A method and culture medium for culturing human pheochromocytoma organoids

Abstract

This invention proposes a method and culture medium for culturing human pheochromocytoma organoids, belonging to the field of organoid culture technology. The method includes the following steps: S1. Pheochromocytoma treatment; S2. ON-TOP seeding of pheochromocytoma and induction culture; S3. Embedding of pheochromocytoma in a gel and maintenance culture. This method is simple, rapid, and efficient. Due to the characteristics of pheochromocytoma, which grows slowly, the ON-TOP embedding method is required to provide a special growth environment and improve the culture success rate. This invention provides a reliable research tool for studying the development and metabolic mechanisms of pheochromocytoma.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to a method and culture medium for culturing human pheochromocytoma organoids. Background Technology

[0002] Pheochromocytomas are rare neuroendocrine tumors of significant clinical importance, characterized primarily by catecholamine production, which can manifest as a range of clinical symptoms. Despite their rarity, the complex clinical presentation and underlying genetic basis of pheochromocytomas make them a major challenge in endocrinology and oncology practice. Early diagnosis, a thorough understanding of the genetic background, and comprehensive treatment strategies are crucial for improving the prognosis of patients with these potentially life-threatening diseases.

[0003] In foreign countries, the rat pheochromocytoma cell line PC12 is frequently used for related in vitro studies. Because the PC12 cell line is rat-derived, its biological characteristics differ somewhat from those of primary cultured human pheochromocytoma cells, and its traits can be altered by the introduction of exogenous DNA or spontaneous phenotypic and genotypic changes. In in vitro experiments studying the pathogenesis of human pheochromocytoma, besides using rat PC12 cells, the human pheochromocytoma cell line KAT45, reported by Venihaki M et al. in 1998, is also used. Its primary cultured cells were obtained from the tumor of a pheochromocytoma patient with Cushing's syndrome. KAT45 cells are similar to PC12 cells in morphology and proliferation, and can secrete norepinephrine (NE), epinephrine (E), and corticotropin-releasing hormone (CRH). However, possibly because this cell line is not representative of typical pheochromocytoma cells, it has not been widely used to date. In 2023, Sun Meili's team successfully established a primary culture method for human pheochromocytoma cells and identified them as having the secretory and expression functions of pheochromocytoma, providing experimental means for further research on the pathogenesis of pheochromocytoma.

[0004] Organoids are tissue analogs with a specific spatial structure formed through three-dimensional in vitro culture. Because they possess highly similar histological features to their corresponding organs and can reproduce their physiological functions, they can closely mimic the physiological structure, function, development, and differentiation processes of in-situ tissues. They are expected to be widely used in research fields such as drug screening, disease modeling, developmental biology, regenerative medicine, precision medicine, and organ transplantation. To date, there have been no reports of successful culturing of pheochromocytoma organoids, either domestically or internationally. Summary of the Invention

[0005] The purpose of this invention is to provide a method and culture medium for culturing human pheochromocytoma organoids. The method and process are simple, rapid, and efficient. Due to the characteristics of pheochromocytoma, which grows slowly, an on-top embedding method is required to provide a special growth environment and improve the culture success rate. This invention provides a reliable research tool for culturing pheochromocytoma organoids, enabling the study of its development and metabolic mechanisms.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for culturing human pheochromocytoma organoids, comprising the following steps:

[0008] S1. Treatment of pheochromocytoma: Remove the tissue, cut it into pieces, wash, digest, centrifuge, discard the supernatant, resuspend, filter, centrifuge, discard the supernatant, add DMEM medium, mix by pipetting, and place on ice to obtain cell suspension;

[0009] S2. Pheochromocytoma ON-TOP seed gel, induction culture: Spot the cell suspension onto the solidified gel droplet, incubate, add pheochromocytoma organoid induction medium, culture, remove the supernatant, add pheochromocytoma maintenance medium, blow away the gel droplet, and collect all liquid into a centrifuge tube.

[0010] S3. Pheochromocytoma embedding method: Centrifuge and discard the supernatant. Add pre-cooled pheochromocytoma maintenance medium to the cell pellet. Use a pipette tip to place the cell-Matrix gel suspension in the center of the well, so that the suspension forms a hemispherical droplet in the middle of the well plate. After the gel is seeded, incubate, then add pheochromocytoma organoid maintenance medium, culture, and change the medium until the organoid matures.

[0011] As a further improvement of the present invention, the digestion conditions in step S1 are as follows: digestion with digestive enzymes at 36-38°C and 150-250 rpm for 5-15 minutes.

[0012] As a further improvement of the present invention, the incubation conditions in step S2 are 36-38°C and the incubation time is 15-25 min.

[0013] As a further improvement of the present invention, the cultivation conditions in step S2 are 36-38℃, 4-6 v / v%CO2, and the time is 1-3 days.

[0014] As a further improvement of the present invention, the pheochromocytoma organoid induction culture medium comprises the following components at the following concentrations: Advance-DMEM F12 culture medium containing 3-7% fetal bovine serum, 3-7% horse serum, 100-200 µg / ml amphotericin B, 0.5-1.5× penicillin / streptomycin, 0.5-1.5× human leukocyte antigen B27, 10-20 ng / ml epidermal growth factor EGF, 20-40 ng / ml bovine basic fibroblast growth factor 2, 1-3 mM L-alanyl-L-glutamine, and 0.5-1.5 mM sodium pyruvate.

[0015] As a further improvement of the present invention, the pheochromocytoma maintenance culture medium comprises the following components at the following concentrations: Advance-DMEM F12 containing 10-20 ng / ml epidermal growth factor EGF, 20-40 ng / ml bovine basic fibroblast growth factor 2, 0.5-1.5× human leukocyte antigen B-27™ additive, and 0.5-1.5× penicillin / streptomycin.

[0016] As a further improvement of the present invention, the incubation conditions in step S3 are 36-38°C, 4-6 v / v% CO2, and 5-15 min.

[0017] As a further improvement of the present invention, the culture conditions in step S3 are 36-38℃ and 4-6 v / v%CO2.

[0018] This invention further protects a pheochromocytoma organoid induction culture medium used in the above-mentioned method for culturing human pheochromocytoma organoids, comprising the following components at the following concentrations: Advance-DMEM F12 culture medium containing 3-7% fetal bovine serum, 3-7% horse serum, 100-200 µg / ml amphotericin B, 0.5-1.5× penicillin / streptomycin, 0.5-1.5× human leukocyte antigen B27, 10-20 ng / ml epidermal growth factor EGF, 20-40 ng / ml bovine basic fibroblast growth factor 2, 1-3 mM L-alanyl-L-glutamine, and 0.5-1.5 mM sodium pyruvate.

[0019] This invention further protects a pheochromocytoma maintenance culture medium used in the above-mentioned method for culturing human pheochromocytoma organoids, characterized in that it comprises the following components at the following concentrations: Advance-DMEM F12 culture medium containing 10-20 ng / ml epidermal growth factor EGF, 20-40 ng / ml bovine basic fibroblast growth factor 2, 0.5-1.5× human leukocyte antigen B-27™ additive, and 0.5-1.5× penicillin / streptomycin.

[0020] The present invention has the following beneficial effects:

[0021] Current reports on pheochromocytoma mostly focus on identification methods and the culture and identification of primary cell lines. There are currently no reports on the culture of pheochromocytoma organoids, either domestically or internationally. The method described in this invention is simple, rapid, and efficient. Due to the characteristics of pheochromocytoma, which grows slowly, an on-top embedding method is required to provide a special growth environment and improve the culture success rate. The pheochromocytoma organoid culture method of this invention provides a reliable research tool for studying the development and metabolic mechanisms of pheochromocytoma.

[0022] The culture medium independently developed in this invention is divided into induction medium and maintenance medium. Due to the growth characteristics of pheochromocytoma, the company has developed induction and maintenance media specifically for this purpose. The induction culture adopts an on-top method to simulate the internal survival environment. After the cells reach the logarithmic growth phase, they are then transferred to an embedding method for maintenance culture, which improves the growth efficiency of organoids and provides complete nutritional support for their growth environment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Light micrograph of human pheochromocytoma organoid (40d);

[0025] Figure 2 HE staining results of human pheochromocytoma tissue and pheochromocytoma organoids. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1

[0028] This embodiment provides a pheochromocytoma organoid induction culture medium comprising the following components at the following concentrations: 5% fetal calf serum (FCS) (Gibco, 16010159), 5% horse serum (Hyclone, HS) (Biotopped, MXO1D), (100-200) µg / ml amphotericin B (Invivogen, ant-pm-1), 1× penicillin / streptomycin (Thermo, 1514s0122), 1× human leukocyte antigen B27 (Gibco, 17504044), (10-20) ng / ml epidermal growth factor EGF (PeproTech, AF-100-15), (20-40) ng / ml bovine basic fibroblast growth factor 2 (bFGF-2) (Dongkang, Y00321), 2 mM L-alanyl-L-glutamine (GlutaMAX) (Thermo, 35050061*), 1mM sodium pyruvate (GIBCO, 11360070*) in Advance-DMEM F12 medium (Gibco, 12634010).

[0029] Example 2

[0030] This embodiment provides a maintenance culture medium for pheochromocytoma organoids, comprising the following components at the following concentrations: Advance-DMEM F12 (Gibco, 12634010) containing (10-20) ng / ml epidermal growth factor EGF (PeproTech, AF-100-15), (20-40) ng / ml bovine basic fibroblast growth factor 2 (bFGF-2) (Dongkang, Y00321), 1X human leukocyte antigen B-27™ additive (vitamin A removed) (Thermo, 12587010), and 1× penicillin / streptomycin (Thermo, 1514s0122).

[0031] Example 3

[0032] This embodiment provides a method for culturing human pheochromocytoma organoids, including the following steps:

[0033] S1. Management of pheochromocytoma:

[0034] (1) Remove the tissue from the preservation solution and cut it into small pieces with a scalpel in a petri dish, each piece approximately 0.5-1 cm in volume. 3 Place the contents into a 50 mL centrifuge tube and wash 10 times with 10-20 mL of pre-cooled PBS.

[0035] (2) Place the centrifuge tube on ice and allow the tissue to settle to the bottom of the tube naturally. Use a pipette tip to discard as much PBS as possible. Replace the Pasteur pipette after each aspiration operation. Use a pipette tip to aspirate the last 2 mL of residual liquid.

[0036] (3) Use sterile forceps to collect the tissue into a clean petri dish.

[0037] (4) Add 50µL of pre-cooled PBS to the tissue sample and chop the tissue into pieces with a disposable sterile scalpel, cutting it into small pieces as finely as possible.

[0038] (5) Observe the morphology and number of cells separated from the tissue under a microscope.

[0039] (6) Add 2 mL of digestive enzyme (OG-TD-001) to a 15 mL centrifuge tube, tilt it at 45° in a constant temperature shaking incubator at 37°C, and digest it at 200 rpm until most of the tissue is digested or loosened (about 10 min).

[0040] (7) Most of the tissue has been digested, meaning that the remaining volume is less than one-fifth of the total volume, or there are no obvious signs of the remaining tissue being digested. Take 1 drop of tissue suspension into well 24. When a large number of 10-20 cell clusters are observed, return the observed tissue suspension as much as possible and add 5 times the volume of digestion liquid DMEM to stop the digestion.

[0041] (8) Centrifuge the above centrifuge tubes at 4℃ and 400 g for 5 min, discard the supernatant, and resuspend in 1 ml of DMEM.

[0042] (9) Place a 100 μm cell sieve on a 50 mL centrifuge tube and transfer the suspension to the sieve using a disposable sterile Pasteur pipette.

[0043] (10) Using a 5 mL syringe needle core, grind the tissue clockwise several times until most of the tissue suspension is filtered out.

[0044] (11) Add 2 ml of DMEM to clean the filter and syringe needle.

[0045] (12) Grind the filter screen surface several times again and clean it repeatedly to ensure that there are no obvious tissue blocks left or only unfilterable connective tissue.

[0046] (13) Collect the filtrate and transfer it to a 15 mL centrifuge tube. Take 100 μL of the filtered digestion liquid and place it in a 24-well plate. Observe it under a microscope to ensure that there are no large tissue fragments remaining. After observation, centrifuge the cell suspension at 400 g for 5 min at 4 °C.

[0047] (14) After centrifugation, discard the supernatant and use your fingers to move the centrifuge tube wall to help loosen the cells (to prevent excessive force when resuspending and to disperse the cell cluster into single cells).

[0048] (15) Add 20 µL of pre-cooled DMEM to the cell pellet and gently mix with a 100 µL pipette tip. Place on ice.

[0049] S2. Pheochromocytoma on-top seed gel, induced culture.

[0050] (16) Half an hour in advance, use a pre-cooled pipette tip to drop 30µL of matrix gel into the middle of the 24-well plate to form a hemispherical droplet, 2-3 drops per well. Place at 37°C and wait for the droplets to solidify. Then, gently drop the cell suspension from (15) onto the solidified droplets. Drop 5µL of cell suspension onto each droplet to ensure full contact with the droplets. Place at 37°C for 20 minutes.

[0051] (17) Remove the 24-well plate from the incubator and add 100 µL of pheochromocytoma organoid induction medium along the sidewall of the well (do not touch it). Be careful not to damage the droplets when adding the medium, and the height of the medium should be lower than the height of the droplets. After adding the medium, put the multi-well plate back into the cell culture incubator (37°C, 5% CO2) for culture.

[0052] (18) Two days later, remove the supernatant from the 24-well plate; take the culture medium at 4°C, use a 1ml pipette to draw 300µL of pheochromocytoma maintenance culture medium, blow away the droplets in the 24-well plate, and collect all the liquid into a 15ml centrifuge tube.

[0053] S3. Pheochromocytoma embedding method for gel implantation and maintenance culture.

[0054] (19) Centrifuge at 4℃ and 400 g for 5 min, discard the supernatant, add 100 µL of pre-chilled pheochromocytoma maintenance medium to the cell pellet, and gently pipette with a 200 µL wide-mouth pipette tip to mix. Then add 50 µL of matrix gel to the cell pellet and gently pipette with a 200 µL pipette tip to mix. Be careful not to form air bubbles; the entire mixing process should be carried out on an ice box to reduce the contact time between your fingers and the reagents and tube walls; before aspirating the matrix gel and suspension, the pipette tip should be pre-chilled several times by blowing and aspirating pre-chilled DMEM.

[0055] (20) When seeding the gel, use a pipette tip to blow the cell-Matrix gel suspension into the center of the wells of a 24-well plate, forming 2-3 hemispherical droplets in the middle of the wells. After seeding, place the 24-well plate in a cell culture incubator (37°C, 5% CO2). After 5 minutes, invert the plate and leave it in the incubator for 20 minutes to allow the cell-Matrix gel suspension to solidify.

[0056] (21) Remove the 24-well plate from the incubator and add 500 µL of pheochromocytoma organoid maintenance medium along the sidewall of the well (do not touch it). Be careful not to damage the gel droplets when adding the medium. After adding the medium, put the multi-well plate back into the cell culture incubator (37°C, 5% CO2) for culture.

[0057] (22) Pheochromocytoma grows slowly. The frequency of medium change should be determined according to the growth status of the organoid and the color of the culture medium. The medium should be changed halfway each time until the organoid matures.

[0058] The image taken with a light microscope 40 days later is as follows: Figure 1 As shown in the figure, the organoid has clear edges, is transparent under the microscope, and has a clearly visible nucleus, indicating that the organoid is growing normally.

[0059] The HE staining results are as follows Figure 2 As shown in the figure, the staining of the organoid nucleus is consistent with that of the tissue nucleus, indicating that the cultured organoid originated from the original tissue and the culture was successful.

[0060] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for culturing human pheochromocytoma organoids, characterized in that, Includes the following steps: S1. Treatment of pheochromocytoma: Remove the tissue, cut it into pieces, wash, digest, centrifuge, discard the supernatant, resuspend, filter, centrifuge, discard the supernatant, add DMEM medium, mix by pipetting, and place on ice to obtain cell suspension; S2. Pheochromocytoma ON-TOP seed gel, induction culture: Spot the cell suspension onto the solidified gel droplet, incubate, add pheochromocytoma organoid induction medium, culture, remove the supernatant, add pheochromocytoma maintenance medium, blow away the gel droplet, and collect all liquid into a centrifuge tube. S3. Pheochromocytoma embedding method: Centrifuge and discard the supernatant. Add pre-cooled pheochromocytoma maintenance medium to the cell pellet. Use a pipette tip to place the cell-Matrix gel suspension in the center of the well, so that the suspension forms a hemispherical droplet in the middle of the well plate. After the gel is seeded, incubate, then add pheochromocytoma organoid maintenance medium, culture, and change the medium until the organoid matures.

2. The method for culturing human pheochromocytoma organoids according to claim 1, characterized in that, The digestion conditions described in step S1 are as follows: digestion with digestive enzymes at 36-38°C and 150-250 rpm for 5-15 minutes.

3. The method for culturing human pheochromocytoma organoids according to claim 1, characterized in that, The incubation conditions described in step S2 are 36-38℃ and 15-25 min.

4. The method for culturing human pheochromocytoma organoids according to claim 1, characterized in that, The cultivation conditions described in step S2 are 36-38℃, 4-6 v / v% CO2, and a time of 1-3 days.

5. The method for culturing human pheochromocytoma organoids according to claim 1, characterized in that, The pheochromocytoma organoid induction medium comprises the following components at the following concentrations: Advance-DMEM F12 medium containing 3-7% fetal bovine serum, 3-7% horse serum, 100-200 µg / ml amphotericin B, 0.5-1.5× penicillin / streptomycin, 0.5-1.5× human leukocyte antigen B27, 10-20 ng / ml epidermal growth factor EGF, 20-40 ng / ml bovine basic fibroblast growth factor 2, 1-3 mM L-alanyl-L-glutamine, and 0.5-1.5 mM sodium pyruvate.

6. The method for culturing human pheochromocytoma organoids according to claim 1, characterized in that, The pheochromocytoma maintenance medium comprises the following components at the following concentrations: Advance-DMEM F12 containing 10-20 ng / ml epidermal growth factor (EGF), 20-40 ng / ml bovine basic fibroblast growth factor 2, 0.5-1.5 × human leukocyte antigen B-27™ additive, and 0.5-1.5 × penicillin / streptomycin.

7. The method for culturing human pheochromocytoma organoids according to claim 1, characterized in that, The incubation conditions described in step S3 are 36-38℃, 4-6 v / v% CO2, and 5-15 min.

8. The method for culturing human pheochromocytoma organoids according to claim 1, characterized in that, The cultivation conditions described in step S3 are 36-38℃ and 4-6 v / v% CO2.

9. A pheochromocytoma organoid induction culture medium used in the method for culturing human pheochromocytoma organoids as described in any one of claims 1-8, characterized in that, The medium contains the following concentrations of components: Advance-DMEM F12 containing 3-7% fetal bovine serum, 3-7% horse serum, 100-200 µg / ml amphotericin B, 0.5-1.5× penicillin / streptomycin, 0.5-1.5× human leukocyte antigen B27, 10-20 ng / ml epidermal growth factor (EGF), 20-40 ng / ml bovine basic fibroblast growth factor 2, 1-3 mM L-alanyl-L-glutamine, and 0.5-1.5 mM sodium pyruvate.

10. A pheochromocytoma maintenance culture medium used in the method for culturing human pheochromocytoma organoids as described in any one of claims 1-8, characterized in that, The product contains the following concentrations of components: Advance-DMEM F12 medium containing 10-20 ng / ml epidermal growth factor (EGF), 20-40 ng / ml bovine basic fibroblast growth factor 2, 0.5-1.5 × human leukocyte antigen B-27™ additive, and 0.5-1.5 × penicillin / streptomycin.

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