Pulmonary artery tissue-derived cell and organoid as well as construction method and application of pulmonary artery tissue-derived cell and organoid

Abstract

According to the pulmonary artery tissue-derived cells and organoids as well as the construction method and application thereof, the pulmonary artery intima exfoliation tissue organoids obtained by the preparation method are clear in structure, various in cell types consistent with the derived tissues, close to the original tissues and convenient for researchers to operate, so that the pulmonary artery intima exfoliation tissue organoids have good practical application value.

Description

Technical Field

[0001] The present invention provides a cell and an organoid derived from pulmonary artery tissue, a construction method and an application thereof, belonging to the technical field of biomedicine, and specifically relating to the field of organoid construction. Background Art

[0002] Pulmonary hypertension (PH) refers to a clinical and pathophysiological syndrome caused by structural or functional changes in the pulmonary vasculature due to various etiologies and different pathogenesis mechanisms, resulting in increased pulmonary vascular resistance and pulmonary artery pressure, and then developing into right heart failure or even death, seriously affecting the quality of life and survival of patients. Clinically, PH is divided into five categories, with a wide range of etiologies and complex pathogenesis mechanisms, involving the combined action of multiple factors and multiple links. Chronic thromboembolic pulmonary hypertension (CTEPH) belongs to the 4th category of PH and is a disease characterized by dyspnea, fatigue, and reduced exercise tolerance. Due to undissolved thromboemboli blocking the proximal pulmonary artery and accompanied by distal pulmonary vascular remodeling, it leads to increased pulmonary vascular resistance, progressive elevation of pulmonary artery pressure, and gradually increasing right heart load, eventually progressing to right heart failure. It is a serious, progressive, and fatal disease. The incidence of CTEPH shows an increasing trend year by year. Due to its insidious onset, rapid progression, and high mortality rate, it has become a global healthcare problem seriously threatening human life and health. Clinically, the diagnosis and treatment of CTEPH patients still face many challenges. Most of the proximal organized thrombi and corresponding intimal thickening can be solved by pulmonary thromboendarterectomy, while the distal lesions that cannot be surgically treated and the residual pulmonary hypertension after surgery may require medical targeted drug therapy.

[0003] A large number of studies have shown that the occurrence and development of CTEPH is a process involving the abnormal participation of multiple genes, multiple cells, multiple factors, and multiple signal transduction systems, but which factors and specific molecular mechanisms have not been fully elucidated. Research has shown that pulmonary artery endothelial cell dysfunction, abnormal proliferation of pulmonary artery smooth muscle cells, and inflammatory responses ultimately lead to pulmonary vascular remodeling and play an important role in the occurrence and development of CTEPH. Therefore, it is very important to construct a disease model that can fully simulate the pathological characteristics of the occurrence and development of CTEPH.

[0004] Currently, CTEPH research mainly relies on animal models and two-dimensional cell culture, but there are many limitations. Commonly used animal models of pulmonary arterial hypertension include the monocrotaline (MCT)-induced model, the hypoxia model, and transgenic models (such as the BMPR2 mutation model). These models simulate the pathological characteristics of pulmonary arterial hypertension to a certain extent, but there are significant differences from the pathophysiological mechanisms of human diseases. They cannot accurately simulate the genetic heterogeneity and pathophysiological characteristics of human pulmonary arterial hypertension, and the drug responses are significantly different 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 arterial hypertension. However, two-dimensional culture cannot simulate the three-dimensional microenvironment of cells in vivo, resulting in the neglect of important factors such as cell-cell interactions, the influence of the extracellular matrix, and mechanical signal transduction. Two-dimensional models are also difficult to simulate the complex vascular remodeling process in diseases, such as vascular wall thickening, vascular lumen stenosis, and the formation of plexiform lesions.

[0005] Organoids are simplified, laboratory-cultured tissue models that mimic various aspects of the complex structure and function in living tissues. They are valuable tools for studying the mechanisms of human tissue development, regeneration, and repair. In recent years, with the rapid development of technologies in the field of biological research, organoid culture technology has emerged. As an emerging three-dimensional culture system, it can better simulate the structure and function of human tissues. However, existing organoid technologies mainly focus on fields such as tumors, the intestine, and the liver. Organoid research for pulmonary arterial hypertension is still in its infancy. There is currently no method for constructing organoids targeting the pathological characteristics of such diseases using existing organoid technologies. Given the limitations of existing research models, it is urgently necessary to develop an organoid model that can accurately simulate the pathological characteristics of pulmonary arterial hypertension, which has important scientific significance and application value. Summary of the Invention

[0006] The inventors of the present invention found that cells derived from pulmonary artery endarterectomy tissues can be used to construct organoids and 2D vascular networks, thereby establishing a method for obtaining cells from pulmonary artery endarterectomy tissues, culturing organoids, and 2D vascular networks. The obtained organoids and 2D vascular networks can better simulate the pathophysiological characteristics of pulmonary arterial hypertension, and have the advantages of a short culture period, rapid proliferation, a high number of cells, and a high viability. They provide a tissue research model close to clinical reality for studying the mechanisms of disease occurrence and development, as well as a new tool for future disease drug screening and personalized treatment.

[0007] In the first aspect of the present invention, a method for preparing cells of pulmonary artery endarterectomy tissues is provided, and the method includes:

[0008] (1) A tissue preparation step of washing the tissue specimen and obtaining cells or tissue fragments;

[0009] (2) Resuspend the cells and tissue fragments with the hydrogel and incubate in a culture vessel until the hydrogel solidifies;

[0010] (3) Add vascular tissue inoculation medium to the culture vessel for culture;

[0011] (4) Add vascular tissue growth medium for culture until it is observed that cells migrate out of the tissue pieces, and then culture for 4 - 10 days;

[0012] (5) Replace the medium with vascular organoid maintenance medium and continue to culture until the cells are almost confluent on the surface of the culture vessel;

[0013] (6) Passage the cells and tissue to obtain P1 - generation cells and further culture them;

[0014] (7) Passage again and remove the tissue fragments to obtain P2 - generation cells;

[0015] (8) Optionally, perform cell cryopreservation.

[0016] In a specific embodiment of the present invention, in step (1), tissue fragments smaller than 1 mm are obtained. 2 are obtained.

[0017] In a specific embodiment of the present invention, in step (3), culture for 24 - 48 h.

[0018] In a specific embodiment of the present invention, in step (4), the period for cells to migrate out of the tissue pieces is 15 - 30 days.

[0019] In a specific embodiment of the present invention, in step (4), after the cells migrate out of the tissue pieces, culture for 7 more days.

[0020] In a specific embodiment of the present invention, in step (4), the medium should be replaced every 4 days.

[0021] In a specific embodiment of the present invention, the culture period of step (5) is 14 - 30 days.

[0022] In a specific embodiment of the present invention, in step (6), culture is carried out on a surface pre - coated with hydrogel.

[0023] The second aspect of the present invention provides the cells obtained by the method of the first aspect; preferably, the cells are P2 - generation cells of step (7) or their sub - passage cells or the cryopreserved cells obtained in step (8) or their sub - passage cells.

[0024] The third aspect of the present invention provides a method for culturing pulmonary artery tissue organoids, and the method comprises the following steps:

[0025] (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;

[0026] (9-2) Inoculate the cell mixture obtained in step (9-1) in the form of droplets into a culture container for incubation;

[0027] (9-3) After the cell mixture becomes a solid cell aggregate, add a vascular organoid formation medium to the culture container for culture;

[0028] (9-4) Transfer the cell aggregate to a culture container with a low attachment surface, preferably an ultra-low attachment surface;

[0029] (9-5) Add a vascular organoid maintenance medium for culture until an organoid tissue is obtained.

[0030] In a specific embodiment of the present invention, the P2 generation cell suspension of the first aspect of the present invention is directly used in step (9-1); in other embodiments, the cryopreserved cells are thawed to obtain a suspension.

[0031] In a specific embodiment of the present invention, 20-50 ul of a 3:1 mixture of hydrogel and collagen I is used in step (9-1), and the number of cells contained is 10,000-50,000.

[0032] In a specific embodiment of the present invention, step (9-3) is cultured for 7 days, and the culture method is to change the medium every other day;

[0033] In a specific embodiment of the present invention, the cell aggregate is transferred by a pipette in step (9-4).

[0034] In a specific embodiment of the present invention, the medium is changed every 4 days in step (9-5), and the culture is carried out for 12-30 days.

[0035] The fourth aspect of the present invention provides a pulmonary artery tissue organoid obtained by the method of the third aspect.

[0036] In a specific embodiment of the present invention, the organoid is spherical and has a 3D network structure.

[0037] In a specific embodiment of the present invention, the organoid contains smooth muscle cells, pericytes and endothelial cells.

[0038] The fifth aspect of the present invention provides a 2D vascular network formed by the cells of the second aspect of the present invention, preferably the 2D vascular network is formed on the surface of a hydrogel.

[0039] The sixth aspect of the present invention provides the use of the organoid of the fourth aspect and the 2D vascular network of the fifth aspect; preferably, the application is to prepare a drug screening kit or a vascular disease research kit; preferably, the drug is used for pulmonary vascular disease, and the vascular disease is pulmonary vascular disease.

[0040] Beneficial technical effects

[0041] The present invention first obtains pulmonary artery intimal denudation tissue cells that can form organoids. The organoids obtained from the cells have a clear structure, are consistent with the source tissue, have diverse cell types, are close to the original tissue, are easy for researchers to operate, and therefore have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure shows the mixture of tissue fragments and cells after the pulmonary artery endarterectomy tissue specimen is processed and cultured. Starting from the first generation, the cells are cultured in 2D form. Figure 1 A is the tissue block after the pulmonary artery endarterectomy tissue specimen is processed; Figure 1 B A mixture of tissue fragments and cells.)

[0043] Figure 2 The cells migrated into the matrix gel and formed a 3D structure. Figure 2 A is a 3D culture tissue block; Figure 2 B is the migration of cells into the matrix to form a 3D structure.

[0044] Figure 3 The second generation of cells formed spheroids, in which a 3D network structure was visible.

[0045] Figure 4 Smooth muscle cells, pericytes, and endothelial cells are visible in the spheroids.

[0046] Figure 5 HE staining was performed on sections of vascular organoids to observe the gross morphology.

[0047] Figure 6 Immunofluorescence staining showed that endothelial cells formed tubular structures.

[0048] Figure 7 It shows that vascular cells can form a network structure on the surface of Matrigel. DETAILED DESCRIPTION

[0049] Unless otherwise specified, the experimental equipment and reagents used in the present invention are conventional reagents in the art and can be obtained through commercial channels.

[0050] The culture container is a multi-well plate conventionally used by those skilled in the art, such as a 6-well plate, a 48-well plate, etc. In order to culture 3D structures, a 48-well ultra-low adsorption plate is used. The ultra-low adsorption plate is also known in the art and can be obtained commercially.

[0051] Unless otherwise specified, the "incubation" or "culturing" described in the present invention is performed statically at 37° C. and 5% CO 2 .

[0052] The hydrogel is used for cell culture, for example, being permeable to dissolved oxygen, permeable to culture medium components, and preferably permeable to metabolic products, for example, for diffusion. The hydrogel preferably has a gel-like structure, for example, the hydrogel can be based on laminin and / or nidogen and / or collagen and / or fibronectin and / or PEG. A preferred hydrogel is matrigel.

[0053] In the present invention, the gel-like protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells is generally referred to as Matrigel. During experimental operation, Matrigel should be processed at 4°C to ensure that it is in a liquid state.

[0054] Typically, all culture media may contain antibacterial agents such as penicillin and / or streptomycin to prevent bacterial contamination.

[0055] The culture medium used in the present invention comprises:

[0056]

[0057]

[0058] Example

[0059] Example 1 Tissue Processing

[0060] The surgically obtained pulmonary artery endarterectomy specimen is separated, cleaned, and trimmed. The yellowish intimal tissue is selected, and bloody thrombus components and muscularized intimal tissue are removed as much as possible. This tissue contains almost no cellular components, which can affect the efficiency of subsequent organoid preparation and hinder its development. The separated, cleaned, and trimmed yellowish intimal tissue is then placed in tissue preservation solution for subsequent cell and organoid preparation.

[0061] Example 2 Organoid Culture

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

[0063] (1) Tissue preparation: Wash the tissue successively 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 an incubator at 5% CO2 and 37 °C for 30 minutes ( Figure 1 ).

[0064] (2) Collect the cells and tissue fragments into a 15-ml centrifuge tube. Centrifuge at 300 g and 4 °C for 5 minutes. Resuspend the cells and tissue fragments (the pellet in the 15-ml tube) with 100 μl of Matrigel, and inoculate the obtained suspension as droplets into a 6-well plate (tissue culture-treated). Incubate the plate in the incubator to allow the Matrigel to turn solid for about 1 hour.

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

[0066] (4) After 48 hours, add vascular tissue growth medium until cells migrate out of the tissue pieces and culture for another 7 days. The medium should be changed every 4 days ( Figure 2 ).

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

[0068] (6) Cell culture: When the cells are approaching confluence, wash the cells and tissue pieces 3 times with cold (4 °C) PBS. After washing, add 3 ml of cell digestion solution to each well and culture in an incubator at 5% CO2 and 37 °C for 15 minutes. Collect the cells and tissue fragments into a 15-ml centrifuge tube. Centrifuge at 300 g and 4 °C for 5 minutes. Resuspend the pellet with vascular organoid maintenance medium and inoculate it into a 6-well plate pre-coated with Matrigel (dilute Matrigel with PBS at 1:100 and add 2 ml of this solution to each well of the 6-well plate and culture in the incubator), to obtain P1 generation cells.

[0069] (7) When the cells are approaching confluence, passage 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, resuspend the cell pellet with cell cryopreservation solution and directly freeze in an -80 °C refrigerator.

[0071] (9) 3D Construction: Prepare the second-generation cells into a cell suspension (the number of cells can be between 10,000 and 50,000) according to the description in the cell culture section (6). Centrifuge at 300 g for 5 min at 4 °C, and resuspend the cell pellet with 30 μl of the mixture (matrix gel and collagen I in a ratio of 3:1). Inoculate the cell suspension and the above mixture in the form of droplets into a 6-well plate (tissue culture-treated). Incubate the plate in an incubator to allow the mixture to turn into a solid state for about 1 - 2 hours. (The mixture should be treated at 4 °C to ensure its liquid state, and the experiment should be carried out as soon as possible when 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 the 7th day. After culturing for 7 days, carefully transfer the dome-shaped gel droplets of the mixture in the culture plate (transfer using a 1 ml pipette, appropriately cut the pipette tip to obtain a large diameter) to a 48-well ultra-low attachment plate, and culture with vascular organoid maintenance medium, and change the medium every 4 days. After transferring to the ultra-low attachment plate for 12 - 30 days, obtain spheroids ( Figure 3 ), and proceed to the next step of staining and identification.

[0072] Example 3 Organoid Staining and Identification

[0073] Staining and Identification: Fix the cell spheroids cultured for 19 - 30 days (the day of inoculation of the mixture is counted as day 0) with 4% paraformaldehyde at 4 °C for 24 hours. Discard the paraformaldehyde and wash 3 times with PBS. Then treat the spheroids with 0.5% Triton X-100 at room temperature for 15 minutes. Discard Triton-X and wash 3 times with PBS. Block with 1% BSA at 4 °C for 24 hours. Dilute the primary antibody with 1% BSA and incubate with the spheroids at 4 °C for 48 hours. The primary antibodies used are: Anti-CD31, Anti-α-SMA, Anti-PDGFRβ. Discard the primary antibody solution and wash 3 times with PBS. Dilute the secondary antibody with 1% BSA and incubate with the spheroids at 4 °C for 48 hours. The secondary antibodies used are: Goat anti-Rabbit IgG(FITC), Goat anti-Rabbit IgG(Alexa Fluor 594), Goat anti-Mouse IgG(FITC), Donkey Anti-Goat IgG(Alexa Fluor 647). Discard the secondary antibody solution and wash 3 times with PBS. Observe and record with a microscope ( Figures 4 - 6 ).

[0074] Example 4 Vascular Network Formation and Identification

[0075] Method for vascular cells to form a vascular network structure on the surface of Matrigel: Add 50 μL of Matrigel to a 96-well plate and spread it evenly. Place it in an incubator for 2 hours. Culture the P2 cells from 1 well (6-well plate) obtained in Example 2 until the confluence reaches 90%. Digest the cells with 2 mL of cell digestion solution for 10 min, and then add culture medium to terminate the digestion. Collect the cells, centrifuge them, and count. Resuspend the cells with vascular organoid maintenance medium and adjust the cell density to 250,000 cells per milliliter. Take 200 μL of cell suspension (50,000 cells) and add it to the well containing Matrigel. Observe once every half hour. When an obvious network is formed, take a photo for recording. Prove the existence of vascular endothelial cells and the functionality of vascular endothelial cells at the two-dimensional culture level ( Figure 7 ).

Claims

1. Preparation method of pulmonary artery intimal exfoliation tissue cells, the method comprising: (1) Tissue preparation step, cleaning the tissue specimen and obtaining cells or tissue fragments; (2) Resuspending the cells and tissue fragments with hydrogel and incubating in a culture container until the hydrogel solidifies; (3) Adding vascular tissue inoculation medium to the culture container for culture; (4) Adding vascular tissue growth medium for culture until cells migrate out of the tissue pieces, and then culturing for 4 - 10 days; (5) Replacing the medium with vascular organoid maintenance medium and continuing to culture until the cells are almost confluent on the surface of the culture container; (6) Subculturing the cells and tissue to obtain P1 - generation cells and further culturing; (7) Subculturing again and removing tissue fragments to obtain P2 - generation cells; (8) Optionally performing cell cryopreservation.

2. The method for preparing cells according to claim 1, wherein: Step (1) Obtain tissue fragments smaller than 1 mm 2 ; and / or, step (3) is cultured for 24 - 48 h; and / or, in step (4), the period for cells to migrate out of the tissue pieces is 15 - 30 days; and / or, in step (4), after cells migrate out of the tissue pieces, they are cultured for 7 days; and / or, in step (4), the medium is changed every 4 days; and / or, the culture period of step (5) is 14 - 30 days; and / or, step (6) is cultured on a surface pre - coated with hydrogel.

3. Cells obtained by the method according to claim 1 or 2; preferably, the cells are P2 - generation cells of step (7) or their sub - cultured cells, or cryopreserved cells obtained in step (8) or their sub - cultured cells.

4. Culture method of pulmonary artery tissue organoids, the method comprising the following steps: (9 - 1) Providing the cells according to claim 3 and adding a mixture of hydrogel and collagen I to obtain a cell mixture; (9 - 2) Inoculating the cell mixture obtained in step (9 - 1) in the form of droplets into a culture container for incubation; (9 - 3) After the cell mixture becomes a solid cell aggregate, adding vascular organoid formation medium to the culture container for culture; (9 - 4) Transferring the cell aggregate to a culture container with a low - attachment surface, preferably an ultra - low - attachment surface; (9 - 5) Adding vascular organoid maintenance medium for culture until organoid tissue is obtained.

5. The culture method of pulmonary artery tissue organoids according to claim 4, step (9 - 1) directly uses P2 - generation cells of step (7) of claim 1; or uses cells obtained after thawing the cryopreserved cells described in step (8) of claim 1.

6. The culture method of pulmonary artery tissue organoids according to claim 4, wherein: in step (9 - 1), a 3:1 mixture of 20 - 50 μl of matrix gel and collagen I is used; and / or, step (9 - 3) is cultured for 7 days, and the culture method is changing the liquid every other day; and / or, [[ID=Z8]]step (9 - 4) transfers the cell aggregate by a pipette; and / or, step (9 - 5) changes the medium every 4 days and cultures for 12 - 30 days.

7. Pulmonary artery tissue organoids obtained by the method according to claim 4.

8. The organoid according to claim 7, wherein the organoid is spherical and has a 3D network structure; and / or, the organoid comprises smooth muscle cells, pericytes and endothelial cells.

9. A 2D vascular network formed based on the cells according to claim 2, preferably the 2D vascular network is formed on the surface of a hydrogel.

10. Use of the organoid according to claim 7 or the 2D vascular network according to claim 9, which is for preparing a drug screening kit or a vascular disease research kit.

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