A culture medium for CPAM organoids

Abstract

The present invention relates to the technical field of culture medium, and specifically to a culture medium for CPAM organoids, comprising three components, namely, Component A, Component B, and Component C, and a plurality of cell growth factors. By coordinating the proportions of various factors and regulating WNT signals, the growth conditions of normal alveolar organoids can be intervened. The culture medium can accurately supply the growth requirements of CPAM organoids, ensure that the size and properties of the organoids are more uniform, and can culture high-quality and large-quantity CPAM organoids in vitro, which is more conducive to simulating the disease properties of CPAM in vitro and more in line with the requirements of subsequent drug screening. At the same time, the cost is effectively controlled, and it is convenient for promotion and application.

Description

Technical Field

[0001] The invention relates to the technical field of culture medium, and in particular to a culture medium for CPAM organoids. Background Art

[0002] Congenital pulmonary airway malformation (CPAM) is also known as congenital cystic adenomatoid malformation (CCAM). It is a congenital lung disease that causes abnormal respiratory function due to congenital abnormalities in the number and structure of alveoli. It is caused by abnormalities in the morphogenesis of lung branching and is a developmental lung disease characterized by excessive proliferation and dilation of terminal bronchioles. Clinically, it usually manifests as shortness of breath, dyspnea, cough, fever, etc., which can lead to respiratory failure and endanger life in severe cases. Based on existing research, it is speculated that the pathogenesis of CPAM may involve signal transduction disorders between epithelial cells and other cells (including endothelial cells, fibroblasts, etc.) during lung embryonic development, resulting in the lack of normal alveolar structure in the lungs and the formation of multicystic lung masses.

[0003] It is currently believed that different types of CPAM originate from different levels of the trachea and bronchial tree and different stages of lung development, and may be affected by intrauterine airway obstruction or atresia. Clinical diagnosis can be divided into four types of CPAM according to the size of the cyst and histological phenotype. Type I accounts for the highest proportion, and its main feature is bronchial lung cysts, which are specifically manifested as complete bronchial occlusion, resulting in cystic masses formed by gas accumulation in bronchial lung tissue. The onset of types II and III is mostly concentrated at the junction of the bronchi / bronchioles, and the main feature is tracheobronchial malformation, which is specifically manifested as congenital malformations of branches at all levels of the tracheobronchial tree and their distal airways, and the normal structure of the hilar region is missing, usually composed of multiple small cysts. IV cases account for a small proportion, and the onset point is usually in the peripheral lung tissue. The main feature is that cystic masses containing fluid can be seen in the lung parenchyma (usually the cyst diameter is greater than 10 cm), leading to gas accumulation, reduced lung capacity and decreased lung compliance.

[0004] Most CPAM children can be detected by prenatal ultrasound at 18 to 22 weeks of pregnancy, with high sensitivity and specificity. It is manifested as high-echo, low (no) echo or mixed echo masses in the lungs on ultrasound images. At present, the best treatment strategy for CPAM is surgical resection, among which the third-generation thoracoscopic precise lesion resection surgery is a commonly used surgical method for treating CPAM. The purpose of the surgery is to remove the diseased tissue to improve the patient's respiratory function. However, clinical analysis shows that some CPAM children have no obvious symptoms after birth. As they age, they cannot be ruled out of the risk of infection and tumors. At present, the recommended treatment methods for CPAM disease are relatively single, and surgical resection should be performed for CPAM children with or without obvious symptoms. Therefore, it is of great significance to simulate the occurrence and development of CPAM disease in vitro and explore its pathogenesis. Organoid models are widely regarded as one of the most effective tools for in vitro experiments to simulate the occurrence of diseases. The successful cultivation of high-quality CPAM organoids in vitro is the key prerequisite for the smooth implementation of a series of subsequent experiments.

[0005] There are currently a variety of culture medium formulas on the market for culturing human alveolar organoids. However, when using the conventional culture scheme for normal alveolar organoids to culture CPAM organoids, it is often difficult to effectively eliminate the interference of normal alveolar organoids, resulting in the inability to accurately simulate the characteristics of CPAM diseases in vitro. At the same time, under long-term large-scale culture conditions, normal alveolar organoids may proliferate from generation to generation, thereby masking the number of CPAM organoids. In addition, CPAM organoids may face problems such as insufficient quantity and poor growth status. The high price of existing human alveolar organoid culture medium also makes the cost of large-scale culture high, which is difficult to meet experimental needs, further limiting the development of related research.

[0006] Therefore, it is of great research significance to develop a low-cost culture medium that can stably culture CPAM organoids with uniform morphology and properties in vitro and in sufficient quantity. This will not only help to more accurately simulate the characteristics of CPAM diseases, but also provide efficient and economical support for related experimental research. Summary of the invention

[0007] The purpose of the present invention is to provide a culture medium for CPAM organoids in view of the problems existing in the prior art. The culture medium can intervene in the growth of normal alveolar organoids by coordinating the proportions of various factors and regulating WNT signals, and can accurately supply the growth requirements of CPAM organoids, and can culture CPAM organoids with uniform morphological properties and a large number in vitro, while effectively controlling costs and facilitating promotion and application.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A CPAM organoid culture medium, wherein each 50 ml of the culture medium comprises a mixture of the following raw materials:

[0010] 80-120 mM TGF-β receptor kinase inhibitor 4-6 ul;

[0011] 8-12 ul of 8-12 mM WNT / β-catenin signaling pathway activator;

[0012] Insulin-transferrin-selenium 400-600 ul;

[0013] Penicillin-Streptomycin-Amphotericin B 400-600 ul;

[0014] 0.8-12 mg / mL human epidermal growth factor 2-4 ul;

[0015] 4-6 ul of 40-60 mg / mL heparin;

[0016] 400-600 mM N-acetylcysteine ​​120-130 ul;

[0017] 8-12 ug / mL human fibroblast growth factor-10 40-60 ul;

[0018] 1.2-1.8 ml of the mixture of Component A, Component B, and Component C;

[0019] The remainder was DMEM / F-12 medium.

[0020] Note: Each 50 ml mixture of Component A, Component B and Component C is prepared by mixing the following ingredients.

[0021] Component A:

[0022] Bovine serum albumin 0.3-0.5 g;

[0023] Biotin 620-630 ul;

[0024] Catalase 620-630 ul;

[0025] Superoxide dismutase 620-630 ul;

[0026] Human holoferritin 620-630 ul;

[0027] Component B:

[0028] Glutathione reduced peptide 2-3 mg;

[0029] L-Carnitine 4-6 mg;

[0030] D-galactose 37-38 mg;

[0031] Putrescine 40-42 mg;

[0032] Component C:

[0033] Ethanolamine 2-3 ul;

[0034] Progesterone 15-16 ul;

[0035] 3,3′,5-triiodo-L-thyronine 2-3 ul;

[0036] L-homoproline 40-60 ul;

[0037] Oleic acid 20-30 ul;

[0038] Linoleic acid 20-30 ul;

[0039] Linolenic acid 20-30 ul;

[0040] Retinyl acetate 12-14 ul;

[0041] DL-α-tocopherol 20-30 ul;

[0042] DL-α-tocopherol acetate 20-30 ul;

[0043] Corticosterone 20-30 ul;

[0044] Sodium selenite 31-32 ul;

[0045] The balance is IMDM medium.

[0046] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0047] The present invention provides a culture medium for CPAM organoids, comprising three components, Component A, Component B, and Component C, and a plurality of cell growth factors. By coordinating the proportions of various factors and regulating WNT signals, the growth conditions of normal alveolar organoids can be intervened. The culture medium can accurately supply the growth requirements of CPAM organoids, ensure that the size and properties of the organoids are more uniform, and can culture high-quality and large-quantity CPAM organoids in vitro, which is more conducive to simulating the disease properties of CPAM in vitro and better meeting the requirements of subsequent drug screening. At the same time, the cost is effectively controlled, which is convenient for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 A display image of the CPAM organoid cultured in Example 1 (4×); Figure 1 The left picture in the figure is a normal alveolar picture. Figure 1 The right picture is a picture of CPAM organoids.

[0049] Figure 2 This is a display of normal alveoli (10×).

[0050] Figure 3 This is a display image of CPAM organoids (10×).

[0051] Figure 4 Immunofluorescence staining of normal tissue and CPAM tissue.

[0052] Figure 5 Immunofluorescence staining images of normal alveolar organoids and CPAM organoids.

[0053] Figure 6 CPAM organoids cultured in normal alveolar organoid medium.

[0054] Figure 7 This is a diagram showing CPAM organoids cultured in the CPAM organoid culture medium of Example 1.

[0055] Figure 8 This is a diagram showing normal alveolar organoids cultured using the CPAM organoid culture medium provided in Example 1. DETAILED DESCRIPTION

[0056] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0057] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0058] The following Example 1 provides a complete implementation:

[0059] Example 1

[0060] 1 Tissue digestion

[0061] (1) First, take the lung tissue and place it in a 15 ml centrifuge tube. Use scissors to cut it into small pieces and place it on ice.

[0062] (2) Add 3 ml of lysis buffer (480 U / ml Collagenase Type I 100 ul + 2 U / ml dispase 1 ml, add DPBS to 10 ml) to the centrifuge tube, mix well, and place in a 37° incubator for 30 min. Take out and invert every 15 min to mix thoroughly (repeat this digestion process three times), then transfer to a C-Tube tube and use program lung-mouse-02-01 to make a single-cell suspension. (This process takes about 20 s).

[0063] (3) Prepare flow cytometry tubes in advance, number them, filter the single-cell suspension using a 70 um filter membrane, transfer it to the flow cytometry tube, rinse it with 1 ml of DMEMF / 12, centrifuge at 4°, 300 g for 5 min, obtain the cell pellet, and discard the supernatant.

[0064] (4) Add 1 ml of lysis buffer to the cell pellet, mix well using a vortex shaker, and place on ice for 5 min.

[0065] (5) After placing on ice for 5 min, centrifuge for 5 min (4°, 300 g) to ensure that the cell pellet turns white, i.e., the cell lysis is successful. Then, add 1 ml FACS buffer (0.5 M EDTA 100 ul + 1 M HEPES 1.25 ml + BSA (powder) 0.25 g + DPBS to 50 ml) to rinse once, and centrifuge for 5 min (4°, 300 g) to obtain a single-cell suspension.

[0066] 2 Magnetic bead sorting

[0067] (1) Dilute the primary antibody (HT2-280 antibody for sorting human AT2 cells) with FACS buffer at a dilution of 1:100, and the total volume used is 100 ul. Incubate the primary antibody for 1 hour in a refrigerator at 4 degrees Celsius, and wash once with 2 ml FACS buffer after the incubation.

[0068] (2) Magnetic bead incubation: Add Anti-Mouse IgM MicroBeads in a biosafety cabinet, add 100 ul of Anti-Mouse IgM MicroBeads to each tube (10 ul beads + 90 ul FACS buffer), incubate in a 4°C refrigerator for 20 min, and centrifuge at 300 g for 5 min.

[0069] (3) After discarding the supernatant of the cell suspension, resuspend it in FACS buffer and load the suspension onto the MACS® Column, which is placed in the magnetic field of the MACS Separator. The magnetically labeled cells pass through the high-gradient magnetic field and are retained in the column. The unlabeled cells flow through the column and are received in the 15 ml centrifuge tube below (waste liquid), while the cells adsorbed by the magnetic beads are the sorted epithelial cells.

[0070] (4) After passing FACS buffer through the magnetic column 2-3 times, add 1-3 ml of buffer to the column, push the column vigorously, and collect the target cells in the flow tube. The cells adsorbed by the magnetic beads are the selected epithelial cells.

[0071] (5) Count the sorted cells and record the number of cells at the four corners of the blood cell counting plate as N (counting principle: count the top but not the bottom, count the left but not the right). The density of the cell suspension obtained is C1=(N / 4)×10 4 Pieces / ml.

[0072] 3 Organoid Plates

[0073] (1) Take 2000-3000 sorted AT2 cells, resuspend them in Organoid-CPAM medium, and mix them with 25 ul matrigel to form a droplet. Note: If it is difficult to ensure the stability of the cell number and cell activity, you can increase the number of cells by about 1000 to improve the success rate.

[0074] (2) Add 50 μl (25 μl AT2 cells + 25 μl matrigel) droplets into one well of a 24-well plate and place in a 37°C incubator for 40 min to allow the matrigel to fix to the bottom of the plate.

[0075] (3) After ensuring that the matrigel is fixed to the bottom of the dish, add 1 ml of Organoid-CPAM medium to each well to cover the cells and culture them in droplets (change the medium every 48 hours).

[0076] The sources of materials for the CPAM culture system are shown in Table 1.

[0077] Table 1 Organoid-CPAM culture system

[0078]

[0079] Taking the preparation of 50 ml Organoid-CPAM medium as an example (components A&B&C are prepared first), the specific formula is as follows:

[0080] Component A: The following reagents are all powders, which can be weighed and dissolved in IMDM media according to the prescribed dosage. The specific formula is as follows:

[0081] (1) Bovine serum albumin 0.4 g (in some embodiments, 0.3-0.5 g).

[0082] (2) Biotin, 100 mg in 10 ml IMDM media. 625 ul (in some embodiments, 620-630 ul).

[0083] (3) Catalase, 100 mg in 10 ml IMDM media. 625 ul (in some embodiments, 620-630 ul).

[0084] (4) Superoxide dismutase, 100 mg in 10 ml IMDM media. 625 ul (in some embodiments, 620-630 ul).

[0085] (5) Human holoferritin, 100 mg in 10 ml IMDM media. 625 ul (in some embodiments, 620-630 ul).

[0086] Component B, the following reagents are all powders, and can be directly weighed and used according to the prescribed dosage. The specific formula is as follows:

[0087] (1) Glutathione reduced peptide 2.5 mg (in some embodiments, 2-3 mg).

[0088] (2) L-Carnitine 5 mg (in some embodiments, 4-6 mg).

[0089] (3) D-galactose 37.5 mg (in some embodiments, 37-38 mg).

[0090] (4) Putrescine 40.25 mg (in some embodiments, 40-42 mg).

[0091] Component C, the following reagents need to be dissolved as required, the solvent is alcohol or sterile water. The specific ratio is as follows:

[0092] (1) 2.5 ul of ethanolamine (in some embodiments, 2-3 ul)

[0093] (2) Progesterone, dissolved in alcohol, at a concentration of 1 mg / ml, stored at -80°C. 5.75 ul (in some embodiments, 15-16 ul).

[0094] (3) 3,3′,5-triiodo-L-thyronine, 10 mg 3,3′,5-triiodo-L-thyronine dissolved in 100 ul DMSO and 4.9 ml ethanol, stored at -80°C. 2.5 ul (in some embodiments, 2-3 ul).

[0095] (4) L-homoproline, dissolved in sterile water, 50 mg / ml, stored at -80°C. 50 ul (in some embodiments, 40-60 ul).

[0096] (5) Oleic acid, dissolved in alcohol, 100 mg / ml, stored at -80°C. 25 ul (in some embodiments, 20-30 ul).

[0097] (6) Linoleic acid, dissolved in alcohol, 100 mg / ml, stored at -80°C. 25 ul (in some embodiments, 20-30 ul).

[0098] (7) Linolenic acid, dissolved in alcohol, 100 mg / ml, stored at -80°C. 25 ul (in some embodiments, 20-30 ul).

[0099] (8) Retinyl acetate, dissolved in alcohol, 20 mg / ml, stored at -80°C. 12.5 ul (in some embodiments, 12-14 ul).

[0100] (9) DL-α-tocopherol, dissolved in alcohol, 100 mg / ml, stored at -80°C. 25 ul (in some embodiments, 20-30 ul).

[0101] (10) DL-α-tocopherol acetate, dissolved in alcohol, 100 mg / ml, stored at -80°C. 25ul (in some embodiments, 20-30ul).

[0102] (11) Corticosterone, dissolved in alcohol, 2 mg / ml, stored at -80°C. 25 ul (in some embodiments, 20-30 ul).

[0103] (12) Sodium selenite, dissolved in sterile water, 1 mg / ml, stored at -80°C. 31.25 ul (in some embodiments, 31-32 ul).

[0104] Finally, all ingredients were diluted to 50 ml with DMEM / F-12.

[0105] Note: CPAM organoids require the addition of ROCK inhibitor (10 mM) 48 hours before establishing primary organoids. Add 0.5 ul ROCK inhibitor (10 mM) to every 1 ml Organoid-CPAM medium.

[0106] The culture results are as follows Figure 1-Figure 3 shown.

[0107] Compared with normal tissues and normal alveolar organoids, there is a KRT17+ / KRT5- cell population in CPAM tissues and organoids. This type of cell is defined as a transitional cell in the process of transformation from AT2 cells to AT1 cells. Therefore, we identified the characteristics of CPAM organoids by staining KRT17 and KRT5. The use of this culture medium can better culture CPAM organoids rich in KRT17+ / KRT5- cell populations. The specific immunofluorescence staining results are shown in the figure. Figure 4-5 shown.

[0108] This organoid formula is also suitable for the cultivation of normal alveolar organoids in children, with the advantages of simple operation, low cost, high survival rate of organoids, etc. Compared with the traditional epithelial-fibroblast co-culture system, it is more time-saving and labor-saving, and has a lower cost than the commercialized alveolar organoid complete culture medium on the market. It can greatly reduce the capital cost of large-scale cultivation of organoids and the construction of a library while ensuring the normal growth of CPAM organoids.

[0109] Table 2 shows the existing normal alveolar organoid culture medium formula.

[0110] Table 2 Normal alveolar organoid culture medium formula

[0111]

[0112] Compared with those grown in normal alveolar organoid culture medium, the organoids cultured in the culture medium of the present invention have a morphology closer to CPAM itself, that is, large solid spheres with fewer vacuolar normal alveolar organoids, which eliminates the interference of normal alveolar organoids for in vitro exploration of the pathogenesis of CPAM disease.

[0113] At the same time, the CPAM organoid culture medium of the present invention is also suitable for the culture of normal alveolar organoids. Compared with the normal alveolar organoid culture medium culture system, the growth and quantity of vacuolar alveolar organoids are not inferior. Figure 6-8 shown.

[0114] In summary, the present invention significantly promotes the growth of CPAM organoids by optimizing the culture medium formula, especially the splitting and adjustment of the B27 component, on the basis of meeting the growth requirements of alveolar organoids (such as providing WNT activators and a series of growth factors, etc.). At the same time, the method effectively controls the experimental cost, greatly reduces the economic investment required for organoid culture, and provides a more economical and efficient solution for related research. Secondly, the present invention optimizes the shortcomings of the existing normal alveolar organoid culture medium formula in culturing CPAM organoids. Under the stimulation of factors such as high concentrations of WNT activators, traditional culture media often cause the growth characteristics of CPAM organoids to deviate, thereby failing to accurately simulate the alveolar pathological characteristics of CPAM diseases. To solve this problem, the present invention successfully promotes the growth of CPAM organoids by precisely regulating the proportion and interaction of each factor in the culture medium, while effectively maintaining its unique biological characteristics. This optimization scheme not only improves the stability and phenotypic consistency of CPAM organoids in in vitro culture, but also more accurately reproduces the pathological characteristics of CPAM diseases, providing a more reliable experimental platform for the study of related disease mechanisms and the development of treatment strategies.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture medium for CPAM organoids, characterized in that: Each 50 ml culture medium is prepared by mixing the following ingredients: 80-120 mM TGF-β receptor kinase inhibitor 4-6 ul; TGF-β receptor kinase inhibitor comes from Selleckchem TGF-β receptor kinase inhibitor with catalog number S1067; 8-12 ul of 8-12 mM Wnt / β-catenin signaling pathway activator; the Wnt / β-catenin signaling pathway activator is derived from Selleckchem Wnt / β-catenin signaling pathway activator with catalog number S1263; Insulin-transferrin-selenium 400-600 ul; Penicillin-Streptomycin-Amphotericin B 400-600 ul; 0.8-12 mg / mL human epidermal growth factor 2-4 ul; 4-6 ul of 40-60 mg / mL heparin; 400-600 mM N-acetylcysteine ​​120-130 ul; 8-12 ug / mL human fibroblast growth factor 10 40-60 ul; 1.2-1.8 ml mixture of Component A, Component B and Component C; The balance was DMEM / F-12 medium; Component A: Bovine serum albumin 0.3-0.5 g; Biotin 100 mg; Catalase 100 mg; Superoxide dismutase 100 mg; Human holoferritin 100 mg; Dissolve together in 10 ml IMDM medium and take 620-630 ul; Component B: Glutathione reduced peptide 2-3 mg; L-Carnitine 4-6 mg; D-galactose 37-38 mg; Putrescine 40-42 mg; Component C: Ethanolamine 2-3 ul; Progesterone, dissolved in alcohol, solubility is 1 mg / ml, take 15-16 ul; 3,3′,5-triiodo-L-thyronine: 10 mg 3,3′,5-triiodo-L-thyronine dissolved in 100 ul DMSO and 4.9 ml alcohol, take 2-3 ul; L-homoproline, dissolved in sterile water, 50 mg / ml, take 40-60 ul; Oleic acid, dissolved in alcohol, 100 mg / ml, take 20-30 ul; Linoleic acid, dissolved in alcohol, 100 mg / ml, take 20-30 ul; Linolenic acid, dissolved in alcohol, 100 mg / ml, take 20-30 ul; Retinyl acetate, dissolved in alcohol, 20 mg / ml, take 12-14 ul; DL-α-tocopherol, dissolved in alcohol, 100 mg / ml, take 20-30 ul; DL-α-tocopherol acetate, dissolved in alcohol, 100 mg / ml, take 20-30 ul; Corticosterone, dissolved in alcohol, 2 mg / ml, take 20-30 ul; Sodium selenite, dissolved in sterile water, 1 mg / ml, take 31-32 ul.

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