A human parathyroid organoid culture medium and a parathyroid organoid culture method

Abstract

The present invention relates to the field of organoid culture technology, in particular to a human parathyroid organoid culture medium and a culture method thereof. In view of the problem of low parathyroid organoid culture efficiency in the prior art, the culture medium provided by the present invention significantly improves the growth efficiency and survival rate of organoids and accelerates the maturation of organoids by optimizing the culture medium formula. The culture medium can effectively support and promote the growth of organoids and is suitable for application scenarios such as disease model establishment, drug screening and cell therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a human parathyroid organoid culture medium and a parathyroid organoid culture method. Background Art

[0002] In the field of biomedical research, organoid culture technology is an innovative method that enables stem cells to form tissues with three-dimensional structures in vitro by simulating the microenvironment of natural organs. These structures replicate the histological characteristics and functions of real organs to a certain extent. Human parathyroid organoids, as a type of organoid, are cultivated from stem cells extracted from parathyroid tissue. They can simulate the functions of the parathyroid gland, including hormone secretion and response to drugs.

[0003] Although organoid technology has shown great potential in simulating human organs and disease research, it still faces some challenges in practical applications. In particular, in the culture of parathyroid organoids, the main problems include difficulty in separation and slow proliferation. These problems limit the efficiency and practicality of parathyroid organoids in scientific research and clinical applications. For example, due to the particularity of parathyroid tissue, it is a technical challenge to isolate a sufficient number of stem cells from patient samples and maintain their stability and functionality. In addition, the growth rate and maturity of organoids also directly affect the cycle and cost of the experiment, and slow growth may lead to delays in research and therapeutic applications.

[0004] Chinese patent CN118773117A discloses a culture medium and a culture method for human parathyroid organoids. The culture medium provided by the invention includes DMEM-F12 basal culture medium and cytokines. The cytokines include conventional cytokines and specific cytokines. The conventional cytokines include B27 protein, A83-01, EGF, triple antibody and ROCK inhibitor; the specific cytokines include sonic hedgehog signaling molecules, activin and p38MAPK inhibitor.

[0005] However, the culture efficiency of human parathyroid organoids in existing technologies needs to be further improved. Summary of the invention

[0006] The object of the present invention is to provide a human parathyroid organoid culture medium and a parathyroid organoid culture method, which can obtain more organoids in a short time.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an organoid culture medium, which uses a basal culture medium as a solvent and comprises the following components:

[0009] N2 supplement, vascular endothelial growth factor-121, angiotensin, B27, 4-hydroxyethylpiperazineethanesulfonic acid, ROCK inhibitor Y-27632, nicotinamide, fibroblast growth factor-10, R-Spondin protein, Noggin protein, epidermal growth factor, and TGF-β receptor inhibitor A 83-01.

[0010] Preferably, the basal culture medium is selected from one or more of DMEM / F12, DMEM, RPMI-1640, Advanced DMEM / F12, and Advanced DMEM.

[0011] Preferably, in the organoid culture medium, the final volume percentage concentration of the N2 supplement is 0.3-0.8%;

[0012] and / or, in the organoid culture medium, the final concentration of vascular endothelial growth factor-121 is 5 to 15 ng / mL;

[0013] And / or, in the organoid culture medium, the final concentration of angiotensin is 0.05-0.2 nmol / mL.

[0014] Preferably, in the organoid culture medium, the final volume percentage concentration of B27 in the organoid culture medium is 0.3-0.8%;

[0015] and / or, in the organoid culture medium, the final concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 5 to 15 mM;

[0016] and / or, in the organoid culture medium, the final concentration of the ROCK inhibitor Y-27632 is 5 to 20 μM;

[0017] and / or, in the organoid culture medium, the final concentration of nicotinamide is 50 to 150 μM;

[0018] and / or, in the organoid culture medium, the final concentration of the fibroblast growth factor-10 is 25 to 75 ng / mL;

[0019] and / or, in the organoid culture medium, the final concentration of the R-Spondin protein is 5 to 15 ng / mL;

[0020] and / or, in the organoid culture medium, the final concentration of the Noggin protein is 10 to 40 ng / mL;

[0021] and / or, in the organoid culture medium, the final concentration of the epidermal growth factor is 10 to 30 ng / mL;

[0022] And / or, in the organoid culture medium, the final concentration of the TGF-β receptor inhibitor A 83-01 is 2.5 to 7.5 μM.

[0023] The present invention also provides use of the above-mentioned organoid culture medium in culturing parathyroid organoids.

[0024] The present invention also provides a method for culturing parathyroid organoids, comprising the following steps:

[0025] The organoid culture medium and matrigel are mixed to obtain a parathyroid organoid culture matrix;

[0026] mixing the parathyroid tissue sample fragments with a digestion solution and performing digestion to obtain a tissue digestion solution;

[0027] The tissue digestion solution is blown and filtered to obtain a precipitate;

[0028] The precipitate was resuspended in cell culture medium and centrifuged to obtain a cell precipitate;

[0029] The cell pellet is inoculated into a parathyroid organoid culture matrix and cultured to obtain parathyroid organoids.

[0030] Preferably, the volume ratio of the organoid culture medium to the matrix gel is 0.8-1.2:0.8-1.2;

[0031] The digestive fluid contains type 2 collagenase and neutral protease;

[0032] The final concentration of type 2 collagenase in the digestion solution is 1.5-2 mg / ml;

[0033] The final concentration of the neutral protease in the digestion solution is 3 to 8 mg / ml;

[0034] The digestion temperature is 35-40°C;

[0035] The digestion time is 1 to 2 hours.

[0036] Preferably, the blowing is performed 5 to 20 times;

[0037] The filtration pore size used in the filtration is 100 μm;

[0038] The cell culture medium is adDMEM / F12+++;

[0039] The centrifugal speed is 200-400g;

[0040] The centrifugation time is 3 to 8 minutes.

[0041] Preferably, the culture time is more than 3 days.

[0042] The present invention also provides parathyroid organoids obtained by the above culture method.

[0043] Beneficial effects of the present invention:

[0044] The culture medium provided by the present invention significantly improves the growth efficiency of organoids, which is crucial for accelerating the development and maturation of organoids. Rapid growth means that more organoids can be obtained in a short time, which is a great advantage for scientific research and clinical applications. The high survival rate of organoids ensures the repeatability and reliability of the experiment, reduces the risk of experimental failure due to cell death, and improves experimental efficiency and resource utilization. The appropriate types and proportions of growth factors and nutrients in the culture medium provide the best growth environment for organoids, which helps to simulate the in vivo environment so that the cultivated organoids are closer to the physiological state of real organs. The ingredients in the culture medium can effectively support the initial growth and development of organoids, promote cell proliferation and differentiation, which is of great significance for studying cell biology and developmental biology.

[0045] In the present invention, the efficient organoid culture method provides a new way to establish a disease model, which helps to deeply understand the pathogenesis of parathyroid-related diseases and provide an experimental platform for new drug development and disease treatment. The high survival rate and rapid growth of organoids provide an ideal model for drug screening and toxicology research, and can more accurately predict the efficacy and side effects of drugs. The culture medium and method of the present invention are conducive to the development of new tissue engineering technologies, providing the possibility of cell therapy for patients with diseases such as hypoparathyroidism. The use of organoids can reduce the dependence on animal models, meet ethical requirements, and provide an experimental model that is closer to the human physiological state. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram of the parathyroid organoid culture results in Example 2 of the present invention;

[0047] Figure 2 This is a diagram of the parathyroid organoid culture results in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0048] The present invention provides an organoid culture medium, which uses a basal culture medium as a solvent and comprises the following components:

[0049] N2 supplement, vascular endothelial growth factor-121, angiotensin, B27, 4-hydroxyethylpiperazineethanesulfonic acid, ROCK inhibitor Y-27632, nicotinamide, fibroblast growth factor-10, R-Spondin protein, Noggin protein, epidermal growth factor, and TGF-β receptor inhibitor A 83-01.

[0050] In the present invention, the N2 supplement is a 100-fold concentrated cell culture additive with a defined chemical composition and sterilized by filtration, which has the function of promoting cell growth and survival. In the prior art, it is generally used for the culture of neurons, neural stem cells, neuroma cells, etc.

[0051] In the present invention, the vascular endothelial growth factor-121 (VEGF-121) is a subtype of the vascular endothelial growth factor (VEGF) family. VEGF is a growth factor that can specifically act on vascular endothelial cells and has activities such as promoting angiogenesis. VEGF-121 is one of the splice variants of VEGF-A, cannot bind to heparin, and is mainly involved in the physiological functions of angiogenesis and enhancing vascular permeability. VEGF-121 exerts its biological effects by binding to its receptors VEGFR-1 and VEGFR-2, promoting the proliferation and migration of vascular endothelial cells and increasing vascular permeability.

[0052] In the present invention, the angiotensin is a type of peptide hormone that causes vasoconstriction and increased blood pressure, and is a key component of the renin-angiotensin system (RAS).

[0053] In the present invention, B27 is a commercial supplement containing multiple neurotrophic factors and hormones, which is generally used in the prior art to optimize the activity and long-term survival rate of prenatal and embryonic neurons, ensure the activity of neural-derived tumor cell lines, and expand EGF-responsive precursor cells from the mouse embryonic striatum and midbrain.

[0054] HEPES is a zwitterionic organic chemical buffer, which is generally used in the prior art to maintain the pH stability of the culture medium.

[0055] ROCK inhibitor Y-27632 is a selective ROCK1 (p160ROCK) inhibitor, which is generally used in the prior art to inhibit the formation of Rho-mediated stress fibers, inhibit G1-S phase cell cycle progression and cytokinesis, help reduce separation-induced apoptosis, and improve cloning efficiency.

[0056] Niacinamide is a form of vitamin B3 that is essential for processes such as cellular energy metabolism and DNA repair.

[0057] FGF-10 (fibroblast growth factor-10) is a cytokine that promotes cell proliferation and differentiation and may play a role in tissue regeneration and repair.

[0058] R-Spondin is a secreted protein that can enhance the activity of the Wnt signaling pathway and plays an important role in the self-renewal and differentiation of stem cells.

[0059] Noggin is an inhibitor of the BMP (bone morphogenetic protein) signaling pathway and plays an important role in regulating embryonic development and tissue formation.

[0060] Epidermal growth factor (EGF) is a cytokine that promotes cell proliferation and differentiation, and is very important for maintaining cell regeneration and repairing damaged tissues.

[0061] A83-01 is an inhibitor of TGF-β (transforming growth factor-β) type I receptor ALK5 / 4 / 7 kinase, which can affect cell proliferation, differentiation and tissue formation.

[0062] In the present invention, these components work together to provide a suitable environment for the culture of organoids, promoting their growth, differentiation and maintenance of function.

[0063] In the present invention, preferably, the basal culture medium is selected from one or more of DMEM / F12, DMEM, RPMI-1640, AdvancedDMEM / F12, and Advanced DMEM.

[0064] Preferably, in the organoid culture medium, the final volume percentage concentration of the N2 supplement is 0.3-0.8%; and / or, in the organoid culture medium, the final concentration of vascular endothelial growth factor-121 is 5-15 ng / mL; and / or, in the organoid culture medium, the final concentration of angiotensin is 0.05-0.2 nmol / mL. Preferably, in the organoid culture medium, the final volume percentage concentration of B27 is 0.3-0.8%; and / or, in the organoid culture medium, the final concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 5-15 mM; and / or, in the organoid culture medium, the final concentration of ROCK inhibitor Y-27632 is 5-20 μM; and / or, in the organoid culture medium, the final concentration of nicotinamide is 50-150 μM; and / or, in the organoid culture medium, The final concentration of the fibroblast growth factor-10 is 25 to 75 ng / mL; and / or, in the organoid culture medium, the final concentration of the R-Spondin protein is 5 to 15 ng / mL; and / or, in the organoid culture medium, the final concentration of the Noggin protein is 10 to 40 ng / mL; and / or, in the organoid culture medium, the final concentration of the epidermal growth factor is 10 to 30 ng / mL; and / or, in the organoid culture medium, the final concentration of the TGF-β receptor inhibitor A 83-01 is 2.5 to 7.5 μM.

[0065] The present invention also provides use of the above-mentioned organoid culture medium in culturing parathyroid organoids.

[0066] The present invention also provides a method for culturing parathyroid organoids, comprising the following steps:

[0067] The organoid culture medium and matrigel are mixed to obtain a parathyroid organoid culture matrix;

[0068] mixing the parathyroid tissue sample fragments with a digestion solution and performing digestion to obtain a tissue digestion solution;

[0069] The tissue digestion solution is blown and filtered to obtain a precipitate;

[0070] The precipitate was resuspended in cell culture medium and centrifuged to obtain a cell precipitate;

[0071] The cell pellet is inoculated into a parathyroid organoid culture matrix and cultured to obtain parathyroid organoids.

[0072] In the present invention, preferably, the volume ratio of the organoid culture medium to the matrix gel is 0.8-1.2:0.8-1.2; the digestion solution contains type 2 collagenase and neutral protease; the final concentration of type 2 collagenase in the digestion solution is 1.5-2 mg / ml; the final concentration of neutral protease in the digestion solution is 3-8 mg / ml; the temperature of the digestion is 35-40°C; the digestion time is 1-2h. Preferably, the number of blowing is 5-20 times; the filter pore size used in the filtration is 100μm; the cell culture medium is adDMEM / F12+++; the centrifugal speed is 200-400g; the centrifugal time is 3-8min. Preferably, the culture time is more than 3d.

[0073] The present invention also provides parathyroid organoids obtained by the above culture method.

[0074] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0075] Example 1 Culture medium preparation

[0076] The following were added to the culture medium DMEM:F12: N2 (0.5X), B27 (0.5X), HEPES (10mM), ROCK inhibitor Y-27632 (10μM), nicotinamide (100μM), FGF-10 (50ng / mL), R-Spondin (10ng / ml) Noggin (25ng / mL), epidermal growth factor (EGF) (20ng / mL), A 83-01 (5μM), VEGF-121 (10ng / mL) and angiotensin (0.1nmol / mL) to obtain organoid culture medium.

[0077] Example 2 Parathyroid Organoid Construction

[0078] The prepared matrix gel (BD: 356231) was placed in a 4°C refrigerator to thaw overnight, and mixed with the organoid culture medium in Example 1 in a 1:1 ratio for later use;

[0079] Add double antibody, glutamine and HEPES into 100 ml D / F12 medium to prepare addDMEM / F12+++, and pre-cool in a 4℃ refrigerator;

[0080] Place the 24-well plate in CO 2 Preheat the incubator (5% CO 2 , 37°C);

[0081] Parathyroid organoid construction procedures:

[0082] Fresh parathyroid tissue samples were collected and immediately placed in tissue protection solution and stored at 4°C.

[0083] Add ice-cold PBS containing 1% double antibody to a 35 mm dish, remove the parathyroid tissue sample, remove normal tissue, fat, blood clots and necrotic parts, and cut the parathyroid sample into 1-2 mm pieces with surgical scissors or a scalpel. 3 Small fragments.

[0084] Transfer the tissue fragments into a 10 ml centrifuge tube, let the tissue settle under gravity for 2-3 minutes, remove the supernatant, and add ice-cold PBS containing 1% double antibody to wash twice.

[0085] Add 3-5 ml of parathyroid organoid digestion solution (1.8 mg / ml type 2 collagenase, 5 mg / ml neutral proteinase) according to the amount of tissue, seal with sealing film, and digest on a shaker at 37°C for 1.5 hours, shaking and mixing on a shaker every 30 minutes.

[0086] After digestion is complete, pipette up and down 10 times with a 1 ml pipette tip to further digest the tissue, filter it using a 100 μm cell strainer, centrifuge at 300 g for 5 min, and remove the supernatant.

[0087] Add 5 ml adDMEM / F12+++ to resuspend, centrifuge at 300 g for 5 min, remove the supernatant, and add the matrix gel-organoid culture medium mixture (10 ml per 10 ml) according to the amount of precipitation. 5 The cells were added with 50 μl of PBS and resuspended.

[0088] Take out the 24-well plate placed in the 37°C incubator 2 hours in advance, add 50 μl of the mixed solution to each well, put it in the 37°C incubator for 5 minutes, and then invert it to solidify for 20 minutes.

[0089] After solidification, add 500 μl of parathyroid organoid culture medium to a 24-well plate and place it in a cell culture incubator for culture. Observe the growth of organoids every day and replace the culture medium every 2 to 3 days. Figure 1 shown.

[0090] Comparative Example 1

[0091] The only difference from Example 2 is that the organoid culture medium is replaced by:

[0092] The culture medium (DMEM:F12) was added with B27 (0.5X), HEPES (10 mM), ROCK inhibitor Y-27632 (10 μM), nicotinamide (100 μM), FGF-10 (50 ng / mL), R-Spondin (10 ng / ml), Noggin (25 ng / mL), epidermal growth factor (EGF) (20 ng / mL), and A 83-01 (5 μM).

[0093] The results are as follows Figure 2 shown.

[0094] In Example 1 of the present invention, the organoids grew very well on the first day, while the organoids in the comparative example died a lot on the first day; in Example 1, the organoids grew very fast and grew larger on the third day, while the organoids in the comparative example 1 grew slowly. It shows that the culture medium provided in Example 1 of the present invention significantly improves the growth efficiency and survival rate of organoids. Specifically, the organoids grew very well on the first day, which shows that the components in the culture medium can effectively support the initial growth and development of organoids, provide cells with necessary nutrients and growth factors, thereby promoting cell proliferation and differentiation; on the third day, the organoids in Example 1 grew very fast and grew larger, which further confirmed the excellent performance of the culture medium of the present invention. The rapid growth rate and large volume indicate that the ratio of growth factors and nutrients in the culture medium is appropriate, which can meet the needs of rapid growth of organoids. Therefore, the culture medium provided in Example 1 of the present invention not only improves the survival rate of organoids, but also promotes their rapid growth through its unique combination of components, showing its significant advantages in organoid culture, and is expected to play an important role in the fields of tissue engineering, disease model research, and drug screening.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An organoid culture medium, characterized in that The basic culture medium is used as the solvent, and the following components are included: N2 supplement, vascular endothelial growth factor-121, angiotensin, B27, 4-hydroxyethylpiperazineethanesulfonic acid, ROCK inhibitor Y-27632, nicotinamide, fibroblast growth factor-10, R-Spondin protein, Noggin protein, epidermal growth factor, and TGF-β receptor inhibitor A 83-01.

2. The organoid culture medium according to claim 1, characterized in that The basic culture medium is selected from one or more of DMEM / F12, DMEM, RPMI-1640, Advanced DMEM / F12, and Advanced DMEM.

3. The organoid culture medium according to claim 1, characterized in that In the organoid culture medium, the final volume percentage concentration of the N2 supplement is 0.3-0.8%; and / or, in the organoid culture medium, the final concentration of vascular endothelial growth factor-121 is 5 to 15 ng / mL; And / or, in the organoid culture medium, the final concentration of angiotensin is 0.05-0.2 nmol / mL.

4. The organoid culture medium according to claim 1, characterized in that In the organoid culture medium, the final volume percentage concentration of B27 in the organoid culture medium is 0.3-0.8%; and / or, in the organoid culture medium, the final concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 5 to 15 mM; and / or, in the organoid culture medium, the final concentration of the ROCK inhibitor Y-27632 is 5 to 20 μM; and / or, in the organoid culture medium, the final concentration of nicotinamide is 50 to 150 μM; and / or, in the organoid culture medium, the final concentration of the fibroblast growth factor-10 is 25 to 75 ng / mL; and / or, in the organoid culture medium, the final concentration of the R-Spondin protein is 5 to 15 ng / mL; and / or, in the organoid culture medium, the final concentration of the Noggin protein is 10 to 40 ng / mL; and / or, in the organoid culture medium, the final concentration of the epidermal growth factor is 10 to 30 ng / mL; And / or, in the organoid culture medium, the final concentration of the TGF-β receptor inhibitor A83-01 is 2.5 to 7.5 μM.

5. Use of the organoid culture medium according to any one of claims 1 to 4 in culturing parathyroid organoids.

6. A method for culturing parathyroid organoids, characterized in that: The following steps are involved: Mixing the organoid culture medium according to any one of claims 1 to 4 with matrigel to obtain a parathyroid organoid culture matrix; mixing the parathyroid tissue sample fragments with a digestion solution and performing digestion to obtain a tissue digestion solution; The tissue digestion solution is blown and filtered to obtain a precipitate; The precipitate was resuspended in cell culture medium and centrifuged to obtain a cell precipitate; The cell pellet is inoculated into a parathyroid organoid culture matrix and cultured to obtain parathyroid organoids.

7. The culture method according to claim 6, characterized in that: The volume ratio of the organoid culture medium to the matrix gel is 0.8-1.2:0.8-1.2; The digestive fluid contains type 2 collagenase and neutral protease; The final concentration of type 2 collagenase in the digestion solution is 1.5-2 mg / ml; The final concentration of the neutral protease in the digestion solution is 3 to 8 mg / ml; The digestion temperature is 35-40°C; The digestion time is 1 to 2 hours.

8. The culture method according to claim 6, characterized in that: The number of blowing and beating is 5 to 20 times; The filtration pore size used in the filtration is 100 μm; The cell culture medium is adDMEM / F12+++; The centrifugal speed is 200-400g; The centrifugation time is 3 to 8 minutes.

9. The culture method according to claim 6, characterized in that: The culture time is more than 3 days.

10. Parathyroid organoid obtained by the culture method according to any one of claims 6 to 9.

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