Pulmonary artery tissue-derived cells, organoids, and methods of construction and use thereof

Abstract

The application provides a pulmonary artery tissue-derived cell, an organoid and a construction method and application thereof. The pulmonary artery intimal stripping tissue organoid structure obtained by the preparation method is clear, has consistent cell types as the source tissue, is close to the original tissue, is convenient for researchers to operate, and thus has good practical application value.

Description

[0001] This application is a divisional application. The original application number is 202510547375.8, the application date is April 28, 2025, and the invention title is: A cell derived from pulmonary artery tissue, an organoid, a method for constructing the same and its application. Technical Field

[0002] This invention provides cells derived from pulmonary artery tissue, organoids, their construction methods, and applications, belonging to the field of biomedical technology, specifically relating to the field of organoid construction. Background Technology

[0003] Pulmonary hypertension (PH) is a clinical and pathophysiological syndrome caused by various etiologies and different pathogenesis mechanisms, resulting in changes in the structure or function of pulmonary vessels, leading to increased pulmonary vascular resistance and pulmonary artery pressure. It can progress to right heart failure and even death, severely impacting patients' quality of life and survival. Clinically, PH is classified into five major categories. Its etiologies are widespread, and its pathogenesis is complex, involving multiple factors and pathways. Chronic thromboembolic pulmonary hypertension (CTEPH) belongs to the fourth category of PH. It is characterized by dyspnea, fatigue, and decreased exercise tolerance. Due to undissolved thrombi blocking the proximal pulmonary artery, accompanied by distal pulmonary vascular remodeling, pulmonary vascular resistance increases, pulmonary artery pressure progressively rises, and right ventricular load gradually increases, eventually progressing to right heart failure. It is a serious, progressive, and fatal disease. The incidence of CTEPH is increasing year by year. Due to its insidious onset, rapid progression and high mortality, it has become a global healthcare problem that seriously endangers human life and health. Clinical diagnosis and treatment of CTEPH patients still face many challenges. Most of the proximal organizing thrombi and corresponding intimal thickening can be resolved by pulmonary thromboendarterectomy, while distal lesions that cannot be operated on and residual pulmonary hypertension after surgery may require targeted medical treatment.

[0004] Numerous studies have shown that the development of CTEPH is a process involving abnormalities in multiple genes, cells, factors, and signal transduction systems; however, the exact factors and specific molecular mechanisms remain unclear. Research indicates that pulmonary artery endothelial cell dysfunction, abnormal proliferation of pulmonary artery smooth muscle cells, and inflammatory responses ultimately lead to pulmonary vascular remodeling, playing a crucial role in the development of CTEPH. Therefore, constructing a disease model that fully mimics the pathological characteristics of CTEPH development is of paramount importance.

[0005] Currently, CTEPH research mainly relies on animal models and two-dimensional cell culture, but these methods have many limitations. Commonly used animal models of pulmonary hypertension include the cynomolgus alkaloid (MCT)-induced model, hypoxia models, and transgenic models (such as the BMPR2 mutation model). These models simulate the pathological features of pulmonary hypertension to some extent, but they differ significantly from the pathophysiological mechanisms of the human disease. They cannot accurately simulate the genetic heterogeneity and pathophysiological characteristics of human pulmonary hypertension, and drug responses differ significantly from those in humans. Two-dimensional cell culture (such as monolayer culture of pulmonary artery endothelial cells and pulmonary artery smooth muscle cells) is a commonly used method for studying the cellular mechanisms of pulmonary hypertension. However, two-dimensional culture cannot simulate the three-dimensional microenvironment of cells in vivo, leading to the neglect of important factors such as cell-cell interactions, the influence of the extracellular matrix, and the transduction of mechanotropic signals. Two-dimensional models also struggle to simulate the complex vascular remodeling processes in the disease, such as vessel wall thickening, luminal stenosis, and the formation of plexiform lesions.

[0006] Organoids are simplified, laboratory-cultured tissue models that mimic various aspects of the complex structure and function of living tissues. They are invaluable tools for studying the mechanisms of human tissue development, regeneration, and repair. In recent years, with the rapid development of technology in the field of biological research, organoid culture technology has emerged. As a new three-dimensional culture system, it can better simulate the structure and function of human tissues. However, existing organoid technologies mainly focus on areas such as tumors, intestines, and livers. Research on organoids for pulmonary hypertension is still in its early stages. Currently, there are no methods for constructing organoids specifically targeting the pathological characteristics of this disease. Given the limitations of existing research models, the development of an organoid model that can accurately simulate the pathological characteristics of pulmonary hypertension is of significant scientific and practical value. Summary of the Invention

[0007] The inventors of this invention have discovered that cells derived from pulmonary artery endarterectomy tissue can be used to construct organoids and 2D vascular networks. This has led to the establishment of a method for obtaining cells from pulmonary artery endarterectomy tissue, culturing organoids and 2D vascular networks. The resulting organoids and 2D vascular networks can better simulate the pathophysiological characteristics of pulmonary hypertension and have the advantages of short culture cycles, rapid proliferation, high cell count, and high viability. This provides a tissue research model that closely reflects clinical practice for studying the mechanisms of disease development and provides novel tools for future drug screening and personalized treatment.

[0008] The first aspect of this invention provides a method for preparing pulmonary artery endarterectomy tissue cells, the method comprising:

[0009] (1) Tissue preparation steps: clean the tissue specimen and obtain cells or tissue fragments;

[0010] (2) Resuspend cells and tissue fragments in hydrogel and incubate in a culture container until the hydrogel solidifies;

[0011] (3) Add vascular tissue inoculation medium to the culture container for culture;

[0012] (4) Add vascular tissue growth medium and culture until cells are observed to migrate out of the tissue slices, then culture for another 4-10 days;

[0013] (5) Replace the culture medium with vascular organoid maintenance medium and continue culturing until the cells almost merge on the surface of the culture container;

[0014] (6) Cells and tissues were passaged to obtain P1 generation cells, which were then further cultured;

[0015] (7) Passage the cells again and remove tissue debris to obtain P2 generation cells;

[0016] (8) Cell cryopreservation may be performed optionally.

[0017] In a specific embodiment of the present invention, step (1) obtains a thickness of less than 1 mm. 2 Fragments of the organization.

[0018] In a specific embodiment of the present invention, step (3) involves culturing for 24-48 hours.

[0019] In a specific embodiment of the present invention, in step (4), the cell migration cycle from the tissue slice is 15-30 days.

[0020] In a specific embodiment of the present invention, in step (4), the cells are cultured for 7 days after migrating from the tissue slice.

[0021] In a specific embodiment of the present invention, in step (4), the culture medium should be changed every 4 days.

[0022] In a specific embodiment of the present invention, the culture period of step (5) is 14-30 days.

[0023] In a specific embodiment of the present invention, step (6) involves culturing on a surface pre-coated with hydrogel.

[0024] The second aspect of the present invention provides cells obtained by the method of the first aspect; preferably, the cells are P2 generation cells of step (7) or their re-passaged cells or cryopreserved cells obtained in step (8) or their re-passaged cells.

[0025] A third aspect of the present invention provides a method for culturing pulmonary artery tissue organoids, the method comprising the following steps:

[0026] (9-1) Provide the cells of the second aspect of the present invention, and add a mixture of hydrogel and collagen I to obtain a cell mixture;

[0027] (9-2) The cell mixture obtained in step (9-1) is seeded into a culture vessel in droplet form for incubation;

[0028] (9-3) After the cell mixture becomes a solid cell aggregate, add the vascular organoid formation medium to the culture container for culture;

[0029] (9-4) Transfer the cell aggregate to a culture vessel with a low adhesion surface, preferably an ultra-low adhesion surface;

[0030] (9-5) Add vascular organoid maintenance culture medium and culture until organoid tissue is obtained.

[0031] In a specific embodiment of the present invention, step (9-1) directly utilizes the P2 generation cell suspension of the first aspect of the present invention; in other embodiments, the frozen cells are thawed to obtain the suspension.

[0032] In a specific embodiment of the present invention, step (9-1) uses a 3:1 mixture of 20-50 μL hydrogel and collagen I, which contains 10,000-50,000 cells.

[0033] In a specific embodiment of the present invention, step (9-3) involves culturing for 7 days, with the culture medium being changed every other day;

[0034] In a specific embodiment of the present invention, step (9-4) involves transferring the cell aggregates using a pipette.

[0035] In a specific embodiment of the present invention, the culture medium is changed every 4 days in step (9-5), and the culture is carried out for 12-30 days.

[0036] The fourth aspect of the present invention provides pulmonary artery tissue organoids obtained by the method of the third aspect.

[0037] In a specific embodiment of the present invention, the organoid is spherical and has a 3D network structure.

[0038] In a specific embodiment of the present invention, the organoid comprises smooth muscle cells, pericytes, and endothelial cells.

[0039] The fifth aspect of the present invention provides a 2D vascular network formed by cells according to the second aspect of the present invention, preferably the 2D vascular network is formed on the surface of a hydrogel.

[0040] The sixth aspect of the present invention provides the use of organoids of the fourth aspect and 2D vascular networks of the fifth aspect; preferably, the application is for preparing drug screening kits or vascular disease research kits; preferably, the drug is used for pulmonary vascular diseases, wherein the vascular disease is a pulmonary vascular disease.

[0041] Beneficial technical effects

[0042] This invention first obtains pulmonary artery endarterial stripping tissue cells that can form organoids. The organoids obtained from these cells have clear structures, are consistent with the source tissue, have diverse cell types, and closely resemble the original tissue, making them easy for researchers to manipulate and thus having good practical application value. Attached Figure Description

[0043] Figure 1 This demonstrates the processing of pulmonary artery endarterectomy tissue specimens. During tissue block culture, the mixture of tissue fragments and cells is cultured in 2D format, starting from passage 1. (Among them...) Figure 1 A is a tissue block after processing of a pulmonary artery endarterectomy specimen; Figure 1 (A mixture of tissue fragments and cells.)

[0044] Figure 2 This demonstrates cell migration into the matrix gel and the formation of 3D structures. (Among them) Figure 2 A is a 3D cultured tissue block; Figure 2 B represents cells migrating into the matrix to form 3D structures.

[0045] Figure 3 The second-generation cells form spheroids, and a 3D network structure can be seen in the spheroids.

[0046] Figure 4 Smooth muscle cells, pericytes, and endothelial cells can be seen in the spheroid.

[0047] Figure 5 The slides of vascular organoids were stained with hematoxylin and eosin (HE) to observe their gross morphology.

[0048] Figure 6 Immunofluorescence staining revealed that endothelial cells formed tubular structures.

[0049] Figure 7 This demonstrates that vascular cells can form a network structure on the surface of the matrix gel. Detailed Implementation

[0050] Unless otherwise specified, the experimental equipment and reagents used in this invention are conventional reagents in the field and can be obtained commercially.

[0051] The culture container is a multi-well plate commonly used by those skilled in the art, such as a 6-well plate or a 48-well plate. In order to cultivate 3D structures, a 48-well ultra-low adsorption plate was used. Ultra-low adsorption plates are also known in the art and are commercially available.

[0052] Unless otherwise specified, the “incubation” or “cultivation” described in this invention is carried out statically at 37°C and 5% CO2.

[0053] Hydrogels are used for cell culture, and are preferably permeable to dissolved oxygen, to culture medium components, and to metabolites, for example, to diffusion. The hydrogel preferably has a gel-like structure; for example, the hydrogel may be based on laminin and / or nestin and / or collagen and / or fibronectin and / or PEG. A preferred hydrogel is a matrix gel.

[0054] In this invention, the gel-like protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells is commonly referred to as Matrigel. During experimental procedures, Matrigel should be treated at 4°C to ensure it remains liquid.

[0055] Typically, all culture media can contain antibacterial agents, such as penicillin and / or streptomycin, to prevent bacterial contamination.

[0056] The culture medium used in this invention includes:

[0057]

[0058] Example

[0059] Example 1: Tissue Processing

[0060] The surgically obtained pulmonary artery endarterectomy tissue specimens were separated, cleaned, and trimmed. The pale yellow intima tissue component was selected, and blood clots and myoblastic intima tissue were removed as much as possible, as this part of the tissue contains almost no cells, which would affect the efficiency of subsequent organoid preparation. The separated, cleaned, and trimmed pale yellow intima tissue component was then placed in tissue preservation solution for subsequent cell and organoid preparation.

[0061] Example 2 Organoid Culture

[0062] Specific steps for preparing organoids from pulmonary artery endarterectomy tissue:

[0063] (1) Tissue preparation: Wash the tissue sequentially with PBS solutions containing 10X, 5X, and 1X antibiotics. Cut the tissue into small pieces (less than 1 mm^2) with scissors. Incubate the sections with 10 ml of cell digestion solution in a 5% CO2 incubator at 37°C for 30 minutes. Figure 1 ).

[0064] (2) Collect cells and tissue fragments into 15 ml centrifuge tubes. Centrifuge at 300 g and 4 °C for 5 minutes. Resuspend the cells and tissue fragments (precipitate in the 15 ml tube) in 100 μl of matrix gel and seed the resulting suspension as droplets into 6-well plates (tissue culture treatment). Incubate the plates in an incubator to allow the matrix gel to invert to solidify for about 1 hour.

[0065] (3) After the matrix gel solidifies, add 3 ml of vascular tissue inoculation medium to each well of the 6-well plate and incubate for 48 hours.

[0066] (4) After 48 hours, add vascular tissue growth medium until cells are observed to migrate from the tissue slice, and then culture for another 7 days. The medium should be changed every 4 days. Figure 2 ).

[0067] (5) After step (4), replace the culture medium with vascular organoid maintenance culture medium for 14-30 days until the cells are almost confluent in the plate.

[0068] (6) Cell Culture: When the cells are nearly confluent, wash the cells and tissue sections three times with cold (4°C) PBS. After washing, add 3 ml of cell digestion solution to each well and incubate for 15 minutes in a 5% CO2, 37°C incubator. Collect the cells and tissue fragments into 15 ml centrifuge tubes. Centrifuge at 300 g, 4°C for 5 minutes. Resuspend the precipitate in vascular organoid maintenance medium and seed it into 6-well plates pre-coated with matrix gel (dilute matrix gel 1:100 with PBS and add 2 ml of this solution to each well of the 6-well plate and incubate in an incubator) to obtain P1 generation cells.

[0069] (7) When the cells are close to confluence, passage them again as described in step (6). After P2 generation, the tissue fragments will be discarded.

[0070] (8) Cell cryopreservation: After treatment with cell digestion solution and centrifugation, the cell pellet is resuspended in cell cryopreservation solution and directly frozen in a -80°C freezer.

[0071] (9) 3D Construction: Prepare a cell suspension of the second-generation cells (cell count can be between 10,000 and 50,000) as described in cell culture section (6). Centrifuge at 300g, 4℃ for 5 min, and resuspend the cell pellet in 30 μl of a mixture (3:1 matrix gel and collagen I). Seed the cell suspension and the above mixture in droplet form into 6-well plates (tissue culture treatment). Incubate the plates in an incubator to allow the mixture to solidify for about 1-2 hours. (The mixture should be treated at 4℃ to ensure its liquid state, and experiments should be performed as soon as possible after treating the mixture). After the mixture becomes solid, add 3 ml of vascular organoid formation medium to each well of the 6-well plate, and change the medium every other day until day 7. After 7 days of culture, the dome-shaped gel droplets of the mixture in the culture plate were carefully transferred (using a 1 ml pipette, with the pipette tip appropriately cut to obtain a large diameter) to a 48-well ultra-low adsorption plate and cultured in vascular organoid maintenance medium, which was changed every 4 days. After 12-30 days in the ultra-low adsorption plate, spherical bodies were obtained. Figure 3 Then proceed to the next step of staining and identification.

[0072] Example 3: Organoid Staining Identification

[0073] Staining and identification: Cell spheroids cultured for 19-30 days (day 0 for mixed inoculation) were fixed with 4% paraformaldehyde at 4°C for 24 hours. The paraformaldehyde was discarded, and the cells were washed three times with PBS. The spheroids were then treated with 0.5% Triton X-100 for 15 minutes at room temperature. The Triton-X was discarded, and the cells were washed three times with PBS. The cells were blocked with 1% BSA at 4°C for 24 hours. The primary antibody was diluted with 1% BSA and incubated with the spheroids at 4°C for 48 hours. Primary antibodies used included: Anti-CD31, Anti-α-SMA, and Anti-PDGFRβ. The primary antibody solution was discarded, and the cells were washed three times with PBS. The secondary antibody was diluted with 1% BSA and incubated with the spheroids at 4°C for 48 hours. Secondary antibodies used: Goat anti-Rabbit IgG (FITC), Goat anti-Rabbit IgG (Alexa Fluor 594), Goat anti-Mouse IgG (FITC), Donkey Anti-Goat IgG (Alexa Fluor 647). The secondary antibody solution was discarded, and the sample was washed three times with PBS. Observation and recording were performed under a microscope. Figure 4-6 ).

[0074] Example 4: Vascular Network Formation and Identification

[0075] Method for forming a vascular network structure on the surface of a matrix gel by vascular cells: 50 μL of matrix gel was added to a 96-well plate and spread evenly. The plate was incubated for 2 hours. P2 cells from one well (6-well plate) obtained in Example 2 were cultured to 90% confluence. The cells were digested with 2 mL of cell digestion solution for 10 min, and culture medium was added to stop the digestion. The cells were collected, centrifuged, and counted. The cells were resuspended in vascular organoid maintenance medium, and the cell density was adjusted to 250,000 cells / mL. 200 μL of cell suspension (50,000 cells) was added to the well containing matrix gel. Observation was performed every half hour. When a clear network was formed, photographs were taken. The presence of vascular endothelial cells and their functional properties were demonstrated in a two-dimensional culture. Figure 7 ).

Claims

1. A pulmonary artery tissue organoid obtained by the following method, wherein the method includes a cell preparation process and a tissue culture process, and the cell preparation process includes the following steps: (1) Tissue preparation steps: clean the tissue specimen and obtain cells and tissue fragments; (2) Resuspend cells and tissue fragments in matrix gel and incubate in a culture container until the matrix gel solidifies; (3) Add vascular tissue inoculation medium to the culture container and culture for 24-48 hours; (4) Add vascular tissue growth medium and culture until cells are observed to migrate out of the tissue slice. The cycle of cell migration out of the tissue slice is 15-30 days, and then culture for another 4-10 days. (5) Replace the culture medium with vascular organoid maintenance medium and continue culturing until the cells converge on the surface of the culture container; (6) Cells and tissues were passaged to obtain P1 generation cells, which were then further cultured; (7) Passage the cells again and remove tissue debris to obtain P2 generation cells; (8) Perform cell cryopreservation; in: The tissue was obtained by separating, cleaning, and trimming pulmonary artery endarterectomy tissue specimens, selecting pale yellow intima tissue components, and removing blood thrombus components and myoblasted intima tissue from the exfoliated tissue. The vascular tissue inoculation medium consists of a 1:1 mixture of ECM endothelial cell culture medium and MSCM mesenchymal stem cell culture medium, as well as 100 ng / ml EGF and 100 ng / ml VEGF. The vascular tissue growth medium consisted of ECM endothelial cell culture medium, 100 ng / ml VEGF and 3 μM CHIR99021; The vascular organoid maintenance culture medium consisted of ECM endothelial cell culture medium and 100 ng / ml VEGF; The tissue culture process includes the following steps: (9-1) The P2 generation cells obtained in step (7) were made into a suspension and a mixture of matrix gel and collagen I was added to obtain a cell mixture; (9-2) The cell mixture obtained in step (9-1) is seeded into a culture vessel in droplet form for incubation; (9-3) After the cell mixture becomes a solid cell aggregate, add the vascular organoid formation medium to the culture container for culture; (9-4) Transferring cell aggregates to culture vessels with ultra-low adhesion surfaces; (9-5) Add vascular organoid maintenance medium and culture until organoid tissue is obtained; in: The organoid is spherical and has a 3D network structure; it contains smooth muscle cells, pericytes, and endothelial cells. The vascular organoid formation medium consists of a 1:1 mixture of ECM endothelial cell culture medium and MSCM mesenchymal stem cell culture medium, and 100 ng / ml VEGF.

2. The pulmonary artery tissue organoid as described in claim 1, wherein: Step (1) Obtain a thickness of less than 1 mm 2 Fragments of the organization; and or, In step (4), the cells are cultured for 7 days after migrating from the tissue slice; and / or, In step (4), the culture medium is changed every 4 days; and / or, The culture period for step (5) is 14-30 days; and / or, Step (6) Incubate on a surface pre-coated with the matrix adhesive.

3. The pulmonary artery tissue organoid as described in claim 1, wherein: Step (9-1) uses a 3:1 mixture of 20-50 μL matrix gel and collagen I; and / or, Step (9-3) involves culturing for 7 days, with the medium changed every other day; and / or, Step (9-4) involves transferring the cell aggregates using a pipette; and / or, Step (9-5): Change the culture medium every 4 days and culture for 12-30 days.

4. Use of the organoid according to claim 1, wherein the use is for preparing a drug screening kit or a vascular disease research kit.

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