A fluorescent and barcode-based alveolar organoid, its construction method, and its application.

Abstract

This invention relates to the field of biology, and in particular to a fluorescent and barcode-based alveolar organoid, its construction method, and its applications. Specifically addressing the issues of specific sorting of AT2 cells, organoid culture, and lineage retracing in disease progression, and for simulating and studying the in vitro pathological process of chronic obstructive pulmonary disease (COPD), this invention utilizes fluorescent lineage tracing technology combined with barcode analysis to construct a fluorescent and barcode-based alveolar organoid. This organoid can track the fate transition of alveolar type II cells (AT2) in disease models, further decoding cell lineages, thereby providing new research evidence for the early diagnosis and treatment of COPD.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to a fluorescent and barcode-based alveolar organoid, its construction method, and its applications. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease characterized by airway obstruction and lung tissue destruction. Its main pathological feature is damage and remodeling of alveolar epithelial cells, characterized by irreversible airflow limitation that worsens over time. COPD is the third leading cause of death worldwide and a leading cause of chronic disability and death globally. The high morbidity and mortality rates of COPD impose a heavy economic burden on patients, their families, and society. Due to continued global environmental degradation and population aging, the burden of COPD is expected to gradually increase in the coming decades. The pathogenesis of COPD is highly complex, involving oxidative stress, inflammation, protease-antiprotease imbalance, and apoptosis.

[0003] The main pathological features of COPD include chronic lung inflammation, airway remodeling, emphysema, and impaired lung function. Chronic lung inflammation is characterized by inflammatory cell infiltration in the walls of bronchi at all levels. Airway remodeling is mainly due to the proliferation of airway smooth muscle cells, activation of fibroblasts into myofibroblasts, and the deposition of extracellular matrix. Emphysema is characterized by enlarged and ruptured alveolar spaces and disruption of the elastic fiber network; as a major pathological feature of COPD, emphysema has always been a focus of COPD research. COPD is a preventable and treatable disease, but because its etiology and pathogenesis are not yet fully understood, there are currently no effective methods to prevent the occurrence or progression of COPD.

[0004] Reliable in vitro models can effectively shorten the time needed to explore the pathogenesis and treatment of COPD; however, existing in vitro models struggle to accurately simulate the microenvironment and cellular behavior of the human lung. The regeneration and repair mechanisms of alveolar type II (AT2) cells are an important research direction in lung diseases, but current technologies have limitations in sorting AT2 cells and studying their lineage retracing. Traditional methods cannot simultaneously achieve specific sorting and dynamic tracking of AT2 cells, limiting systematic research on abnormal AT2 cell lineage regeneration and repair during disease progression. Therefore, it is necessary to find a method capable of specific sorting and dynamic tracking of AT2 cells. Summary of the Invention

[0005] The purpose of this invention is to provide an alveolar organoid for fluorescence and barcode lineage tracking, its construction method, and its applications, in order to solve the problems existing in the prior art. This invention develops an alveolar organoid carrying a barcode specifically fluorescently labeled with the AT2 cell lineage, which can simulate the structural characteristics of alveolar tissue. Combining fluorescence lineage tracking and barcode technology, it can accurately track the fate and functional changes of AT2 cells in vitro, providing a new research tool for COPD research.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for constructing alveolar organoids based on fluorescence and barcode lineage tracing, comprising the following steps:

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

[0009] After crossbreeding the reporter mice, SPC-Cre ERT2 was obtained. + / - R26RtdTomato + / + Lineage tracing mice;

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

[0011] The SPC-Cre ERT2 + / - R26R tdTomato + / + After mating the pedigree-tracking mice with the barcode-tracked pedigree-tracking mice carrying rtTA / HMF9, SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous dual-label pedigree-tracking mice were obtained.

[0012] Lung tissue from mice was traced using SPC-Cre ERT2, R26R tdTomato, rtTA, and HMF9-barcode heterozygous double-label lineages. Type II alveolar cells were isolated and cultured to obtain the alveolar organoids.

[0013] The culture medium used was Advance DMEM / F12 as the base medium, and also included 1×B27, 1×penicillin-streptomycin, 5-10μM Y-27632, 2-5μM CHIR-99021, 10μM SB-431542, 1μM MBIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

[0014] More preferably, the culture medium used is based on Advance DMEM / F12 medium, and further includes 1×B27, 1×penicillin-streptomycin, 10μMY-27632, 3μM CHIR-99021, 10μM SB-431542, 1μMBIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

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

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

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

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

[0019] More preferably, during the digestion process, the lung tissue is blown around every 10-15 minutes, which can effectively increase the lysis efficiency.

[0020] Preferably, the centrifugation conditions are: 500 rcf for 10 min;

[0021] The conditions for the incubation of the red lysate were: incubation at 4°C for 5 minutes.

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

[0023] This invention provides alveolar organoids constructed using the above-described construction method.

[0024] This invention provides the application of the above-mentioned alveolar organoids in screening or preparing drugs for the prevention or treatment of chronic obstructive pulmonary disease.

[0025] This invention provides a method for inducing lineage fluorescence and barcode mutations in the above-mentioned alveolar organoids, using tamoxifen and doxycycline to induce the culture of the alveolar organoids;

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

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

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

[0029] More preferably, the culture medium used is based on Advance DMEM / F12 medium, and further includes 1×B27, 1×penicillin-streptomycin, 10μMY-27632, 3μM CHIR-99021, 10μM SB-431542, 1μMBIRB796, 10ng / mL NOGGIN, 50ng / mL EGF, 10ng / mL FGF-7, 10ng / mL FGF-10, 10ng / mL RSpondin-1 and 50ng / mL NRG 1.

[0030] This invention provides the application of the above-described culture medium in culturing alveolar organoids.

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

[0032] 1. The AT2 lineage was specifically sorted using SPC-tdTOMATO fluorescent labeling, improving sorting efficiency and accuracy.

[0033] 2. The alveolar organoids obtained using the construction method of this invention can be used to observe the differentiation lineage of AT2 in vitro under tamoxifen induction; under doxycycline induction, barcodes can be used for mutation accumulation and cell lineage tracing, providing a reliable platform for exploring disease mechanisms.

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

[0035] 4. This invention also optimizes the culture medium scheme for mouse alveolar organoids, resulting in higher organoid formation efficiency.

[0036] In summary, this invention specifically addresses the issues of specific sorting of AT2 cells, organoid culture, and lineage retracing in disease progression, and is used to simulate and study the in vitro pathological process of COPD. By combining fluorescence lineage tracing technology with barcode analysis technology, a fluorescence and barcode lineage tracing alveolar organoid has been constructed. This organoid can track the fate transition of alveolar type II cells (AT2) in disease models, further decoding cell lineages, thereby providing new research evidence for the early diagnosis and treatment of COPD. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 To track the breeding of mice using a double-marker pedigree;

[0039] Figure 2 This is a schematic diagram of the magnetic bead sorting method;

[0040] Figure 3 This is a schematic diagram of the flow sorting method;

[0041] Figure 4 The images show the in vitro induction of alveolar organoids by different concentrations of tamoxifen; where A represents the fluorescence and bright-field images of alveolar organoids treated with different concentrations of tamoxifen; and B represents the quantitative map of organoid formation.

[0042] Figure 5 Fluorescence image for organoid culture using magnetic bead sorting;

[0043] Figure 6 The images show the flow cytometry sorting of AT2 cells for organoid culture; where A is a gating diagram of flow cytometry sorting; and B is a fluorescence image of organoid culture using flow cytometry sorting.

[0044] Figure 7 The image shows the growth of organoids under different Dox-induced concentrations; where Alveolar Maintain Phase D2 is the second day of the cell maintenance phase, Alveolar Maintain Phase D5 is the fifth day of the cell maintenance phase, Alveolar Maintain Phase D9 is the ninth day of the cell maintenance phase, and Alveolar Differentiation D9 is the ninth day of the cell differentiation phase.

[0045] Figure 8 A statistical chart of organoid formation numbers;

[0046] Figure 9 A statistical chart of organoid diameters;

[0047] Figure 10 This refers to barcode mutation scenarios;

[0048] Figure 11 Bright-field diagrams of organoid growth after the addition of different cytokines during the organoid proliferation culture period.

[0049] Figure 12 A statistical chart of organoid numbers;

[0050] Figure 13 The growth of alveolar organoids (A) and the quantitative graph of organoid formation efficiency (B) in the CSE exposure group and the Control group;

[0051] Figure 14 Sequencing results of alveolar organoids exposed in CSE;

[0052] Figure 15 Figures showing organoid growth after culturing organoids for 10 days using different sorting methods;

[0053] Figure 16 Bright-field images of alveolar and non-alveolar organoids. Detailed Implementation

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

[0055] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0056] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0057] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0059] Unless otherwise specified, the materials used in this invention are all readily available to those skilled in the art; the steps used in this invention are all well-known to those skilled in the art.

[0060] Example 1: Construction of Hybrid Mice

[0061] ① Type II alveolar epithelial cell lineage tracking mouse (SPC-Cre ERT2) + / - R26RtdTomato + / + Breeding of pedigree-tracing mice

[0062] In this embodiment, heterozygous mice were obtained by mating positive mice with wild-type mice, and homozygous mice were obtained by mating heterozygous mice with each other. These mice were then induced with tamoxifen, and the AT2 cell lineage was identified using PCR and fluorescence observation. Type II alveolar epithelial cells induced by tamoxifen specifically emitted red fluorescence; this modification is heritable and can be passed on to daughter cells, providing a powerful tool for studying cell fate.

[0063] The specific steps are as follows:

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

[0065] ② Construction of barcode mice (barcode lineage tracking mice carrying rtTA / HMF9 or barcode lineage tracking mice carrying rtTA and HMF9)

[0066] Heterozygous mice were obtained by mating homozygous tool mice and homozygous barcode mice, and then induced with doxycycline. The transcriptional status of the mice and barcodes was identified by PCR and fluorescence observation. After doxycycline induction, mutations accumulated in the two barcode regions during cell division, and these mutations could be inherited by daughter cells, providing a powerful tool for studying cell development and lineage tracing.

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

[0068] ③ Breeding of dual-marker pedigree tracking mice (SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous dual-marker pedigree tracking mice)

[0069] Specific steps are as follows Figure 1 As shown: SPC-Cre ERT2 obtained from the above two breeding steps + / - R26RtdTomato + / + Lineage-tracking mice were crossbred with barcode-labeled lineage-tracking mice carrying rtTA / HMF9 to obtain SPC-Cre ERT2, R26RtdTomato, rtTA, HMF9-barcode heterozygous dual-label lineage-tracking mice (SPC-Cre ERT2). + / - R26RtdTomato + / - rtTA + / - HMF9-barcode + / - Heterozygous mice were used. SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous dual-label lineage-tracking mice were induced with tamoxifen (same as the type II alveolar epithelial cell lineage-tracking mouse model); simultaneously, they were induced with doxycycline (same as the barcode mouse model). PCR and fluorescence microscopy were then used to identify the expression of red fluorescence and the transcription of RNA barcodes in type II alveolar epithelial cells. Results showed that type II alveolar epithelial cells specifically emit red fluorescence, and the barcode region accumulates mutations during cell division. This dual-label lineage-tracking mouse model can provide more accurate and comprehensive data support for studying the fate, differentiation, and damage repair capabilities of type II alveolar epithelial cells.

[0070] Example 2: Method for culturing alveolar organoids

[0071] AT2 cells from mice were sorted using 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 opening the mouse's thoracic cavity, 5 mL of DPBS solution was infused into the right lung using a 10 mL syringe with a 22 GG needle to remove a large number of residual red blood cells. Non-lung tissue (such as trachea and mucosa) was removed as much as possible. The lung tissue was cut into a homogenate and digested using tissue digestive enzymes (tissue collagenase I: 2 mg / mL; dispersin: 5 mg / mL; DNase: 0.01 mg / mL; DMEM-F12 medium: 3 mL / mL) (digestion at 37℃±0.5℃ for 30-45 min; in this invention, digestion at 37℃ for 30 min; during digestion, the lung tissue was pipetted every 10-15 min to effectively increase lysis efficiency). The mixture was centrifuged at 500 rcf for 10 min, then filtered using a filter-type cell sieve, and red blood cells were lysed. This was done by incubating the lysed lung single-cell fluid with 3 mL of red blood cell lysis buffer (Solepro) at 4℃ for 5 min, and then sorting AT2 cells using magnetic bead sorting or flow cytometry.

[0074] The steps of the magnetic bead sorting method are as follows: Figure 2 As shown, specifically, CD45 magnetic beads were incubated in lung single-cell fluid after sieving red blood cell splitting. The incubation conditions were as follows: the volume ratio of lung single-cell fluid after sieving red blood cell splitting, magnetic beads, and magnetic bead buffer was 10:1. 7 Cells: 10 μL: 90 μL; incubated at 4°C for 25 min; CD45 cells were negatively selected. - Cells. Reusing CD45 - Cells were incubated with CD326 magnetic beads under the following conditions: the volume ratio of lung single-cell fluid after cytosolic leukemia screening, magnetic beads, and magnetic bead buffer was 10:1. 7 Cells: 10 μL: 90 μL; incubated at 4°C for 20 min; CD326 cells were positively selected. + Cells. Then at 10 3 -10 4 CD326 +Cells were seeded onto plates at a ratio of 1:1 for Matrigel and DMEM / F12 in the droplets. The plates were cured at 37±0.5℃ for 20-30 min. After solidification, 1 mL of alveolar organoid culture medium (based on Advance DMEM / F12 medium, also containing 1×B27, 1×penicillin-streptomycin, 10 μMY-27632, 3 μM CHIR-99021, 10 μM SB-431542, 1 μMBIRB796, 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, with Y-27632 added 4 days prior to culture) was added. Alveolar organoids were successfully constructed; in this example, culture was performed for 14 days.

[0075] The steps of the flow sorting method are as follows: Figure 3 As shown, specifically: SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous double-labeled lineage-tracing mice were pre-administered with tamoxifen at a concentration of 100 mg / kg for 5 consecutive days. Five days after drug withdrawal, AT2 cells were sorted, and AT2 cell isolation was performed as described above. Subsequently, CD45 magnetic beads were incubated with lung single cells after sieving for red blood cell splitting. The incubation conditions were: a volume ratio of lung single cell fluid after sieving for red blood cell splitting to magnetic beads and magnetic bead buffer of 10:1. 7 Cells: 10 μL: 90 μL; incubated at 4°C for 25 min; CD45 cells were negatively selected. - Cells. CD45 - Cells were collected in sorting medium (based on Advance DMEM / F12 medium, also containing 1×B27, 1X GlutaMAX, 1XP / S, and 10μMY-27632), and then tdTOMATO+AT2 cells were sorted using excitation light at 581nm. The results are as follows. Figure 6 As shown. By Figure 6 It can be seen that flow cytometry is feasible for sorting AT2 cells, and the gating scheme of flow cytometry sorting is also demonstrated. (Using 10...) 3 -10 4 The cells were seeded into plates at a ratio of tdTOMATO+AT2 cells / droplet, with a Matrigel and DMEM / F12 volume ratio of 1:1 in the droplets. The plates were cured at 37±0.5℃ for 20-30 min. After solidification, 1 mL of alveolar organoid culture medium was added, and the plates were cultured for 7-14 days, successfully constructing alveolar organoids. In this example, the culture period was 18 days. CD326 +The cells consist of total lung epithelial cells, mostly AT2 cells, but also include bronchiolar epithelial cells such as basal cells, ciliated cells, and goblet cells. AT2 cells can be sorted using SPC-tomaoto, improving the specificity of alveolar organoids.

[0076] Example 3: Concentration Determination of In Vitro Drug-Induced Linear Fluorescence and Barcode Mutation

[0077] (1) Tamoxifen induction concentration exploration:

[0078] Different concentrations (50 nM, 250 nM, 500 nM, 1 μM, 2 μM) of tamoxifen were added in vitro to induce SPC-CRE tdTOMATO fluorescence expression in the alveolar organoids prepared in Example 2. Induction was performed for 24 h or 48 h, and the results after culturing to day 7 are as follows: Figure 4 and Figure 5 As shown in the figure. The results showed that tamoxifen induction conditions of 250-500 nM concentration for 24-48 h resulted in the observation of td-TOMATO fluorescent expression after 7 days of culture, without significantly causing cytotoxicity.

[0079] (2) Exploration of doxycycline-induced concentration:

[0080] Barcode mutations in the alveolar organoids prepared in Example 2 were induced in vitro with doxycycline at concentrations of 1 μg / mL, 4 μg / mL, or 8 μg / mL for 10 days. An investigation was then conducted, with alveolar organoids not induced by doxycycline serving as a control. Results are as follows: Figures 7-10 As shown in the figure. The results showed that treatment with doxycycline at a concentration of 1 μg / mL up to day 10 resulted in an average of 2-3 mutations in the HMF9 barcode, meeting the requirements for lineage tracing without significantly affecting organoid growth. Furthermore, after culturing to AMM D10, 22 organoids were selected for first-generation sequencing, revealing 2-8 accumulated mutations in 23 organoid samples, primarily GtoA and CtoT mutations. This base mutation system induced C / G and T / A mutations, and the sequencing results also conformed to the mutation principles. This demonstrates that at the in vitro organoid level, DOX can induce mutation accumulation in the barcode fragment, and this mutation system can effectively accumulate mutations for reverse lineage tracing. Therefore, doxycycline at 1 μg / mL does not affect organoid growth and can induce barcode sequence mutations for cell lineage tracing.

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

[0082] Example 4: Optimization of mouse alveolar culture medium

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

[0084] The CD326+ cells screened in Example 2 were seeded into different alveolar organoid culture media, with an inoculum size of 10-1. 4 / drop, after incubation at 37℃ for 7 days, an investigation was conducted, and the specific groups were: 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 as the basal medium, which also contained 1×B27, 1×penicillin-streptomycin, 10μMY-27632, 3μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL EGF and 10ng / mL LFGF-10;

[0086] The original culture medium + FGF7 consisted of Advance DMEM / F12 as the basal medium, and also contained 1×B27, 1×penicillin-streptomycin, 10μMY-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] The original culture medium + FGF7 + RS consisted of Advance DMEM / F12 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 10ng / mL RSpondin-1;

[0088] The original culture medium + FGF7 + NRG1 consisted of Advance DMEM / F12 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 LGF-7, and 50ng / mL NRG1.

[0089] The original culture medium +FGF7+RS+NRG1 (+FGF7+RS+NRG1) was based on Advance DMEM / F12 medium and also contained 1×B27, 1×penicillin-streptomycin, 10μM Y-27632, 3μM CHIR-99021, 10μM MSB-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.

[0090] The results are as follows Figure 11 and Figure 12 As shown in the figure. 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 three cigarettes (Double Happiness brand), remove the filters, and connect the cigarettes to one end of a Boehringer Ingelheim (BTI) absorption tube. Connect the other end of the three BTI absorption tubes connected in series to a 50mL syringe, ensuring the apparatus is sealed. Each of the three BTI absorption tubes contains 5mL of PBS as the cigarette smoke absorption solution, used to collect the mainstream smoke from the burning cigarette. After lighting the cigarette, aspirate the syringe at a rate of 50mL / min to fully dissolve the cigarette smoke in the PBS, resulting in a 100% CSE solution.

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

[0094] Whole lung cells from dual-labeled lineage-tracing mice were extracted using the flow cytometry method provided in Example 2. Tamoxifen was administered at a concentration of 100 mg / kg for 5 consecutive days, followed by a 5-day drug withdrawal period before AT2 cell sorting. AT2 cell isolation was performed as described in Example 2. Subsequently, CD45 magnetic beads were incubated with lung single cells after sieving for red blood cell fragmentation. The incubation conditions were as follows: the volume ratio of lung single cell fluid after sieving for red blood cell fragmentation to magnetic beads and magnetic bead buffer was 10:1. 7 Cells: 10 μL: 90 μL; incubated at 4°C for 25 min; CD45 cells were negatively selected. - Cells. CD45 - Cells were collected in sorting medium (based on Advance DMEM / F12 medium, also containing 1×B27, 1×GlutaMAX, 1×P / S, and 10μMY-27632), and then tdTOMATO cells were sorted using excitation light at 581 nm. + AT2 cells). (At 10) 4 tdTOMATO + AT2 cells were cultured in plates at a cell / droplet ratio using alveolar organoid culture medium. Two groups were selected: a Control group and a CSE-exposed group. Both groups were seeded with the same number of cells. In the CSE-exposed group, 0.2% cigarette extract was added to the medium. After 10 days of culture, the growth of alveolar organoids was observed. The results are as follows: Figure 13 As shown, Figure 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 decreased significantly after 10 days of CSE exposure, those that did form alveolar organoids are suitable for studying the in vitro pathological process of COPD.

[0095] Subsequently, after culturing CSE-exposed alveolar organoids to AMM D10 (day 10 of the proliferation phase), 20 organoids were selected for first-generation sequencing. The results are as follows: Figure 14 As shown in the figure. The results indicate that 4-9 out of 20 organoid samples had accumulated mutations, demonstrating that DOX can induce mutation accumulation in barcode fragments during CSE exposure to simulate the COPD process.

[0096] Example 6

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

[0098] Method 1: After digestion, centrifugation, filtration, and cleavage of mouse whole lung cells, 10 cells were taken. 4 One cell / droplet plate.

[0099] Method 2: After obtaining whole lung cells, digestion, centrifugation, filtration, and red blood cell splitting, CD45 cells were sorted using magnetic bead sorting. - Cells, at 10 4 Seed plate with 1 cell / droplet.

[0100] Method 3: After obtaining whole lung cells, digestion, centrifugation, filtration, and red blood cell splitting, CD45 cells were sorted using magnetic bead sorting. - Cells were incubated with CD326 magnetic beads, and CD45 were sorted out. - CD326 + Cells, and with 10 4 One cell / droplet plate.

[0101] Method 4: After obtaining whole lung cells, digestion, centrifugation, filtration, and red blood cell splitting, CD45 was separated using magnetic bead sorting. - Cells were then sorted using flow cytometry to separate tdTOMATO. + AT2 cells, at 10 4 Cell / droplet seeding plate, magnetic bead sorting and flow cytometry sorting are the same as in Example 2.

[0102] The digestion, centrifugation, filtration, and red blood cell splitting steps in methods 1-4 are the same as in Example 2.

[0103] After culturing organoids using different sorting methods for 10 days, the organoid growth diagrams were observed, and the results are as follows: Figure 15 As shown. By Figure 5 It can be seen that, in Method 1, after whole-lung digestion, centrifugation, filtration, and red blood cell splitting, total cells were seeded into plates, resulting in low organoid formation efficiency and dominant growth of fibroblasts (growing in a reticular pattern); in Method 2, after whole-lung digestion, centrifugation, filtration, and red blood cell splitting, CD45 cells were sorted out. - Post-cell seeding resulted in low organoid formation efficiency; Method 3: After digestion, centrifugation, filtration, and red blood cell splitting of whole lung cells, CD45 was sorted out. - CD326 + The cells produced a relatively high number of organoids, but contained a large number of non-alveolar organoids, resulting in lower organoid purity. Method 4: After digestion, centrifugation, filtration, and red blood cell splitting of whole lung cells, tdTOMATO was sorted out. + The AT2 cells produced alveolar organoids of appropriate quantity and size, with high purity.

[0104] Example 7

[0105] With 10 4 tdTOMATO + AT2 cells (isolated in Example 6) were seeded into plates at a ratio of 1:1 to droplets and cultured in alveolar organoid medium for 10 days to obtain alveolar organoids. Simultaneously, CD45 was used as the catalyst. - CD326 +The cells (isolated in Example 6) were non-alveolar cells, and the culture conditions were the same as those for tdTOMATO. + Bright-field images of AT2 cells, alveolar organoids, and non-alveolar organoids are shown below. Figure 16 As shown. The results show that the alveolar organoids are regular, translucent spherical shapes, similar to... Figure 15 Growth diagram of the tdTOMATO+AT2 group; non-alveolar organoids are irregularly folded and sac-like, similar to... Figure 15 CD45 in - CD326 + The organoids in the group are not translucent.

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing alveolar organoids based on fluorescence and barcode lineage tracing, characterized in that, Includes the following steps: SPC-Cre ERT2 tool mice and R26R tdTomato reporter mice were mated to obtain reporter mice; After crossbreeding the reporter mice, SPC-Cre ERT2 was obtained. + / - R26R tdTomato + / + Lineage tracing mice; The rtTA tool mice were mated with HMF9-barcode mice to obtain barcode lineage tracking mice carrying rtTA / HMF9; The SPC-Cre ERT2 + / - R26R tdTomato + / + After mating the pedigree-tracking mice with the barcode-carrying pedigree-tracking mice carrying rtTA / HMF9, SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous dual-label pedigree-tracking mice were obtained; the SPC-Cre ERT2, R26R tdTomato, rtTA, HMF9-barcode heterozygous dual-label pedigree-tracking mice were then administered tamoxifen. Lung tissue from mice with SPC-Cre ERT2, R26R tdTomato, rtTA, and HMF9-barcode heterozygous dual-label lineages after drug withdrawal was collected. Alveolar type II cells were isolated and cultured to obtain alveolar organoids. The isolation of alveolar type II cells included digestion, centrifugation, filtration, cleavage, and sorting of the lung tissue. The sorting was performed using magnetic bead sorting to separate CD45 cells. - Cells were then sorted using flow cytometry to separate tdTOMATO. + AT2 cells; The culture medium used was prepared from Advance DMEM / F12 medium, 1×B27, 1×penicillin-streptomycin, 5-10μM Y-27632, 2-5μM CHIR-99021, 10μM SB-431542, 1μM BIRB796, 10ng / mL NOGGIN, 50ng / mL LEGF, 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 digestive enzymes used in the digestion included 2 mg / mL tissue collagenase I, 5 mg / mL dispersin, 0.01 mg / mL DNase, and 3 mL DMEM-F12 medium. The digestion temperature is 36.5℃-37.5℃, and the time is 30-40 minutes.

3. The construction method according to claim 1, characterized in that, The centrifugation conditions were: 500 rcf for 10 min; The conditions for the pyrolysis of the red were: pyrolysis at 4°C for 5 minutes.

4. Alveolar organoids constructed using the construction method according to any one of claims 1-3.

5. A method for inducing fluorescence or barcode mutations in the alveolar organoid lineage as described in claim 4, characterized in that, The alveolar organoids were induced and cultured using tamoxifen or doxycycline. The concentration of tamoxifen was 250 nM-500 nM, and the induction culture time was 24-48 h. The concentration of doxycycline was 1 μg / mL, and the induction culture time was 10 days.

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