, 4) incubating for 30min at room temperature, avoiding light, during which 15s vortexing for 5min each time, 5) immediately centrifuging in a mini-bench centrifuge for 10min at 13000rpm, 6) carefully pumping out supernatant, ensuring no water droplet residue, visible wall or bottom purple or pink deposition, 7) adding 1mL of end dissociation solution, 8) vortexing for 15s, 9) incubating for 5min at room temperature, avoiding light, ensuring sufficient dissolution, 10) transferring to a new cuvette, 11) immediately placing into a spectrophotometer for detection (wavelength 656 nm): obtaining absorbance readings of the sample, 12) obtaining the corresponding glycosaminoglycan content (μg) of the sample according to the standard curve described above

(5) Concentration calculation

Samples according to the standard curve described above correspond to glycosaminoglycan content (μg)/0.050 (sample volume; mL) =μg glycosaminoglycan/mLMethod 5, histological examination 1, paraffin section preparation

(1) Fresh lung tissue and three lung decellularized tissues are fixed in 4% paraformaldehyde for 24h, or three-dimensional models cultured for 1,5 and 7 days are respectively fixed in 4% paraformaldehyde for 24h. (2) Gradient dehydration, taking out the tissue blocks, and sequentially putting the tissue blocks into 70%,80%,90%,95% and 100% ethanol solutions for 15min each. (3) Transparent, taking out the tissue block, soaking in xylene twice for 10 min/time. (4) And (5) waxing, namely taking out the tissue blocks, and soaking the tissue blocks in paraffin solution for 1h each time. (5) Embedding, namely taking out the tissue blocks, embedding the tissue blocks in the middle of an embedding box, and solidifying the tissue blocks in a freezing table. (6) Slicing, and slicing after the wax block is completely solidified, wherein the thickness is 4 mu m. (7) And (5) spreading, namely placing the cut wax sheet into a sheet bleaching machine for spreading. (8) And fishing out the slice, and fishing out the slice by using the glass slide and marking the slice. And (9) baking the slide glass, and placing the slide glass on a slide baking machine for 2-3 h. (10) the slides were placed in a 37℃incubator overnight. (11) And (5) baking the slide glass, placing the slide glass in a baking oven for 2-3 hours, and loading the slide glass into a box for standby.

2. HE staining

(1) Dewaxing, immersing the slices in xylene twice for 10min each, immersing in 100%,95%,70%, and 60% ethanol for 5min each in sequence, and dewaxing to distilled water for 5min. (2) hematoxylin staining for 5min. (3) differentiation with 1% hydrochloric acid for 5s, washing with tap water for 5min. (4) 1% ammonia water is blued, and tap water is washed for 5min. (5) eosin staining for 5min, washing with tap water for 5min. (6) Dehydrated, and immersed in 60%, 75%, 95% and 100% ethanol for 2s each in sequence. (7) immersing in xylene twice, 5 min/time. And (8) sealing the neutral resin.

3. EVG staining

(1) Dewaxing, immersing the slices in xylene twice for 10min each, immersing in 100%,95%,70%, and 60% ethanol for 5min each in sequence, and dewaxing to distilled water for 5min. (2) Verhoeffs hematoxylin staining for 30min, washing with tap water for 5min. (3) The 2% ferric trichloride solution differentiated until a black fiber gray background was observed under the mirror, and was immersed in distilled water for 5s. (4) Iodine is removed by 5% sodium thiosulfate for 1min, and the solution is soaked in distilled water for 5s. (5) eosin staining for 5min, washing with tap water for 5min. (6) Van Gieson's solution counterstaining for 5min. (7) Dehydrated, and immersed in 60%, 75%, 95% and 100% ethanol for 2s each in sequence. (8) immersing in xylene twice, 5 min/time. (9) neutral resin sealing sheet.

4. Alcian Blue staining

(1) Dewaxing, immersing the slices in xylene twice for 10min each, immersing in 100%,95%,70%, and 60% ethanol for 5min each in sequence, and dewaxing to distilled water for 5min. (2) soaking in Alcian acidizing fluid for 3min. (3) staining with Alcian staining solution for 30min. (4) flushing with running water. (5) re-dyeing the core-setting red dyeing liquid for 5min. (6) washing with running water for 1min. (7) Dehydrated, and immersed in 60%, 75%, 95% and 100% ethanol for 2s each in sequence. (8) immersing in xylene twice, 5 min/time.

5. Immunohistochemical staining

(1) Dewaxing, immersing the slices in xylene twice for 10min each, immersing in 100%,95%,70%, and 60% ethanol for 5min each in sequence, and dewaxing to distilled water for 5min. (2) 3% hydrogen peroxide is incubated for 15min at room temperature, tap water is used for soaking and washing for 5s in sequence by distilled water. (3) immersing in EDTA at pH8.0, and raising the temperature to 140℃for Wen Xiufu min. (4) PBS was washed 3 times, 5 min/time. (5) Goat serum/rabbit serum was added dropwise, and the mixture was kept in an incubator at 37℃for 20 minutes. (6) Respectively dripping an Anti-Rabbit Anti-Collagen Type I, a Rabbit Anti-Collagen Type III, a Rabbit Anti-Collagen Type IV, a Rabbit Anti-Elastin, a Rabbit Anti-fibre select and a Rabbit Anti-LAMININ, and placing in a refrigerator at 4 ℃ overnight. (7) PBS wash 3 times, 5 min/time. (8) Respectively dripping the secondary antibodies (see main reagent) corresponding to the primary antibodies, and placing the primary antibodies in a 37 ℃ incubator for incubation for 20min. (9) PBS was washed 3 times, 5 min/time. (10) DAB color development, observation under a mirror and termination at proper time. (11) hematoxylin staining nuclei for 3min, washing with running water. (12) differentiation with 1% hydrochloric acid for 5s, washing with running water. (13) 1% ammonia water bluing and running water flushing. (14) eosin dip dyeing for 5min, and washing with running water. (15) Dehydrated, and immersed in 60%, 75%, 95% and 100% ethanol for 2s each in sequence. (16) immersion in xylene twice, 5 min/time. (17) neutral resin sealing sheets.

6. Proteomic analysis

1) Drawing materials

After decellularization, the lung decellularized matrix material was taken at 30mg×3, and the control commercial Matrigel was taken at 30mg×3 and stored at low temperature (-80 ℃).

2) Protein extraction

Taking out the sample stored at low temperature, weighing a proper amount of the sample, putting the sample into a precooling mortar, adding liquid nitrogen and fully grinding the sample into powder. The samples of each group were each added with an appropriate amount of lysis buffer (8M urea, 1% protease inhibitor) and sonicated. Centrifugation at 12000g for 10min at 4℃to remove cell debris, the supernatant was transferred to a new centrifuge tube, and protein concentration was determined using the BCA kit.

3) Protein concentration determination

Taking 5 mu L of protein sample, adding 0 mu L, 5 mu L, 10 mu L, 15 mu L and 20 mu L of standard substance into sample holes of an ELISA strip, adding sample diluent to complement to 20 mu L, and detecting 3 compound holes respectively;

adding 5 mu L of protein sample to be detected into sample holes of the ELISA strip, adding sample diluent to complement to 20 mu L, and detecting 3 complex holes respectively;

200 mu L of BCA working solution is added into each hole, and the mixture is stood for reaction for 30min at 37 ℃;

a570 (optimal absorption wavelength is 562nm, other wavelengths between 540 and 595nm are applicable) is measured by an enzyme-labeled instrument;

the protein concentration of the sample was calculated from the standard curve and the sample volume used.

4) Pancreatin enzymolysis

And (3) taking an equal amount of each sample protein for enzymolysis, and regulating the final volume to be uniform by using a lysate. Slowly adding 20% TCA, mixing thoroughly, standing at 4deg.C for precipitation for 2 hr. 4500g, centrifuging for 5min, discarding supernatant, washing the precipitate with pre-cooled acetone for 2-3 times. The precipitate was dried, and after adding 200mM TEAB, the precipitate was broken up by sonication, and pancreatin was added at a ratio of 1:50 (protease: protein) for overnight enzymolysis. Dithiothreitol (DTT) was added to give a final concentration of 5mM and reduced at 56℃for 30min. Iodoacetamide (IAA) was added to a final concentration of 11mM and incubated at room temperature for 15min (protected from light).

5) Liquid chromatography-mass spectrometry analysis

And (3) dissolving the peptide segment after enzymolysis by using a liquid chromatography mobile phase A, separating by using an EASY-nLC 1200 ultra-high performance liquid system, and stabilizing the flow rate at 550.00nL/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.

TABLE 3 liquid chromatography parameters

The separated peptide fragment is injected into an NSI ion source (2.3 kV) for ionization, the mass spectrum analysis adopts Exporis 480, and the detection and analysis of the peptide fragment parent ion and the secondary fragments thereof use high-resolution Orbitrap. The scanning range of the primary mass spectrum is set to 400-1200 m/z, and the scanning resolution is 60000.00; the fixed starting point of the scanning range of the secondary mass spectrum is set to be 100m/z, and the scanning resolution is 15000.00. The data acquisition mode uses a data dependent scanning (DDA) procedure. In order to improve the effective utilization of mass spectrometry, the parameters were set as follows: automatic Gain Control (AGC) is 100%, signal threshold is 5E4ions/s, and maximum injection time is 50ms; the dynamic exclusion time for tandem mass spectrometry was set to 20s to avoid repeated scans of parent ions.

6) Database search

Secondary mass spectrometry data was retrieved using PD 2.4. The database is Rattus_norvegicus_10116 (29951 sequences), and a reverse database is added in the database to calculate false positive rate (FDR) caused by random matching, and meanwhile, a common pollution database is added in the database to eliminate the influence of pollution proteins in the identification result; the search parameters were set as follows: the enzyme cutting mode is Trypsin/P; the number of the missed cut sites is 2; the minimum length of the peptide fragment is 7 amino acid residues; the maximum modification number of the peptide fragment is 5; the First parent ion mass error tolerance of the First search is 20.0ppm, the First parent ion mass error tolerance of the Main search is 5ppm, and the mass error tolerance of the second fragment ion is 20.0ppm. Cysteine alkylation Carbamidomethyl (C) was set as a fixed modification, [ ' Acetyl (Protein N-term), ' Oxidation (M) ', ' Deamidation (NQ) ' ] as a variable modification. The quantification method was set to LFQ, and FDR for protein identification, PSM identification was set to 1%.

7) Bioinformatics analysis

Subcellular localization: eukaryotic cells are subdivided into functionally distinct membrane-bound regions. 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 analyzed subcellular localization using Wolfpsort subcellular localization prediction software. Wolfpsort software is an updated version of PSORT/PSORT II for predicting eukaryotic sequences.

Method 6, statistical analysis

Data processing and analysis were performed using SPSS 17.0 and GraphPad Prism8 software, and each set of measurement data was expressed as "mean.+ -. Standard deviation". Statistical differences between the two groups were analyzed by t-test; the comparison between more than two groups was analyzed using One-Way ANOVA in combination with the Least Significant Difference (LSD) test. p <0.05 is a significant difference.

Method 7, cell culture

7.1 cell resuscitation

(1) And taking the Calu-3 and A549 cell cryopreservation tube out of the liquid nitrogen tank, and rapidly melting the cells in a water bath kettle with constant temperature of 37 ℃.

(2) The cell suspension was transferred to a 15mL centrifuge tube, 1mL of complete medium was added, well mixed, centrifuged, and the supernatant was discarded.

(3) Cells were resuspended by adding 1 XPBS, centrifuged and the supernatant discarded.

(4) Adding complete culture medium to re-suspend cells, inoculating to a culture dish, shaking, and culturing in a cell culture box.

7.2 passage of cells

(1) And (5) cleaning and replacing the liquid in time according to the growth condition of the cells, and passaging when the cell density reaches about 80%.

(2) The culture medium is discarded, the cells are washed by 1 XPBS, and the cells growing on the wall are added with a proper amount of 0.25 percent trypsin for digestion, and the cells are semi-adhered or directly blown down.

(3) After the cells are rounded, adding a complete culture medium to stop digestion, fully blowing and sucking the cells to completely fall off, transferring the cells to a 15mL centrifuge tube for centrifugation, and discarding the supernatant.

(4) Cells were resuspended by adding 1 XPBS, centrifuged and the supernatant discarded.

(5) Adding complete culture medium to re-suspend the cells, selecting proper proportion according to the cell quantity, inoculating to a new culture dish, shaking uniformly, and placing into a cell culture box for culture.

7.3 cryopreservation of cells

(1) And (5) freezing and preserving the cells after the cells grow to the logarithmic phase.

(5) Adding 1.5mL of cell cryopreservation liquid at 4 ℃, uniformly mixing and transferring to a cryopreservation tube, marking the tube wall, and cryopreserving cells by adopting a gradient cryopreservation method.

Method 8, immunofluorescence

(1) Dewaxing, immersing the slices in xylene twice for 10min each, immersing in 100%,95%,70%, and 60% ethanol for 5min each in sequence, and dewaxing to distilled water for 5min. (2) 3% hydrogen peroxide is incubated for 15min at room temperature, tap water is used for soaking and washing for 5s in sequence by distilled water. (3) immersing in EDTA at pH8.0, and raising the temperature to 140℃for Wen Xiufu min. (4) PBS wash 5 times, 5 min/time. (5) Goat serum/rabbit serum was added dropwise, and the mixture was kept in an incubator at 37℃for 10 minutes. (6) The primary Anti-CDH1/E-cadherin Antibody, anti-TJP1 Antibody, anti-Prosurfactant Protein C Antibody and Anti-ACE2 Anti-body were added dropwise, respectively, and placed in a refrigerator at 4deg.C overnight. (7) PBS wash 5 times, 5 min/time. (8) Alex Flour594AffiniPure Donkey Anti-Rabbit IgG was added dropwise, and incubated in an incubator at 37℃for 20min, protected from light. (9) PBS was washed 3 times, 5 min/time, and protected from light. And (10) placing the glycerol sealing sheet in a refrigerator at 4 ℃ for light-shielding preservation.

Method 9, RT-qPCR detection Gene expression

(1) RNA extraction

1) Extraction of Calu-3 cell Total RNA: the cells to be extracted are washed by precooled PBS, 1mL of LTrilol is added to each 10cm dish, shaking is carried out, the lysate is fully contacted with the cells to lyse the cells, after about 1min, the lysate is sucked out by a pipette and transferred to a 15mL centrifuge tube, and after blowing and sucking are carried out evenly, the cells are kept stand for 5min at room temperature.

2) Extraction of Calu-3 cell Total RNA in Matrigel-based three-dimensional model: the Matrigel matrix ball to be extracted is washed by precooled PBS, the Matrigel matrix ball is directly resuspended by Trizol, after about 1min, the lysate is sucked out by a pipette and transferred to a 15mL centrifuge tube, and the mixture is kept stand for 5min at room temperature after blowing and sucking uniformly.

3) Extraction of Calu-3 cell Total RNA in an in vitro lung three-dimensional model based on acellular matrix: the acellular matrix to be extracted is washed by precooled PBS and is taken out and placed in a 1.5mLEP tube, trizol is added, the matrix is crushed by a small ball mill, and the mixture is left to stand for 5min at room temperature after the matrix is crushed fully.

4) The lysate was transferred to a fresh 1.5mLEP tube, 1/5 of its volume of chloroform was added, vigorously shaken for 30s, and allowed to stand at room temperature for 3min. Centrifuge at 12000rpm for 15min at 4 ℃.

5) The upper colorless liquid was carefully aspirated and transferred to a new 1.5mL EP tube, an equal volume of isopropanol was added, mixed upside down and allowed to stand at room temperature for 10min. A small amount of white RNA precipitate was visible by centrifugation at 12,000rpm for 10min at 4 ℃.

6) The supernatant was discarded, 1mL of 75% pre-chilled ethanol (formulated with DEPC water) was added, vortexed, and centrifuged at 7500rpm for 5min at 4 ℃.

7) The supernatant was discarded, the EP tube was placed in an ultra clean bench and dried until the white precipitate became clear, and then dissolved in an appropriate amount of DEPC water, and the obtained RNA was used in the subsequent experiments or stored in a-80℃refrigerator for use.

(2) Reverse transcription cDNA

1) RNA concentration was measured and 500ng was quantified.

2) Thawing the reverse transcription reagent at room temperature, rapidly placing on ice after thawing, preparing mixed solution to remove genome DNA, fully mixing, centrifuging briefly, and placing in a constant-temperature water bath at 42 ℃ for incubation for 3min. And then placed on ice.

3) Preparing a reverse transcription reaction system 4), adding Mix in the reverse transcription reaction into the reaction solution of the gDNA removal step, and fully and uniformly mixing.

5) Incubation was performed at 42℃for 15min and at 95℃for 3min, and the resulting cDNA was placed on ice for subsequent experiments or low temperature storage.

(3) And (3) carrying out real-time fluorescent quantitative PCR (4) statistical analysis, and calculating the relative expression quantity of the mRNA of the related genes in the cells according to different groups of CT values.

Method 10, western Blotting

(1) Protein sample preparation

1) RIPA lysate is prepared and protease inhibitor and phosphatase inhibitor are added 2min before use.

2) Extraction of Calu-3 cell Total protein: the cells to be extracted were digested, centrifuged, the supernatant removed, washed with 1 XPBS, centrifuged, RIPA resuspended, lysed on ice for 15-20min (flick or vortex10 s every 5 min), centrifuged at 12000rpm at 4℃for 20min and the supernatant aspirated to a new tube.

3) Extraction of cellular proteins in Matrigel-based three-dimensional models: the medium was discarded, washed three times with 1 XPBS, the Matrigel matrix pellet was resuspended directly by RIPA, lysed on ice for 15-20min (flick every 5min or votex10 s), centrifuged at 12000rpm at 4℃for 20min and the supernatant aspirated to a new tube.

4) Extraction of cellular proteins in an in vitro lung three-dimensional model based on acellular matrix: the culture medium is discarded, 1 xPBS is washed three times, the lung decellularized matrix is taken out and placed in a 1.5mLEP tube, RIPA is added, the matrix is crushed by a small ball mill, the matrix is cracked for 15-20min (uniformly flicked every 5min or votex10 s) after the matrix is crushed fully, the mixture is centrifuged at 12000rpm at 4 ℃ for 20min, and the supernatant is sucked out to a new tube.

(2) Protein concentration determination by BCA method

A. Diluting BSA standard B. Preparing BCA working solution to detect protein sample concentration

1) Reagents a and B were mixed at 50:1, adding the mixed working solution into a 96-well plate, wherein 200 mu L of working solution is needed for each well, and three wells are arranged for each sample to serve as a control.

2) The diluted BSA standard substance and the sample to be tested are added into a 96-well plate, 10 mu L of each well is fully and evenly mixed with the working solution, and then the mixture is placed in a 37 ℃ incubator for light-proof reaction for 40min.

3) The 96-well plate was taken out, OD at 562nm was measured using an enzyme-labeled instrument, and a standard curve of protein concentration was drawn. Protein concentration was calculated from the measured OD value of the protein sample.

4) And adding 5×loading buffer according to the total Loading volume of each lane to obtain 1×concentration, calculating the required protein sample volume according to 30 μg protein sample/lane, supplementing with RIPA when the total Loading amount is less than 20 μL, fully mixing, placing in a constant temperature metal bath at 95 ℃ for denaturation for 5min, and then placing in a refrigerator at-20 ℃ for standby.

(3) Preparing SDS-PAGE concentrated gel and separating gel

1) The prepared glass plate is inserted into the glue maker, and inspection is performed to ensure that no liquid leakage occurs.

2) Preparing concentrated gel and separating gel. 3) Adding the prepared separating gel between glass plates of a gel maker by using a 1mL liquid transfer device, adding absolute ethyl alcohol at 1/4 of the positions of the glass plates, flattening, standing at room temperature for about 40min to solidify, pouring the absolute ethyl alcohol, and sucking the absolute ethyl alcohol by using absorbent paper.

4) The prepared concentrated gel was added to the upper 1/4 of the gap between the glass plates of the gel maker using a 1mL pipette, carefully added to avoid bubble generation, and then quickly inserted into a vertical sample comb, and allowed to set by standing at room temperature for about 40 min.

(4) SDS-PAGE electrophoresis

1) And (3) installing an electrophoresis tank: and (3) mounting the solidified glue making plate into an electrophoresis apparatus, and adding 1 x Running Buffer inside and outside the electrophoresis tank to ensure that the electrophoresis tank is free from liquid leakage and vertically pulling out the sample comb.

2) Beginning Loading, 2. Mu. L Prestained Protein Ladder was added to each of the two lanes, then prepared protein samples were added sequentially to the middle lane, 20. Mu.L per lane, and unused comb wells were filled in with Loading buffer.

3) The electrophoresis apparatus is set at 120V to start electrophoresis, and the electrophoresis is completed when the sample runs to the bottom of the separation gel, and the power supply is disconnected.

(5) Wet transfer film

1) The PVDF film is cut according to the size of the glue and soaked in absolute methanol for 20s, and then the PVDF film, the filter paper and the sponge are soaked in a 1×running buffer together for standby.

2) And cutting the gel, taking out the gel plate in the electrophoresis tank, carefully separating the two glass plates by using matched slices, cutting off redundant gel according to the Marker position, cutting the corner mark, and flushing with running water to catch up with the gel surface.

3) Taking out the soaked sponge, spreading the soaked sponge on the black surface of the film transferring clamp, taking out the soaked filter paper, spreading the soaked filter paper on the rubber surface, reversing the rubber plate, taking down the rubber plate, placing the filter paper and the rubber on the sponge of the black surface of the film transferring clamp, spreading the PVDF film on the rubber surface (the front surface of the mark is made on the contact surface of the film and the rubber before the film is spread), spreading the PVDF film, spreading the filter paper on the PVDF film, spreading the sponge on the filter paper, ensuring that no air bubble exists in the three-layer system, and finally clamping by the clamp.

4) And (3) starting to transfer the film, placing the black surface of the transfer head towards the cathode into a transfer tank of an ice bath, pouring a precooled Trans buffer, enabling liquid to overflow the clamping plate, covering a cover of the transfer tank, setting the voltage to be 60V, and transferring the film for 3h.

(6) Closure

1) And taking out the PVDF film after finishing film transfer, and soaking the PVDF film into TBST.

2) Blocking with TBST containing 5% skimmed milk powder was performed for 1h at room temperature.

(7) Incubation with primary antibody

1) Diluting the antibody with an antibody-mating diluent.

2) Soaking PVDF film in TBST, marking target strip area with pen, placing PVDF film in preservative film, cutting film with steel ruler and knife, and soaking in TBST.

3) After the PVDF membrane was blotted dry (to prevent excessive dilution), it was placed in an Antibody incubation bag, an appropriate amount of Anti-ACE2 Antibody was added, and it was placed in a refrigerator at 4℃overnight for incubation.

(8) Mouse Anti-beta-action incubation

1) The antibodies were diluted with TBST containing 5% nonfat dry milk.

2) PVDF membrane was placed in antibody dilutions and incubated for 1h in a shaker at room temperature.

3) TBST membrane wash was performed for 3X 15min after incubation was completed.

(9) ECL method development

1) And uniformly mixing the solution A and the solution B in the chemiluminescent kit in a ratio of 1:1.

2) The liquid on the PVDF film was sucked up with filter paper, placed in an imager tray, and the ECL mixture was carefully added dropwise to the front side of the PVDF film, the exposure was adjusted, and the film was photographed.

Method 11, matrigel-based in vitro three-dimensional model establishment

(1) Two-dimensional cultured Calu-3 cells were washed, digested, centrifuged, the supernatant was discarded, complete medium was added, resuspended in cell suspension, and cell counts were performed.

(2) After cell counting, centrifugation, supernatant removal, re-suspension with Matrigel and inoculation into 24-well plates (inoculation density: 2.5X10) ⁵ cells/mL), 30 μl per well.

(3) The 24-hole plate is placed in a cell culture box for 20min, and after the matrigel is converted into gel, 2mL of complete culture medium is added for three-dimensional culture, and liquid is changed once every 24 h.

Example 1 preparation of the acellular matrix of the lung

The preparation method comprises the following steps:

the heart-lung tissue taken out was placed in a 10cm cell culture dish, an indwelling needle (No. 22) was introduced into the pulmonary artery along the cardiac catheter, the indwelling needle was fixed by an arterial clamp, PBS perfusion was performed on the lung by indwelling at a speed of 1mL/min with a peristaltic pump, and air bubbles were discharged. Then, 40ml of LPBS was injected into the lungs along the trachea to expand the lungs, lavage out residual blood and expel the lung air. After lavage, the trachea is closed with an arterial clip, leaving the PBS solution in the lungs for 30min. After 30min, the tracheal artery clamp was removed and the lungs were further deflated and the perfusion with PBS was continued for 15min. After 15min, the lung was decellularized by the following three methods, respectively. The decellularization device is shown in FIG. 1.

Method one, triton X-100+SDS treatment group: 1% Triton X-100+ was perfused for 2.5h (first 1h: 3000. Mu.L/min, later 1.5h: 5000. Mu.L/min), 0.1% SDS was perfused for 3h (5000. Mu.L/min), and PBS was eluted for 12h (5000. Mu.L/min).

Method two, triton X-100+chaps treatment group: 1% Triton X-100 was perfused for 2.5h (first 1h: 3000. Mu.L/min, later 1.5h: 5000. Mu.L/min), 8mM Chaps was perfused for 3h (5000. Mu.L/min), and PBS was eluted for 12h (5000. Mu.L/min).

Method three, triton X-100+SDS+chaps combination treatment group: 1% Triton X-100 was perfused for 2.5h (first 1h: 3000. Mu.L/min, later 1.5h: 5000. Mu.L/min), 0.1% SDS was perfused for 1.5h (5000. Mu.L/min), 8mM CHAPS was perfused for 1.5h (5000. Mu.L/min), and PBS was eluted for 12h (5000. Mu.L/min).

Morphology observation:

after three methods of decellularization, lung tissue is not substantially altered. Along with the progress of the elution procedure, the three methods for removing the cells from the lung can remove the blood and the cell components in the natural lung tissues, and the lung tissues are gradually transparent. The decellularized lung prepared by the Triton+SDS+chaps combined treatment group has optimal transparency, and can clearly observe dense bronchus, blood vessels and other vascular structures existing in lung lobes, so that the method can better retain the tissue structure of the lung. Whereas only a small number of vascular structures could be observed in the triton+chaps and triton+sds groups (fig. 2, scale bar=10 mm).

Example 2 SEM examination results

The SEM result of the cut-off section of the lung tissue and the decellularized lung tissue shows that the microstructure of the natural lung is organized in order, and the lung cells are distributed in a network structure formed by extracellular matrixes. The micro-tissue structure of the decellularized lung of the Triton+SDS treatment group is seriously damaged, and no visible cell component exists; the micro-tissue structure of the decellularized lung of the triton+chaps treated group remained better, but part of the cell residue was visible in the extracellular matrix network structure; the Triton + SDS + Chaps combination treatment group was not only effective in preserving the micro-tissue structure of the natural lung, but also without significant cellular components (fig. 3, scale bar = 50 μm).

The results show that the Triton+SDS+chaps combined elution method has the best effect on the three-dimensional structure retention of the extracellular matrix of the natural lung.

Example 3 HE staining and DNA quantitative analysis

HE staining results showed that compared to natural lungs, triton+sds treated groups had no visible cell residues, but the lung tissue structures were severely destroyed with almost no intact retention of alveoli and vascular structures. Lung tissue structure remained better in triton+chaps treated groups, but there was more cell residue. The Triton + SDS + Chaps combination treatment group was able to better preserve the tissue structure of the natural lung and also effectively remove cellular components (fig. 4, scale bar = 200 μm).

As a result of DNA quantification, the three decellularized treatment groups can remove more than 95% of DNA components (p < 0.01) in the natural lung compared with the natural lung. 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. By comparison, the three decellularized treated groups had DNA content of triton+chaps group > triton+sds+chaps group > triton+sds, with significant differences between groups (p < 0.01) (fig. 5, representing p < 0.01).

Example 4 Alcian Blue staining and GAG quantitative analysis

Alcian blue staining can blue stain polysaccharide components in the extracellular matrix and red stain cell components. The staining results showed that the tissue structure of polysaccharide in decellularized matrix of triton+sds treated group was severely destroyed and the polysaccharide component was lost more without obvious visible cell component compared to natural lung tissue. The tissue structure of polysaccharide in decellularized matrix of triton+chaps treated group remained better but there was some cell residue. The tissue structure of polysaccharide in decellularized matrix of triton+sds+chaps combined treatment group remained intact, polysaccharide content was rich, and no cell residue was visible (fig. 6, scale bar=100 μm).

GAG quantification results showed that GAG content was significantly reduced in all three decellularized groups compared to native lung tissue (p < 0.01). GAG content of Triton+chaps group is 37.91 + -1.52 μg/mg, GAG content of Triton+SDS+chaps group is 23.48+ -0.93 μg/mg, and GAG content of Triton+SDS group is 11.11+ -0.78 μg/mg. By comparison, the three decellularized treatment groups had GAG content of triton+chaps group > triton+sds+chaps group > triton+sds, with significant differences between groups (p < 0.01) (fig. 7, representing p < 0.01).

EXAMPLE 5 immunohistochemical staining of structural proteins

We performed immunohistochemical staining of collagen components in the major structural proteins of extracellular matrix in rat lungs and decellularized lungs. The results indicate that the acellular matrix obtained in the three acellular treatment groups all expressed type I, type III and type IV collagen. Wherein, the network structure formed by collagen fibers in the lung acellular matrix of the Triton+SDS treatment group is seriously damaged; lung decellularized matrix from triton+chaps treatment and triton+sds+chaps combined treatment better retained collagen fiber network structure, but there was more cell residue in triton+chaps treatment (fig. 8, scale bar=100 μm). The results show that the lung acellular matrix obtained by the Triton+SDS+chaps combined treatment group has a better collagen fiber network structure.

Example 6 histological staining of functional proteins

The EVG dyeing can make the collagen fiber red and the elastic fiber blue-black. The staining results show that the lung acellular matrix of the triton+sds treated group is mainly elastic fiber, the content of collagen fiber is less, and the fiber structure is seriously damaged. The lung acellular matrix of the Triton+chaps treatment group is mainly collagen fiber, the content of elastic fiber is low, and the fiber structure is well preserved. The lung decellularized matrix of the Triton + SDS + Chaps combination treatment group was rich in both collagen and elastic fibers and the fiber structure remained intact (fig. 9a, scale bar = 100 μm). The results of Elastin immunohistochemical staining were consistent with those of elastic fibers in EVG staining, again demonstrating that the composition and structure retention of elastic fibers in the lung decellularized matrix of the Triton + SDS + Chaps combination treatment group was relatively intact (fig. 9B). Immunohistochemical staining results of Fibronectin and Laminin show that the acellular matrix obtained in the three acellular treatment groups all express Fibronectin and Laminin. Likewise, the composition and structural retention of fibronectin and laminin in the lung decellularized matrix of the Triton+SDS+chaps combination treatment group was relatively intact (FIGS. 9C-D).

Example 7 proteomic ComponentsAnalysis

Proteomic analysis of lung acellular matrix

The differences in protein composition between the lung decellularized matrix and Matrigel were further analyzed by proteomics studies. All identified proteins can be categorized as intracellular and extracellular proteins according to the analytical methods reported by huanjin Bi et al. The analysis showed that the total protein number of D-Lung was greater than Matrigel, wherein 3333 proteins, including 742 extracellular proteins, were identified in total from the D-Lung sample; 3127 proteins, including 588 extracellular proteins, were identified from Matrigel samples. Venn diagram shows that the common protein of D-Lung and Matrigel includes 2234 intracellular proteins and 533 extracellular proteins (FIG. 10A). We further analyzed the subcellular localization of proteins and found that the amounts of plasma membrane proteins, extracellular matrix proteins, nucleoproteins, cytoplasmic proteins and other proteins in D-Lung and Matrigel were less different in percentage from each other in all identified proteins, accounting for 11.7% and 10.5% of the total protein amounts, respectively (fig. 10B). GO analysis showed that the percentage of the relevant proteins in D-Lung was higher than Matrigel in both the Cellular Component (CC), biological Process (BP) and Molecular Function (MF) classes (FIG. 10C).

Differential expression of Matrisome subclass proteins in D-Lung and Matrigel

To analyze more closely the compositional differences of D-Lung and Matrigel, we used the matriname database to make further comments on the extracellular matrix proteins in the recognition proteins. The matriname database can divide extracellular matrix proteins into core ECM proteins including collagen, ECM glycoproteins, and proteoglycans, and ECM-related proteins including ECM accessory proteins, ECM modulators, and secreted factors.

We matched 225 proteins in the matriname database, with 115 core ECM proteins including 22 collagen, 83 ECM glycoproteins, 10 proteoglycans; 107 ECM-related proteins, including 29 ECM accessory proteins, 52 ECM-modulating factors, and 26 secreted factors.

The number of proteins in D-Lung that matched the matriname database was greater than that in Matrigel, with the exception of ECM regulator and ECM glycoprotein, the number of extracellular matrix subclass proteins in D-Lung was higher than Matrigel (FIG. 11A). We further analyzed the differential expression of the proteins and identified a total of 1170 differentially expressed D-Lung proteins, including 964 upregulated proteins, as compared to Matrigel. After matching with Matrigel, the average expression levels of collagen, ECM accessory protein, ECM glycoprotein and proteoglycan in D-Lung protein were significantly higher than Matrigel, ECM regulator expression levels were lower than Matrigel, and secretion factor expression levels were not significantly different (fig. 11B). Volcanic images showed significant differences in the levels of matriprime subclass protein expression of D-Lung and Matrigel, especially significant upregulation of collagen expression, such as Col1a1, col4a4, col5a2, col6a6, and Col12a1, etc., with minor protein downregulation, such as Col8a1 (fig. 11C). The heat map shows that the different subclasses of matrisomeproteins in D-Lung are expressed more frequently than Matrigel, with more pronounced differences in collagen, ECM accessory proteins, ECM glycoproteins, proteoglycans, and secreted factor subclasses (fig. 11D).

Example 8 establishment of in vitro Lung tissue model and detection of cell State based on the acellular matrix of the Lung

Method for establishing in-vitro lung tissue model

(1) And (3) paving: the prepared lung decellularized matrix is cut into small discs with the diameter of 1cm and the thickness of 3mm, and the small discs are spread on a 24-pore plate, and 1 disc/pore is formed.

(2) And (3) freeze-drying: the spread 24-pore plate is placed in a refrigerator at the temperature of minus 80 ℃ for overnight, taken out the next day and placed in a freeze dryer for freeze drying for about 5 hours.

(3) And (3) radiation sterilization: the cobalt 60 is adopted to carry out irradiation sterilization (irradiation dose is 10-15 kGy) on the freeze-dried matrix, and the freeze-dried matrix can be preserved for long term for standby after sterilization.

(4) Two-dimensional cultured Calu-3 cells were washed, digested, centrifuged, the supernatant was discarded, complete medium (DMEM medium) was added, resuspended into cell suspension, and cell counting was performed.

(5) CellsAfter counting, centrifugation, discarding the supernatant, resuspension the cells with complete medium and seeding in 24-well plates after irradiation (seeding density: 2.5X10) ⁵ Individual cells/mL), 50 μl per well.

(6) The 24-well plate is placed in a cell culture box for 1.5h, so that the cell suspension and the acellular matrix are fully combined, and then 2mL of complete culture medium is added for three-dimensional culture, and the liquid is changed once every 24 h.

Cell state detection

After human lung epithelial cells Calu-3 are inoculated into rat lung decellularized matrix material, the materials are obtained at different time points, HE staining is carried out, and the growth state of the Calu-3 in the rat lung decellularized matrix material is observed. As a result, it was found that cells were able to adhere to the surface of the lung decellularized matrix material and grow along the lung matrix edges after 1 day of culture with Calu-3 cells. After 3 days of culture, calu-3 cells grew well overall and grew on alveolar-like scaffolds in decellularized matrix material. After 5 days of culture, the number of Calu-3 cells in the lung acellular matrix material increased significantly, and were able to grow on the acellular matrix alveolar structure and form an alveolar-like structure. In addition, calu-3 cells in a division proliferation state can be observed as indicated by the arrow (FIG. 12). The result shows that the rat lung acellular matrix material has good biocompatibility and can support the three-dimensional culture of Calu-3 cells. Calu-3 cells are inoculated into rat lung acellular matrix material, and can form an alveolus-like bionic structure after 5 days of culture, which indicates that the lung acellular matrix material can be used for in vitro establishment of human three-dimensional lung tissue model.

EXAMPLE 9 proliferation of Calu-3 cells in vitro three-dimensional lung tissue model

Detection method

(1) Calu-3 cells were grown at 4X 10 ⁴ The density of each mL was inoculated into two-dimensional culture and two three-dimensional models, respectively, and 96-well plates were used.

(2) Cells cultured in two dimensions were subjected to cell viability assay on Day 2 (noted Day 0) after inoculation; three-dimensional model cell viability assays were performed on Day 3 (noted Day 0) after cell seeding.

(3) The medium was discarded and 100. Mu.L of complete medium containing 10% CellTiter-Blue was added to each well.

(4) The 96-well plate is put back into an incubator to react for 3 hours, and then the 96-well plate is placed in an enzyme-labeled instrument for detection.

(5) Cell viability was examined sequentially from day 1 to day 8 post inoculation.

Detection result

After human lung epithelial cells Calu-3 are inoculated into rat lung decellularized matrix material and Matrigel, proliferation of Calu-3 cells in the two materials is detected by using CellTiter-Blue kit, and a growth curve is drawn. The results show that the growth rate of Calu-3 cells in the lung decellularized matrix material is significantly higher than that of Matrigel and its growth rate in two-dimensional culture (p < 0.01). The above results further demonstrate that the lung decellularized matrix material has good biocompatibility and better supports growth and proliferation of human lung epithelial cells Calu-3 compared to Matrigel (fig. 13).

Example 10 expression of Lung function proteins in vitro three-dimensional Lung tissue models

Human lung epithelial cells Calu-3 were inoculated into rat lung decellularized matrix material and cultured for 5 days to establish an in vitro three-dimensional lung tissue model. The expression of CDH1, TJP1 and SFTPC proteins in an in vitro three-dimensional lung tissue model was detected using immunofluorescence staining. The results show that Calu-3 cells present positive expression of these epithelial function related proteins, CDH1, TJP1 and SFTPC, on alveolar-like structures in the lung decellularized matrix material, indicating that rat lung decellularized matrix is capable of supporting the function of human lung epithelial cells Calu-3. A three-dimensional lung tissue model based on rat lung acellular matrix is expected to mimic part of the function of human lung epithelial tissue in vivo (fig. 14).

EXAMPLE 11 mRNA expression of Lung function Gene in vitro three-dimensional Lung tissue model

To compare the differences in gene expression levels of Calu-3 cell functional proteins CDH1, TJP1 and SFTPC in an in vitro three-dimensional lung tissue model with that of Matrigel-based three-dimensional models, we examined the mRNA expression levels of Calu-3 cell CDH1, TJP1 and SFTPC genes in two-dimensional culture using RT-qPCR. The results show that the mRNA expression level of CDH1, TJP1, SFTPC genes in the in vitro three-dimensional lung tissue model based on lung acellular matrix was significantly higher than in the two-dimensional culture group and the three-dimensional Matrigel-based model group (p < 0.01). The results indicate that lung decellularized matrix was better able to maintain the function of human lung epithelial cells Calu-3 compared to Matrigel (fig. 15).

Example 12 identification of ACE2 expression in vitro three-dimensional lung tissue model

Studies have shown that ACE2 on the surface of host cells is the major receptor protein that binds to the S protein of SARS-CoV-2 and mediates viral entry into host cells. To investigate whether a three-dimensional lung tissue model based on lung acellular matrix could be used for the establishment of a three-dimensional infection model in vitro of SARS-CoV-2. We examined ACE2 protein expression in an in vitro three-dimensional lung tissue model by immunofluorescence staining. The results show that Calu-3 cells present positive expression of ACE2 protein on alveolar-like structures in the lung decellularized matrix material, indicating that rat lung decellularized matrix is capable of supporting ACE2 expression by human lung epithelial cells Calu-3. Thus, a three-dimensional lung tissue model based on lung acellular matrix has the potential to model SARS-CoV-2 in vitro three-dimensional infection (FIG. 16A).

To compare differences in Calu-3 cell ACE2 gene expression levels in an in vitro three-dimensional lung tissue model with a Matrigel-based three-dimensional model, we examined mRNA expression levels of Calu-3 cell ACE2 genes in an in vitro three-dimensional lung tissue model based on lung decellularized matrix using RT-qPCR. The results showed that the mRNA expression level of ACE2 gene was significantly higher in the three-dimensional lung tissue model in vitro based on the lung acellular matrix than in the two-dimensional culture group and the three-dimensional model group based on Matrigel (p < 0.01). It was shown that lung decellularized matrix was better able to maintain mRNA expression of ACE2 gene of human lung epithelial cells Calu-3 compared to Matrigel. (FIG. 16B). WB was consistent with RT-qPCR results, further showing that ACE2 protein expression levels of Calu-3 cells in lung decellularized matrix material were significantly higher than in the two-dimensional culture and Matrigel three-dimensional model group (p <0.01, fig. 16 CD). The above results demonstrate that Calu-3 cells in an in vitro three-dimensional lung tissue model based on lung decellularized matrix may be more susceptible to SARS-CoV-2 infection than two-dimensional models and Matrigel three-dimensional models.

EXAMPLE 13 establishment of SARS-CoV-2 pseudovirus infection model

The experimental method comprises the following steps:

1. SARS-CoV-2 pseudovirus infection

(1) Culturing a three-dimensional model to be infected: in vitro three-dimensional models inoculated with A549 and Calu-3 cells were cultured to day five for pseudovirus infection.

(2) Viral infection: the complete culture medium in the model to be infected is sucked, cells are infected according to MOI 1, the culture dish is incubated for 12 hours at 37 ℃ for slow shaking to promote virus infection, the liquid is changed after 12 hours of infection, and the complete culture medium is added for continuous culture for three days.

(3) SARS-CoV-2D614G, alpha variant, delta variant and Omicron variant pseudoviruses are provided by the military medical institute Wang Xuejun teacher laboratory. The above pseudoviruses were produced by co-transfection of 293T cells with the lentiviral double reporter plasmid pLDR, the lentiviral packaging plasmid psPAX2 (12260) and the SARS-CoV-2S plasmid. Wherein the lentiviral reporter plasmid of the D614G, alpha Omicron variant pseudovirus carries dual reporter genes eGFP and luc2 luciferase, and the lentiviral reporter plasmid of the Delta variant pseudovirus carries dual reporter genes mcherry and luc2 luciferase.

2. Histological identification of SARS-CoV-2 pseudovirus infection three-dimensional model

(1) The infected in vitro three-dimensional model was paraffin-embedded and sectioned (method same as 2.5.1) and prepared for immunofluorescent staining.

(2) Dewaxing, immersing the slices in xylene twice for 10min each, immersing in 100%,95%,70%, and 60% ethanol for 5min each in sequence, and dewaxing to distilled water for 5min.

(3) 3% hydrogen peroxide is incubated for 15min at room temperature, tap water is used for soaking and washing for 5s in sequence by distilled water.

(4) Immersing in EDTA at pH8.0, and repairing at 140 deg.C under high pressure for 3min.

(5) PBS was washed 5 times, 5 min/time.

(6) Goat serum/rabbit serum was added dropwise, and the mixture was kept in an incubator at 37℃for 10 minutes.

(7) Anti-ACE2 Anti-body was added dropwise and placed in a refrigerator at 4℃overnight.

(8) PBS was washed 5 times, 5 min/time.

(9) According to the fluorescence carried by pseudoviruses, a fluorescent secondary antibody Alex Flour594 AffiniPure Donkey Anti-Rabbit IgG or Alex Flour488 AffiniPure Donkey Anti-Rabbit IgG is added in a differential mode, and the mixture is placed in a 37 ℃ incubator for incubation for 20min and protected from light.

(10) PBS was washed 3 times, 5 min/time, and protected from light.

(11) And (5) placing the glycerol sealing sheet in a refrigerator at 4 ℃ for light-shielding preservation.

3. Analysis of infection Rate of SARS-CoV-2 pseudovirus

Different pseudovirus infections were obtained from DL-A549 groups, three-dimensional model specimens after infection of DL-Calu3 group and M-Calu3 group, each specimen was observed under a fluorescence microscope from 5 sections, five fields of view were randomly selected for each section, and the number of eGFP positive cells (eGFP) was calculated by Image Pro Image analysis software, respectively ⁺ cells) or number of eRFP positive cells (eRFP) ⁺ cells) and Total cell number (Total cells). Infection efficiency of SARS-CoV-2D614G type, alpha and Omicron variant pseudovirus using eGFP ⁺ Is expressed as a percentage of the green fluorescently labeled cells of DAPI labeled cells (eGFP ⁺ cells/Total cells); infection efficiency of SARS-CoV-2Delta variant Using mcherry ⁺ Is expressed as a percentage of red fluorescent-labeled cells relative to DAPI-labeled cells (mcherry ⁺ cells/Total cells)。

Experimental results:

to investigate the feasibility of a lung acellular matrix based in vitro three-dimensional lung tissue model as a model for SARS-CoV-2 in vitro infection, we used four SARS-CoV-2 pseudoviruses (D614G, alpha, delta and Omicron) to perform in vitro infection (MOI=1.0) on a lung acellular matrix three-dimensional model with a Matrigel three-dimensional model, and compared the infection efficiencies of each pseudovirus under the two three-dimensional models. Given that a549 cells do not express ACE2 (a key receptor for SARS-CoV-2 infected cells), this experiment set up a three-dimensional lung tissue model of seeding a549 cells in the lung decellularized matrix as a negative control.

Immunofluorescence staining showed that Calu-3 cells in the three-dimensional model in vitro based on the decellularized matrix of the lung easily formed alveolar-like structures, whereas Calu-3 cells in the Matrigel three-dimensional model grew in multiple aggregates to form cytoballs, lacking the biomimetic structure of the lung tissue. Furthermore, the higher ACE2 expression of Calu-3 cells in the three-dimensional model in vitro based on lung acellular matrix than in the Matrigel three-dimensional model further corroborated the previous RT-qPCR and WB results. Since the cells infected with SARS-CoV-2 pseudovirus can express the fluorescent reporter gene carried by the pseudovirus and thus have fluorescent signals, we observed and compared the infection efficiency of the SARS-CoV-2 pseudovirus of D614G, alpha, delta and omacron in two three-dimensional models, respectively.

As a result, it was found that SARS-CoV-2 pseudovirus was unable to infect A549 cells in the lung acellular matrix, but was able to infect Calu-3 cells in the lung acellular matrix and Calu-3 cells in the Matrigel three-dimensional model (FIGS. 17-20). The quantitative analysis results show that the infection rate of the four SARS-CoV-2 pseudoviruses in the lung acellular matrix three-dimensional model is obviously higher than that of the Matrigel group (p <0.05, figure 21), and the in vitro three-dimensional model based on the lung acellular matrix has higher SARS-CoV-2 pseudovirus infection rate compared with the Matrigel three-dimensional model 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 lung three-dimensional model based on acellular matrix, comprising the steps of freeze-drying and sterilizing the acellular matrix, adding cells and a cell culture medium like the acellular matrix, and performing cell culture;

2) PBS was infused into the lungs along the trachea, expanding both lungs;

3) The arterial clamp seals the trachea, leaving the PBS solution in the lungs;

4) Continuing to perfuse with PBS after deflation;

2. The method of claim 1, wherein said Triton X-100 is 1% Triton X-100, said SDS is 0.1% SDS, and said CHAPS is 8mM CHAPS;

preferably, 1% Triton X-100 in step 5) is perfused for 1-4h; preferably, 2-3 hours; more preferably, 2.5h;

preferably, 0.1% SDS in said step 5) is perfused for 0.5-3h; preferably, 1-2h; more preferably, 1.5h;

preferably, the 8mM CHAPS of step 5) is perfused for 0.5-3 hours; preferably, 1-2h; more preferably, 1.5h;

preferably, the PBS elution in step 5) lasts at least 8 hours; preferably, at least 10 hours; more preferably, at least 12 hours; more preferably, 12h;

preferably, 1% Triton X-100 in step 5) is perfused for 2.5h,0.1% SDS is perfused for 1.5h,8mM CHAPS is perfused for 1.5h, and PBS is eluted for 12h;

more preferably, 1% Triton X-100 is perfused for 2.5h in step 5), the first 1h:3000 μL/min, and the second 1.5h:5000 μL/min;0.1% SDS perfused for 1.5h, 5000. Mu.L/min); 8mM CHAPS perfused for 1.5h, 5000. Mu.L/min; PBS was eluted for 12h at 5000. Mu.L/min.

3. The method according to claim 1, wherein the lung acellular matrix is cut into small pieces with the thickness of 3mm and/or the diameter of 1cm, and then the small pieces are subjected to freeze-drying and sterilization treatment.

4. The method of claim 1, wherein the cardiopulmonary tissue is taken from a model organism;

preferably, the model organism comprises a mammal or a non-mammal;

preferably, the model organism comprises a rat, mouse, sheep, cow, horse, pig, alpaca, rabbit, fish or monkey;

preferably, the above step 1) is performed by peristaltic pump in PBS perfusion;

more preferably, the peristaltic pump controls a flow rate of 1mL/min;

preferably, the PBS is left in the lung for at least 20min in step 3) above;

specifically, the preparation is carried out by taking 30min of stay as an example in the invention;

preferably, the PBS infusion in step 4) above lasts for 0-30min; preferably, 10-20min; more preferably, 15min.

5. The method according to claim 1, wherein the specific operation steps of lyophilization in the model building method are: placing the lung acellular matrix tablet into a refrigerator at-80deg.C overnight, taking out the tablet the next day, and freeze-drying in a freeze dryer;

6. The method of claim 1, wherein the sterilization step of the modeling method comprises sterilization means known in the art, including chemical sterilization, radiation sterilization, dry heat sterilization, and wet heat sterilization;

Preferably, the lung acellular matrix is sterilized by radiation sterilization;

preferably, the radiation in the radiation sterilization comprises Co ⁶⁰ Radiation, cesium ¹³⁷ Radiation, ultraviolet, X-rays, and gamma rays;

preferably, the sterilization step in the modeling method is to use Co ⁶⁰ Sterilizing;

7. The method of claim 1, wherein the lung acellular matrix is contained in a suitable containerThe cell culture is carried out in the container, and the cell density is 2-3×10 when inoculating cells ⁵ Individual cells/mL;

8. The method of claim 1, wherein the step of culturing the cells comprises culturing the cells for 1-2 hours after adding the cells and the medium, and then supplementing the medium to continue the culturing; preferably, after 1.5h, the culture medium is replenished for further culture;

preferably, the cell culture Medium comprises TeSR-E8, mTER 1, E8, dalberg modified eagle Medium, minimal essential Medium, eagle minimal Medium, F-10, F-12, alpha-minimal essential Medium, G-minimal essential Medium, IMPM, amnioMax, novel second generation amniotic fluid Medium, chang's Medium, mesem matrix-XF Medium, RPMI1640, ham's F12, DMEM/F12, ham's F-12 KMedeum, hepatozYME-SFM, william's EMedium, waymouth's Medium, or Hepatocyte Culture Medium;

Preferably, the cells comprise lung-related cells;

preferably, the lung-related cells comprise: perivascular cells of the lung, smooth muscle cells of the microvasculature, fibroblasts of the pulmonary artery, cells of the lung cancer, endothelial cells of the pulmonary vein;

preferably, the lung cancer cells include any type of lung cancer cells, including commercial cell lines or cells isolated from the lung of a lung cancer patient;

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

10. The use of the in vitro lung three-dimensional model prepared by the method of claim 1 in simulating in vitro a viral infection;

preferably, the virus comprises a human-caused disease virus; the human-caused disease virus comprises a middle east respiratory syndrome coronavirus, a severe acute respiratory syndrome coronavirus 2, an H5 subtype avian influenza virus, a canine coronavirus, an Ebola virus, an Zika virus and variants of the above viruses;

preferably, the virus comprises severe acute respiratory syndrome coronavirus 2 and D614G, alpha, delta, omicron, XBB, ba.5 variants thereof.