Fluorescence and bar code pedigree tracking alveolar organ and construction method and application thereof

Abstract

The invention relates to the field of biology, in particular to a fluorescence and bar code pedigree tracking alveolar organ and a construction method and application thereof. The invention particularly aims at the problems of specific sorting of AT2 cells, culture of organoids and pedigree retracking in the disease process, and is used for simulating and researching the in-vitro pathological process of chronic obstructive pulmonary disease (COPD). The alveolar organ with fluorescence and bar code pedigree tracking is constructed and can track the fate transformation of alveolar type II cells (AT2) in a disease model, and the cell pedigree is further decoded, so that a new research basis is provided for early diagnosis and treatment of COPD (chronic obstructive pulmonary disease).

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to an alveolar organoid with fluorescence and barcode lineage tracking, and a construction method and application thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease, which is mainly manifested by airway obstruction and lung tissue destruction. Its main pathological feature is the damage and remodeling of alveolar epithelial cells, which is characterized by irreversible respiratory airflow limitation and worsens over time. COPD is the third leading cause of death in the world and the main cause of chronic disability and death in the world. The morbidity and mortality of COPD are high, which brings a heavy economic burden to patients themselves, their families and society. Due to the continuous deterioration of the global environment and the aging of the population, the burden of COPD will gradually increase in the next few decades. The pathogenesis of COPD is very complex, including oxidative stress, inflammation, protease-antiprotease imbalance and cell apoptosis.

[0003] The main pathological features of COPD include: chronic lung inflammation, airway remodeling, emphysema and impaired lung function. Chronic lung inflammation is manifested by the infiltration of inflammatory cells in the bronchial walls at all levels. Airway remodeling is mainly due to the proliferation of airway smooth muscle cells, the activation of fibroblasts into myofibroblasts, and the deposition of extracellular matrix. The pathological manifestations of emphysema are enlargement and rupture of alveolar spaces and destruction of elastic fiber networks. As the main pathological feature of COPD, emphysema has always been the focus of COPD research. COPD is a preventable and treatable disease, but because its etiology and pathogenesis are still unclear, there is currently no way to effectively prevent the occurrence or progression of COPD.

[0004] Reliable in vitro models can effectively shorten the exploration of the pathogenesis and treatment of COPD, but existing in vitro models are difficult to accurately simulate the microenvironment and cell behavior of the human lung. The regeneration and repair mechanism of alveolar type II (AT2) cells is an important research direction for lung diseases, but existing technologies have certain limitations in sorting AT2 cells and studying their lineage retracing. Traditional methods are difficult to achieve specific sorting and dynamic tracking of AT2 cells at the same time, which limits the systematic study of abnormal regeneration and repair of AT2 cell lineages during the disease process. Therefore, it is necessary to find a method that can specifically sort and dynamically track AT2 cells. Summary of the invention

[0005] The purpose of the present invention is to provide an alveolar organoid with fluorescence and barcode lineage tracing and its construction method and application to solve the problems existing in the above-mentioned prior art. The present invention develops an alveolar organoid carrying AT2 cell lineage-specific fluorescent marker barcode, which can simulate the structural characteristics of alveolar tissue, and combines fluorescence lineage tracing and barcode technology to accurately track the fate and functional changes of AT2 cells under in vitro conditions, providing a new research tool for COPD research.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing alveolar organoids with fluorescence and barcode lineage tracing, comprising the following steps:

[0008] The SPC-Cre ERT2 tool mice and R26R tdTomato reporter mice were mated to obtain reporter mice;

[0009] The reporter mice were mated with each other to obtain SPC-Cre ERT2 + / - ,R26RtdTomato + / + lineage-tracing mice;

[0010] The rtTA tool mice were mated with HMF9-barcode mice to obtain barcoded lineage-tracing mice carrying rtTA / HMF9;

[0011] The SPC-Cre ERT2 + / - , R26R tdTomato + / + After mating the lineage tracing mice with the barcoded lineage tracing mice carrying rtTA / HMF9, SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-marked lineage tracing mice were obtained;

[0012] Taking lung tissue from SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-labeled lineage tracing mice, isolating alveolar type II cells, and culturing them to obtain the alveolar organoids;

[0013] The culture medium used in the culture is based on Advance DMEM / F12 medium, and also includes 1×B27, 1×penicillin-streptomycin, 5-10μM Y-27632, 2-5μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

[0014] Further preferably, the culture medium used in the culture is Advance DMEM / F12 medium as the basal medium, and also includes 1×B27, 1×penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

[0015] Further preferably, when taking the lung tissue of the SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-marked lineage tracing mouse, non-lung tissue (such as trachea, mucosa) should be removed as much as possible, and the lung tissue should be minced into a homogenate.

[0016] Preferably, the separation of alveolar type II cells comprises the steps of digesting, centrifuging, filtering, lysing and sorting the lung tissue.

[0017] Preferably, the digestive enzymes used in the digestion include 2 mg / mL tissue collagenase I, 5 mg / mL dispase, 0.01 mg / mL DNase and 3 mL DMEM-F12 culture medium;

[0018] The digestion temperature is 36.5°C-37.5°C and the digestion time is 30-40 minutes.

[0019] Further preferably, during the digestion process, the lung tissue is blown every 10-15 minutes to effectively increase the lysis efficiency.

[0020] Preferably, the centrifugal condition parameters are: centrifugation at 500 rcf for 10 min;

[0021] The erythrolysis condition parameters are: incubation at 4° C. for 5 minutes.

[0022] Preferably, the sorting method includes magnetic bead sorting and flow sorting.

[0023] The present invention provides alveolar organoids constructed using the above-mentioned construction method.

[0024] The present invention provides the use of the alveolar organoids in screening or preparing drugs for preventing or treating chronic obstructive pulmonary disease.

[0025] The present invention provides a method for inducing the above-mentioned alveolar organoid lineage fluorescence and barcode mutation, using tamoxifen and doxycycline to induce and culture the alveolar organoid;

[0026] The concentration of tamoxifen is 250nM-500nM, and the induction culture time is 24-48h;

[0027] The concentration of doxycycline is 1 μg / mL, and the induction culture time is 10 days.

[0028] The present invention provides a culture medium for culturing alveolar organoids, wherein the culture medium is based on Advance DMEM / F12 culture medium and further comprises 1×B27, 1×penicillin-streptomycin, 5-10μM Y-27632, 2-5μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

[0029] Further preferably, the culture medium used in the culture is Advance DMEM / F12 medium as the basal medium, and also includes 1×B27, 1×penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

[0030] The present invention provides use of the above-mentioned culture medium in culturing alveolar organoids.

[0031] The present invention discloses the following technical effects:

[0032] 1. Use SPC-tdTOMATO fluorescent marker to specifically sort out the AT2 lineage and improve sorting efficiency and accuracy.

[0033] 2. The alveolar organoids obtained by the construction method of the present invention can be used to observe the differentiation lineage of AT2 in vitro under the induction of tamoxifen; under the induction of doxycycline, the barcodes can accumulate mutations and perform cell lineage tracking, providing a reliable platform for the exploration of disease mechanisms.

[0034] 3. Reduce experimental costs: Using organoid models to explore disease mechanisms is reproducible and reliable, avoiding the shortcomings of expensive animal experiments and long experimental cycles.

[0035] 4. The present invention also optimizes the culture medium scheme of mouse alveolar organoids to make the formation efficiency of organoids higher.

[0036] In summary, the present invention is particularly aimed at the specific sorting of AT2 cells and the culture of organoids and the lineage retracing problem in the disease process, and is used to simulate and study the in vitro pathological process of COPD. By combining the fluorescence lineage tracing technology with the barcode analysis technology, a fluorescence and barcode lineage tracing alveolar organoid is constructed, which can track the fate transformation of alveolar type II cells (AT2) in the disease model and further decode the cell lineage, thereby providing a new research basis for the early diagnosis and treatment of COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 Breeding for double-marker lineage tracing mice;

[0039] Figure 2 Schematic diagram of magnetic bead sorting method;

[0040] Figure 3 Schematic diagram of flow sorting method;

[0041] Figure 4 In vitro induction of tamoxifen at different concentrations; A is the fluorescence image and bright field image of alveolar organoids treated with tamoxifen at different concentrations; B is the quantitative image of organoid formation;

[0042] Figure 5 This is a fluorescent image of organoid culture using magnetic bead sorting;

[0043] Figure 6 AT2 cells were flow-sorted and cultured for organoids; A is a gate image of flow sorting; B is a fluorescence image of organoid culture using flow sorting;

[0044] Figure 7 It is the growth diagram of organoids under different Dox induction concentrations; among them, Alveolar Maintain Phase D2 is the second day of the cell maintenance period, Alveolar Maintain Phase D5 is the fifth day of the cell maintenance period, Alveolar Maintain Phase D9 is the ninth day of the cell maintenance period, and Alveolar Differentiation D9 is the ninth day of the cell differentiation period;

[0045] Figure 8 Statistical graph of organoid formation;

[0046] Fig. 9 This is a statistical diagram of organoid diameter;

[0047] Fig.10 is the barcode mutation situation;

[0048] Fig.11 This is a bright field image of organoid growth during the proliferation period of the organoid culture after adding different cytokines;

[0049] Fig.12 This is a statistical chart of the number of organoids;

[0050] Fig.13 Growth of alveolar organoids in the CSE-exposed group and the Control group (A) and quantification of organoid formation efficiency (B);

[0051] Fig.14 Sequencing results of alveolar organoids exposed to CSE;

[0052] Fig.15 Organoid growth diagram after culturing organoids for different sorting methods to day 10;

[0053] Fig.16 Bright field images of alveolar and non-alveolar organoids. DETAILED DESCRIPTION

[0054] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0055] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0056] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0057] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0058] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0059] Unless otherwise specified, the materials used in the present invention can be purchased by a person skilled in the art; and the steps used in the present invention are steps well known to a person skilled in the art.

[0060] Example 1 Hybrid Mouse Construction

[0061] ①, Type II alveolar epithelial cell lineage tracing mice (SPC-Cre ERT2 + / - ,R26RtdTomato + / + Breeding of lineage-traced mice

[0062] In this example, positive mice and wild-type mice were mated to obtain heterozygous mice, and heterozygous mice were mated to obtain homozygous mice, and tamoxifen was induced, and AT2 cell lineage was identified by PCR technology and fluorescence observation. Tamoxifen induced type II alveolar epithelial cells to specifically emit red fluorescence, and this modification can be inherited to daughter cells, providing a powerful tool for studying cell fate.

[0063] The specific steps are:

[0064] The SPC-Cre ERT2 tool mice (wild type) and R26R tdTomato reporter mice (positive mice) were mated to obtain reporter mice; the reporter mice were mated with each other to obtain SPC-Cre ERT2 + / - ,R26RtdTomato + / + Lineage tracing mice, SPC-Cre ERT2 + / - , R26R tdTomato + / + The lineage-tracing mice were induced with tamoxifen (administered at a concentration of 100 mg / kg for 5 consecutive days and then stopped for 5 days), and then the AT2 cell lineage was identified by PCR technology and fluorescence observation. The results showed that SPC-Cre ERT2 + / - ,R26RtdTomato + / + Type II alveolar epithelial cells (AT2 cells) in lineage-tracing mice can specifically emit red fluorescence.

[0065] ② Construction of barcoded mice (barcoded lineage-tracing mice carrying rtTA / HMF9 or barcoded lineage-tracing mice carrying rtTA and HMF9)

[0066] Homozygous tool mice and homozygous barcode mice were selected to mate to obtain heterozygous mice, and then doxycycline was induced. The transcription of mice and barcodes was identified by PCR technology and fluorescence observation. After doxycycline induction, the two barcode regions will accumulate mutations during cell division, and the mutations can be inherited to daughter cells, providing a powerful tool for studying cell development and lineage tracing.

[0067] The rtTA tool mice (commercially available Tet-on system mice) were mated with HMF9-barcode mice (the construction method of the mice was the same as the construction method in the Chinese patent "CN202010285948.1", and the barcode sequence in the mouse system involved in the patent was also given) to obtain barcoded lineage-tracing mice carrying rtTA / HMF9. The barcoded lineage-tracing mice carrying rtTA / HMF9 were induced with Doxycycline (at a concentration of 0.1%, administered daily in the feed), and then the transcription of the mice and the barcode was identified by PCR technology and fluorescence observation. The results showed that after Doxycycline induction, the barcode region would accumulate mutations during cell division.

[0068] ③. Breeding of double-marked lineage tracing mice (SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-marked lineage tracing mice)

[0069] Specific steps are as follows Figure 1 As shown: The SPC-Cre ERT2 obtained by the above two steps of breeding + / - ,R26RtdTomato + / + Lineage-traced mice were mated with barcoded lineage-traced mice carrying rtTA / HMF9 to obtain SPC-Cre ERT2, R26RtdTomato, rtTA, HMF9-barcode heterozygous double-marked lineage-traced mice (SPC-Cre ERT2 + / - ,R26RtdTomato + / - ,rtTA + / - , HMF9-barcode + / - Heterozygous mice). SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-marked lineage tracing mice were induced with tamoxifen (same as breeding of type II alveolar epithelial cell lineage tracing mice); Doxycycline was induced at the same time (same as barcode mouse construction), and then the red fluorescence expression of type II alveolar epithelial cells and RNA barcode transcription in mice were identified by PCR technology and fluorescence microscopy. The results showed that type II alveolar epithelial cells specifically emit red fluorescence, and the barcode region accumulates mutations during cell division. This double-marked lineage tracing mouse model can provide more accurate and comprehensive data support for studying the fate, differentiation, and damage repair ability of type II alveolar epithelial cells.

[0070] Example 2 Method for culturing alveolar organoids

[0071] AT2 cells of SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-marked lineage-tracing mice were sorted out by magnetic bead sorting or flow cytometry and cultured into organoid models.

[0072] AT2 cells were isolated from lung tissue using the following steps:

[0073] After the mouse chest cavity was opened, 5 mL of DPBS solution was perfused into the right lung of the mouse using a 10 mL syringe with a 22GG needle to remove a large number of red blood cells remaining in the lung; non-lung tissues (such as trachea and mucosa) were removed as much as possible, the lung tissue was sheared into a homogenate, and digested with tissue digestion enzymes (tissue collagenase I: 2 mg / mL; dispase: 5 mg / mL; DNase: 0.01 mg / mL; DMEM-F12 culture medium: 3 mL / mL) (digestion at 37°C ± 0.5°C for 30-45 min. The present invention is digested at 37°C for 30 min. During the digestion process, the lung tissue is blown every 10-15 min to effectively increase the lysis efficiency), centrifuged at 500 rcf for 10 min, and then filtered using a filter cell sieve, and lysed red blood cells, that is, 3 mL of red blood cell lysis solution (Solabo Company) was used for incubation at 4°C for 5 min to obtain a lung single cell fluid after sieving and lysing red blood cells, and then AT2 cells were sorted using a magnetic bead sorting method or a flow sorting method.

[0074] The steps of magnetic bead separation are as follows Figure 2 Specifically, the CD45 magnetic beads were incubated with the sieved erythrocyte single cell solution, and the incubation condition parameters were: the volume ratio of the sieved erythrocyte single cell solution, magnetic beads and magnetic bead buffer was 10 7 Cells: 10μL: 90μL; incubate at 4℃ for 25min; negatively select CD45 - Cells. Reuse CD45 - The cells were incubated with CD326 magnetic beads. The incubation parameters were as follows: the volume ratio of the sieved red lung single cell solution, magnetic beads, and magnetic bead buffer was 10 7 Cells: 10μL: 90μL; incubate at 4℃ for 20min; positively select CD326 + cells. Then 10 3 -10 4 CD326 +The ratio of cells / gel droplets was seeded, wherein the volume ratio of Matrigel to DMEM / F12 in the gel droplets was 1:1. The gel droplets were solidified at 37±0.5°C for 20-30 minutes, and after the gel droplets solidified, 1 mL of alveolar organoid culture medium was added (based on Advance DMEM / F12 culture medium, also containing 1×B27, 1×penicillin-streptomycin, 10 μM Y-27632, 3 μM CHIR-99021, 10 μM SB-431542, 1 μM BIR B796, 10 ng / mL NOGGIN, 50 ng / mL EGF, 10 ng / mL FGF-7, 10 ng / mL FGF-10, 10 ng / mL RSpondin-1 and 50 ng / mL NRG-β1, wherein Y-27632 was added 4 days before culture) for 7-14 days to successfully construct alveolar organoids. In this embodiment, the culture was performed for 14 days.

[0075] The steps of flow cytometry are as follows: Figure 3 As shown in the figure, specifically: SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-labeled lineage tracing mice were given tamoxifen at a concentration of 100 mg / kg in advance for 5 consecutive days. AT2 cells can be sorted 5 days after drug withdrawal. AT2 cell separation is the same as above. Then, the lung single cells after sieve erythrocytes were incubated with CD45 magnetic beads. The incubation condition parameters were: the volume ratio of the lung single cell fluid after sieve erythrocytes, magnetic beads and magnetic bead buffer was 10 7 Cells: 10μL: 90μL; incubate at 4℃ for 25min; negatively select CD45 - cells. - The cells were collected in sorting medium (Advance DMEM / F12 medium as the base medium, also containing 1×B27, 1X GlutaMAX, 1XP / S, 10μMY-27632), and then tdTOMATO+AT2 cells were sorted using excitation light at 581nm. The results are shown in Figure 6 As shown. Figure 6 It can be seen that flow cytometry is feasible for sorting AT2 cells, and the gating scheme of flow cytometry is also shown. 3 -10 4 The volume ratio of Matrigel to DMEM / F12 in the gel droplets was 1:1. The gel droplets were solidified at 37±0.5℃ for 20-30min. After the gel droplets solidified, 1mL of alveolar organoid culture medium was added and cultured for 7-14 days. Alveolar organoids were successfully constructed. In this example, the alveolar organoids were cultured for 18 days. CD326 +The cells are total lung epithelial cells, most of which are AT2 cells, but also contain bronchiolar epithelial cells such as basal cells, ciliated cells, goblet cells, etc. AT2 cells can be sorted out using SPC-tomaoto to improve the specificity of alveolar organoids.

[0076] Example 3 In vitro drug-induced lineage fluorescence and concentration exploration of barcode mutations

[0077] (1) Exploration of tamoxifen induction concentration:

[0078] Different concentrations of tamoxifen (50 nM, 250 nM, 500 nM, 1 μM, 2 μM) were added in vitro to induce the SPC-CRE tdTOMATO fluorescence expression of the alveolar organoids prepared in Example 2. The induction was performed for 24 h or 48 h. The results of culturing on the 7th day were as follows: Figure 4 and Figure 5 The results showed that the tamoxifen induction condition was 250-500 nM concentration for 24-48 h, and td-TOMATO fluorescence expression could be observed after 7 days of culture without causing significant cytotoxicity.

[0079] (2) Exploration of doxycycline induction concentration:

[0080] Doxycycline at a concentration of 1 μg / mL, 4 μg / mL or 8 μg / mL was added in vitro to induce the barcode mutation of the alveolar organoids prepared in Example 2. The barcode mutation was induced for 10 days and then investigated. At the same time, alveolar organoids not induced with doxycycline were used as controls. The results are shown in FIG. Figure 7-10 As shown. The results showed that when doxycycline was treated at a concentration of 1 μg / mL until the 10th day, it was found that the HMF9 barcode had an average of 2-3 mutations, which met the needs of lineage tracing and did not significantly affect the growth of organoids. Moreover, after culturing to AMM D10, 22 organoids were selected for first-generation sequencing, and it was found that 2-8 mutations accumulated in 23 organoid samples, mainly GtoA and CtoT mutations. In addition, the base mutation system induces C / G and T / A mutations, and the sequencing results are also in line with the mutation principle. It is proved that at the in vitro organoid level, DOX can induce barcode fragments to accumulate mutations, and the mutation system can also effectively accumulate mutations for reverse lineage tracing. It can be seen that under the induction of 1 μg / mL doxycycline, the growth of organoids is not affected, and barcode sequence mutations can be induced for cell lineage tracing.

[0081] In summary, adding different exposure factors or drugs to the alveolar culture medium and observing the changes in alveolar organoids and the accumulation of barcode mutations has excellent application prospects in studying the occurrence and development mechanisms of lung diseases and tracking the fate of stem cells.

[0082] Example 4 Optimization of mouse alveolar culture medium

[0083] To promote the formation of alveolar organoids, the following experiments were performed:

[0084] The CD326+ cells screened in Example 2 were inoculated into different alveolar organoid culture media for culture at a seeding rate of 10 4 / drop, and after culturing at 37°C for 7 days, the cells were investigated and grouped into the following groups: original culture medium, original culture medium + FGF7 (+FGF7), original culture medium + FGF7 + RS (+FGF7 + RS), original culture medium + FGF7 + NRG1 (+FGF7 + NRG1), and original culture medium + FGF7 + RS + NRG1 (+FGF7 + RS + NRG1);

[0085] The original culture medium was: Advance DMEM / F12 medium as the basal medium, and also contained 1×B27, 1× penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, and 10ng / mL FGF-10;

[0086] Original medium + FGF7 was: Advance DMEM / F12 medium as the basal medium, and also contained 1×B27, 1× penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-10, and 10ng / mL FGF-7;

[0087] Original medium + FGF7 + RS is: Advance DMEM / F12 medium as the basal medium, and also contains 1×B27, 1× penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-10, 10ng / mL FGF-7, and 10ng / mL RSpondin-1;

[0088] The original medium + FGF7 + NRG1 was: Advance DMEM / F12 medium as the basal medium, and also contained 1×B27, 1× penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-10, 10ng / mL FGF-7, and 50ng / mL NRG1;

[0089] The original culture medium +FGF7+RS+NRG1 (+FGF7+RS+NRG1) is: Advance DMEM / F12 medium as the base medium, and also contains 1×B27, 1×penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

[0090] The results are as follows Fig.11 and Fig.12 The results showed that FGF7, RS and NRG1 can all promote the growth of alveolar organoids, and their combination can significantly promote the formation and growth of alveolar organoids.

[0091] Example 5

[0092] Extraction method of cigarette smoke extract (CSE): Take 3 cigarettes (Double Happiness brand) and remove the filter tips. Connect the cigarettes to one end of a large bag absorber tube. Connect the other end of the 3 series-connected large bag absorbers to a 50mL syringe and ensure that the device is airtight. Each of the 3 series-connected large bag absorbers contains 5mL PBS as a cigarette smoke absorption liquid, which is used to collect the mainstream smoke from burning cigarettes. After lighting the cigarette, draw the syringe at a speed of 50mL / min to fully dissolve the cigarette smoke in PBS. This is a 100% CSE solution.

[0093] Before use, the CSE stock solution was filtered through a microporous membrane with a diameter of 0.22 μm to remove bacteria and particles.

[0094] The whole lung cells of the double-labeled lineage-tracing mice were extracted according to the flow sorting method provided in Example 2. (Tamoxifen was administered at a concentration of 100 mg / kg for 5 consecutive days. AT2 cell sorting was performed 5 days after drug withdrawal. AT2 cell separation was performed as in Example 2. Then, the lung single cells after sieve lysed red blood cells were incubated with CD45 magnetic beads. The incubation condition parameters were: the volume ratio of the lung single cell fluid after sieve lysed red blood cells, magnetic beads and magnetic bead buffer was 10 7 Cells: 10μL: 90μL; incubate at 4℃ for 25min; negatively select CD45 - cells. - The cells were collected in sorting medium (based on Advance DMEM / F12 medium, also containing 1×B27, 1×GlutaMAX, 1×P / S, 10μMY-27632), and then tdTOMATO was sorted using excitation light at 581nm. + AT2 cells). 4 tdTOMATO + AT2 cells were plated at a ratio of 1:1 cell / gel droplet. The culture medium used was alveolar organoid culture medium. The cells were divided into a control group and a CSE-exposed group. The two groups were plated with the same number of cells. 0.2% cigarette extract was added to the culture medium of the CSE-exposed group. After 10 days of culture, the growth of alveolar organoids was observed. The results are shown in Figure 2. Fig.13 As shown, Fig.13 The image shows the growth of alveolar organoids after ten days of exposure to CSE. The results show that although the formation efficiency of alveolar organoids was significantly reduced after the tenth day of CSE exposure, the alveolar organoids that can be formed can be used to study the in vitro pathological process of COPD.

[0095] After that, the CSE-exposed alveolar organoids were cultured to AMM D10 (10th day of proliferation period), and 20 organoids were selected for first-generation sequencing. The results are as follows Fig.14 The results showed that 4-9 mutations accumulated in 20 organoid samples, proving that DOX can induce mutation accumulation of barcode fragments in CSE exposure-simulated COPD process.

[0096] Example 6

[0097] In order to compare the formation efficiency of alveolar organoids under different sorting methods, the following four sorting methods were used:

[0098] Method 1. After digestion, centrifugation, filtration and red lysis of mouse lung cells, 10 4 cells / plate.

[0099] Method 2: After digestion, centrifugation, filtration, and red lysis of whole lung cells, CD45 - Cells, 10 4 Cells / droplet were plated.

[0100] Method 3: After digestion, centrifugation, filtration and red lysis of whole lung cells, CD45 - cells and continued to incubate with CD326 magnetic beads to sort out CD45 - CD326 + cells, and 10 4 cells / plate.

[0101] Method 4: After digestion, centrifugation, filtration, and red lysis of whole lung cells, CD45 - Cells were then separated by flow cytometry to isolate tdTOMATO + AT2 cells, with 10 4 Cells / droplet plate, magnetic bead sorting method and flow sorting method are the same as in Example 2.

[0102] The digestion, centrifugation, filtration and red lysis steps in Method 1-Method 4 are the same as those in Example 2.

[0103] After culturing organoids with different sorting methods for 10 days, the growth graph of the organoids was observed. The results are as follows Fig.15 As shown. Figure 5 It can be seen that method 1: after whole lung digestion, centrifugation, filtration, and erythrocyte lysis, total cell plate was plated, the efficiency of organoid formation was low, and fibroblasts (growing in a reticular pattern) grew preferentially; method 2: after whole lung digestion, centrifugation, filtration, and erythrocyte lysis, CD45 - After the cells were plated, the efficiency of organoid formation was low; Method 3: Whole lung cells were digested, centrifuged, filtered, and lysed, and CD45 - CD326 + Cells, the number of organoids formed is better, but it contains more non-alveolar organoids and the purity of organoids is low; Method 4: After digestion, centrifugation, filtration, and red lysis of whole lung cells, tdTOMATO + AT2 cells, the number and size of alveolar organoids formed were appropriate, and the purity of alveolar organoids was high.

[0104] Example 7

[0105] Take 10 4 tdTOMATO + AT2 cells (isolated in Example 6) / gel droplets were plated and cultured in an alveolar organoid culture medium for 10 days to obtain alveolar organoids. - CD326 +The cells (isolated in Example 6) are non-alveolar cells, and the culture conditions are the same as those of tdTOMATO + Bright field images of AT2 cells, alveolar organoids, and non-alveolar organoids. Fig.16 The results showed that alveolar organoids were regular, transparent spheres. Fig.15 Growth graph of tdTOMATO+AT2 group in the figure; non-alveolar organoids are irregular folded cysts, Fig.15 CD45 - CD326 + Opaque organoids in the group.

[0106] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing alveolar organoids with fluorescence and barcode lineage tracing, characterized in that: The following steps are involved: The SPC-Cre ERT2 tool mice were mated with the R26RtdTomato reporter mice to obtain reporter mice; The reporter mice were mated with each other to obtain SPC-Cre ERT2 + / - ,R26RtdTomato + / + Lineage-tracing mice; The rtTA tool mice were mated with HMF9-barcode mice to obtain barcoded lineage-tracing mice carrying rtTA / HMF9; The SPC-Cre ERT2 + / - , R26R tdTomato + / + After mating the lineage tracing mice with the barcoded lineage tracing mice carrying rtTA / HMF9, SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-marked lineage tracing mice were obtained; Taking lung tissue from SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-labeled lineage tracing mice, isolating alveolar type II cells, and culturing them to obtain the alveolar organoids; The culture medium used in the culture is based on Advance DMEM / F12 medium, and also includes 1×B27, 1×penicillin-streptomycin, 5-10μM Y-27632, 2-5μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

2. The construction method according to claim 1, characterized in that: The method for isolating alveolar type II cells comprises the steps of digesting, centrifuging, filtering, lysing and sorting the lung tissue.

3. The construction method according to claim 2, characterized in that: The digestion enzymes used in the digestion include 2 mg / mL tissue collagenase I, 5 mg / mL dispase, 0.01 mg / mL DNase and 3 mL DMEM-F12 culture medium; The digestion temperature is 36.5°C-37.5°C and the digestion time is 30-40 minutes.

4. The construction method according to claim 2, characterized in that: The centrifugal condition parameters are: 500 rcf centrifugation for 10 min; The red lysis condition parameters are: lysis at 4°C for 5 minutes.

5. The construction method according to claim 2, characterized in that: The sorting methods include magnetic bead sorting and flow sorting.

6. Alveolar organoids constructed using the construction method described in any one of claims 1-5.

7. Use of the alveolar organoids according to claim 6 in screening or preparing drugs for preventing or treating chronic obstructive pulmonary disease.

8. A method for inducing lineage fluorescence and barcode mutations in alveolar organoids according to claim 6, characterized in that: Inducing and culturing the alveolar organoids using tamoxifen and doxycycline; The concentration of tamoxifen is 250nM-500nM, and the induction culture time is 24-48h; The concentration of doxycycline is 1 μg / mL, and the induction culture time is 10 days.

9. A culture medium for culturing alveolar organoids, characterized in that: The culture medium is based on Advance DMEM / F12 medium and also includes 1×B27, 1×penicillin-streptomycin, 5-10μM Y-27632, 2-5μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG1.

10. Use of the culture medium according to claim 9 in culturing alveolar organoids.

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