A culture medium and culture method for preparing forest musk gland organoids for musk

Abstract

The present application relates to the technical field of organoid culture, and specifically discloses a culture medium for musk gland organoids and its preparation method and application. The present application discloses a culture medium, which uses DMEM / F12 as the basic culture medium; contains the following concentrations of components as functional additives: albumin, B‑27, HEPES, glutamine, N‑acetyl‑L‑cysteine, niacinamide, growth factor, Wnt3a, A83‑01, Forskolin, penicillin, streptomycin, hydrocortisone, insulin; the growth factor is EGF and bFGF with a weight ratio of 350: (11‑21). The present application also discloses a method for preparing the above-mentioned culture medium and its application in musk gland organoid culture. The culture medium of the present application can be used to obtain musk gland organoids with stem cell characteristics, and the cell activity is high.

Description

Technical Field

[0001] The present application relates to the technical field of organoid culture, and in particular to a culture medium and a culture method for preparing musk gland organoids. Background Art

[0002] Musk is a traditional precious Chinese medicinal material and a raw material for the spice industry. It is the dried secretion from the scent sacs of adult male musk deer (Moschus berezovskii Flerov), horse musk deer (Moschus sifanicus Przewalski) or musk deer (Moschusmoschiferus Linnaeus), which are animals of the Cervidae family. Musk has the effects of invigorating the mind, promoting blood circulation and unblocking meridians, and reducing swelling and relieving pain. It is used to treat common, frequently occurring and difficult diseases such as fever, coma, stroke, phlegm, pain, swelling, scrofula, and injuries from falls. It has a long history of medicinal use of more than 2,000 years and has played a long and huge role in the medical care of the Chinese nation.

[0003] Musk deer is one of my country's unique economic animals, distributed in more than 10 provinces in Northeast, North, Northwest and Southwest China. my country has the most species and resources of musk deer, and its musk production ranks first in the world. However, due to long-term hunting of musk deer for musk, musk resources are seriously insufficient, the amount of musk available for purchase is decreasing, the price is expensive, and the quality is difficult to guarantee.

[0004] In order to solve the clinical medication problems caused by the shortage of musk, alleviate the contradiction between supply and demand of raw materials in traditional Chinese medicine factories, and protect the ecological balance, it is very necessary and urgent to carry out scientific research and development of natural musk substitutes.

[0005] Since forest musk deer are first-class protected animals in China, it is very difficult to obtain approval for breeding and it is expensive. In addition, they are timid and have a strong stress response. Experimental sampling can easily cause harm to musk deer or even death. Therefore, current in vitro scent secretion research is basically focused on muskrats, and the research on forest musk deer's in vitro scent production is limited to two-dimensional culture. In terms of the research on in vitro scent production using cell engineering, there is no research on the culture of forest musk gland organoids, and there are only a few reports on the two-dimensional culture of forest musk gland scent cells. However, the two-dimensional culture of forest musk deer's scent cells will cause the cells to lose their biological characteristics and cannot realize the scent secretion function in vivo.

[0006] There is a lack of research on the three-dimensional organoid culture of musk glands, and there is no report on the production of the main active ingredients of musk. Summary of the invention

[0007] In order to solve the technical problems existing in the above-mentioned prior art, the present application provides a culture medium and a culture method for preparing musk glandular organoids.

[0008] The present application provides a musk gland organoid culture medium, wherein the culture medium uses DMEM / F12 as a basic culture medium;

[0009] The culture medium contains the following components as functional additives at concentrations: 0.05-0.2 mg / mL albumin, 0.5-5 mg / mL B-27, 5-15 mM HEPES, 0.05-0.1 mg / mL glutamine, 1-5 mM N-acetyl-L-cysteine, 5-15 mM niacinamide, 300-400 ng / mL growth factor, 60-120 ng / mL Wnt3a, 0.2-1.6 μM A83-01, 200-400 μM Forskolin, 60-140 U / mL penicillin, 0.05-0.25 mg / mL streptomycin, 25-75 ng / mL hydrocortisone, 20-50 ng / mL insulin;

[0010] The growth factors are EGF and bFGF in a weight ratio of 350:(11-21).

[0011] The present application uses the above-mentioned concentrations of albumin, B-27, HEPES, glutamine, N-acetyl-L-cysteine, niacinamide, growth factor, Wnt3a, A83-01, FSK, penicillin, streptomycin, hydrocortisone, and insulin as main raw material components, which can effectively improve the culture medium ratio, promote the proliferation and differentiation of organoid cells, increase the renewal rate of organoid cells, and improve the speed and quality of culture.

[0012] Preferably, the culture medium comprises the following components at concentrations: 0.1-0.2 mg / mL albumin, 1-3 mg / mL B-27, 8-12 mM HEPES, 0.05-0.1 mg / mL glutamine, 1-5 mM N-acetyl-L-cysteine, 7-13 mM niacinamide, 330-380 ng / mL growth factor, 80-100 ng / mL Wnt3a, 0.6-1.2 μM A83-01, 280-320 μM Forskolin, 90-120 U / mL penicillin, 0.1-0.2 mg / mL streptomycin, 25-75 ng / mL hydrocortisone, and 20-50 ng / mL insulin.

[0013] Preferably, the growth factors are EGF and bFGF in a weight ratio of 350:(13-17).

[0014] Preferably, the insulin concentration is lower than the hydrocortisone concentration.

[0015] Preferably, the culture medium contains the following components as functional additives at the following concentrations: 8-20 μg / mL prostaglandin, 0.5-5 μg / mL dexamethasone.

[0016] Furthermore, the culture medium contains the following components as functional additives at the following concentrations: 15-20 μg / mL prostaglandin, 0.5-2.5 μg / mL dexamethasone.

[0017] Through experimental analysis, it can be known that the present application selects prostaglandins and dexamethasone of the above concentrations as functional additives, adds them to the culture medium, and uses the culture medium to culture forest musk gland organoids, which can further improve the culture effect of forest musk gland organoid cell proliferation and differentiation.

[0018] In a second aspect, the present application provides a method for preparing the above-mentioned musk gland organoid culture medium, which specifically comprises the following steps:

[0019] Weigh the corresponding weight of each raw material, fully dissolve it in 950 mL of basal culture medium DMEM / F12, adjust the pH to 6.6-7.2, add sterile deionized water to make up to 1000 mL, and then filter it with a filter membrane with a pore size of 0.22 μm to obtain the culture medium, and store it at 4°C for use.

[0020] In a third aspect, the present application provides the use of the above-mentioned forest musk gland organoid culture medium in culturing forest musk gland organoids.

[0021] In a fourth aspect, the present application provides a forest musk glandular organoid, which is obtained by culturing using the above-mentioned forest musk glandular organoid culture medium.

[0022] In a fifth aspect, the present application provides a method for culturing musk glandular organoids, which is cultured using the above-mentioned culture medium, and specifically comprises the following steps: in the musk glandular organoid culture medium, at a temperature of (0.4-4)×10 5 The forest musk gland organoids were inoculated with an inoculation density of cells / mL, and then cultured at 37°C and saturated humidity of 5% CO2.

[0023] In summary, the technical solution of this application has the following effects:

[0024] The present application uses DMEM / F12 as the basic culture medium, albumin, B-27, HEPES, glutamine, N-acetyl-L-cysteine, niacinamide, growth factors, Wnt3a, A83-01, FSK, penicillin, streptomycin, hydrocortisone, and insulin as the main raw material components, and then prepares a culture medium for culturing musk gland organoids, which can promote the proliferation and differentiation of organoid cells, increase the renewal rate of organoid cells, and improve the speed and quality of culture. The present application further improves the culture effect of the culture medium on musk gland organoids by screening the types and amounts of growth factors and functional additives.

[0025] The technical solution of the present application provides a culture medium and a culture method for forest musk gland organoids, which can be cultured under three-dimensional culture conditions to obtain forest musk gland organoids. The forest musk gland organoids have a morphology similar to that of forest musk glands and maintain strong stem cell characteristics. A large number of forest musk gland organoids can be expanded in a short period of time, which has made basic scientific research for the early realization of in vitro production of musk main substances by forest musk cells and has broad application prospects.

[0026] The forest musk gland organoid cells obtained through three-dimensional culture in the present application have high vitality. The forest musk gland organoids formed after a period of culture can express CK14 and Ki67, which are cell proliferation markers, indicating that the forest musk gland organoid cells have strong proliferation ability; they can express αSMA and SOX9 proteins, which shows that the forest musk gland organoids obtained by culture still have stem cell characteristics; and compared with the isolated forest musk gland cells, the expression of stem components is increased and the expression of differentiation components is decreased, indicating that the forest musk gland stem cells are enriched, and a large number of forest musk gland seed cells can be expanded in a short period of time, establishing a method for effectively culturing and obtaining forest musk gland organoids.

[0027] The musk gland organoids obtained through three-dimensional culture in the present application are similar to the in vivo musk gland tissue in morphology and function, maintain and retain the biological characteristics of the in vivo musk gland, and can produce substances containing the main active ingredients of natural musk, such as musk ketone and steroids, after agonist stimulation, laying the foundation for the industrial production of in vitro musk with high efficacy and safety, and the application prospects are very broad.

[0028] The present application provides for the first time a three-dimensional culture medium for culturing forest musk deer LD cells to obtain forest musk gland organoids, which adopts a mixed culture method of matrix gel and cells, and adds other components on the basis of Advanced DMEM / F-12 culture medium. The cultured forest musk gland organoid cells have high activity and high stemness components. The traditional two-dimensional culture of forest musk LD cells causes the scent gland cells to lose their biological characteristics in vivo, while the forest musk gland organoids generated by three-dimensional culture in the present invention are organoid tissues composed of forest musk gland stem cells and mature cells, which are similar to the in vivo forest musk gland tissue in morphology and structure, and maintain and retain the biological characteristics of the in vivo forest musk gland. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a microscopic observation image (100x) of the musk gland organoid in Example 2 at 0 hours after inoculation.

[0030] Figure 2 This is a microscopic observation image (100x) of the musk gland organoid in Example 2 after 4 days of inoculation and culture.

[0031] Figure 3 This is a morphological picture (10x) of the musk gland organoids at 4 days after inoculation and culture in Example 2 without adipogenic induction staining.

[0032] Figure 4 This is the morphological image of the oil red staining effect of the musk gland organoid at 4 days of inoculation and culture in Example 2 (10x) DETAILED DESCRIPTION

[0033] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as being included in the scope of protection claimed in the present application.

[0034] The sources of the reagents used in this application are specifically as follows: Advanced DMEM / F12 (Cat. No. 12634-010), B-27 (Cat. No. 17504044), HEPES (Cat. No. 15630080), and glutamine (Cat. No. 35050061) were purchased from Thermo Scientific, N-acetyl-L-cysteine ​​(Cat. No. A9165), and niacinamide (Cat. No. N0636) were purchased from Sigma-Aldrich, and A83-01 (Cat. No. 2939) was purchased from Tocris; they and the reagents can all be obtained commercially.

[0035] Preparation Example

[0036] Preparation Example 1-11

[0037] Preparation Examples 1-11 respectively provide a musk gland organoid culture medium.

[0038] The difference between the above preparation examples is that the concentration of each component in the musk gland organoid culture medium is shown in Table 1.

[0039] The method for preparing the culture medium comprises the following steps:

[0040] According to the 1L specification of the culture medium, according to the final concentration of each component in Table 1, weigh the corresponding weight of albumin, B-27, HEPES, glutamine, N-acetyl-L-cysteine, niacinamide, growth factor (a mixture of EGF and bFGF with a weight ratio of 350:17), insulin, hydrocortisone, prostaglandins, dexamethasone, Wnt3a, A83-01, Forskolin, penicillin, and streptomycin respectively; fully dissolve them in 950mL of the basic culture medium DMEM / F12, adjust the pH to 6.6-7.2, add sterile deionized water to make up to 1000mL, and then filter with a filter membrane with a pore size of 0.22μm to obtain the musk gland organoid culture medium, and store it at 4℃ for use.

[0041] Table 1 Concentration of each component in the culture medium of musk gland organoids in preparation examples 1-11

[0042]

[0043]

[0044] Preparation Examples 12-19

[0045] Preparation Examples 12-19 respectively provide a musk gland organoid culture medium.

[0046] The difference between the above-mentioned preparation examples and Preparation Example 2 is that the concentrations of the components in the functional additives are different, as shown in Table 2.

[0047] Table 2 Concentration of each component in the functional additives in the culture medium of musk gland organoids in preparation examples 12-19

[0048]

[0049] Preparation Examples 20-22

[0050] Preparation Examples 20-22 respectively provide a musk gland organoid culture medium.

[0051] The differences between the above-mentioned preparation examples and preparation example 2 are as follows:

[0052] In Preparation Example 20: a mixture of EGF and FGF10 at a weight ratio of 350:17 was used as the growth factor.

[0053] In Preparation Example 21: an equal amount of thyroid stimulating hormone is used instead of insulin.

[0054] In Preparation Example 22: an equal amount of dexamethasone was used instead of hydrocortisone.

[0055] Example

[0056] Examples 1-16

[0057] Embodiments 1-16 respectively provide a method for culturing musk gland organoids.

[0058] The difference between the above embodiments is that the culture medium used in the culture of musk gland organoids is different, and the culture medium used in Examples 1-16 is derived from Preparation Examples 1-8 and 12-19, respectively.

[0059] The method for culturing musk gland organoids in the above embodiment specifically comprises the following steps:

[0060] Digestion: The obtained musk gland tissue was washed and then cut into tissue fragments of about 1 mm in size with ophthalmic scissors; washed twice with physiological saline containing 100U / ml penicillin and 100μg / ml streptomycin, and then added with a digestion solution containing trypsin-EDTA (0.25%) and no phenol red, placed in a 37°C water bath for incubation for 5 minutes, and terminated with PBS.

[0061] Cell preparation: The digested tissue was centrifuged at 1500rpm in a desktop centrifuge for 5min. After centrifugation, the supernatant was discarded and added with complete culture medium (DMEM / F12 basal culture medium, containing 10% fetal bovine serum, 10ng / ml EGF, 100U / ml penicillin-streptomycin). The number of tissues was observed under a microscope, inoculated into a 35mm culture dish, placed in a 37°C, 5% CO2 cell culture incubator for incubation for 48h, and the tissue adhesion was observed under an inverted microscope; after most of the tissues adhered to the wall, the complete culture medium was replaced, and after continuing to culture for 48h, the tissues migrated out of the forest musk gland cells. When the cells grew and converged, trypsin containing EDTA was used for digestion and passage, and cell amplification was performed by one-to-three passages to obtain a large number of primary forest musk gland cells.

[0062] Cell culture: Take a 35 mm culture dish of forest musk gland cells in the growth stage and digest them to obtain 1-5×10 5 The cells were mixed with the purchased finished matrix gel at a weight ratio of 2:1, inoculated into a 24-well plate, and the musk gland organoid culture medium in the corresponding preparation example was added. Three-dimensional cell culture was performed at 37°C and saturated humidity of 5% CO2. The growth was checked after 48 hours, and the culture medium was changed every 2 days.

[0063] Comparative Example

[0064] Comparative Examples 1-6

[0065] Comparative Examples 1-6 respectively provide a method for culturing musk gland organoids.

[0066] The difference between the above comparative examples and Example 1 is that the culture medium used in the method for culturing musk gland organoids is different, and the culture medium used in comparative examples 1-7 is derived from preparation examples 9-11 and 20-22, respectively.

[0067] The methods for culturing musk gland organoids in the above comparative examples are the same as those in Example 1.

[0068] Performance testing

[0069] (1) Appearance

[0070] The morphology of the musk gland organoids in Example 2 at different inoculation and culture times was observed using an inverted microscope (100x).

[0071] like Figure 1-2 This is a morphological diagram of the musk gland organoid in Example 2; Figure 1 This is the morphology of the musk gland organoids in Example 2 at 0h of inoculation and culture. Figure 2 This is the morphology of the musk gland organoids in Example 2 after 4 days of inoculation and culture.

[0072] Depend on Figure 1-2 It can be seen that during the organoid culture process, the number of cells gradually increased, and the organoids gradually became larger during the culture process. After 4 days of culture, the cell size could reach 50-70um, and the organoid cells had a high degree of morphological consistency. The above morphological observation results show that the musk gland organoids inoculated and cultured using the culture medium provided in the present application grew and developed rapidly, and the cell morphology was uniform.

[0073] (2) Expression of cell proliferation markers (CK14, Ki67)

[0074] Detection method: Take the musk gland organoids in the example and comparative example at 4 days of inoculation and culture, adjust the cell density to 1×10 5 / mL, aspirate 100μL cell suspension, add fluorescently labeled antibodies CK14 and Ki67 respectively, and incubate at 4℃ in the dark for 30min. After incubation, add 1% bovine serum albumin, centrifuge at 1500rpm for 5min, and discard the supernatant. Add 100μL PBS solution, vortex mix, centrifuge at 1500rpm for 5min, discard the supernatant, and repeat 2 times. Add 0.2mL PBS solution and vortex mix, and use flow cytometer to detect the corresponding cell surface proliferation markers (%).

[0075] Test results: as shown in Table 3.

[0076] (3) Adipogenic differentiation rate

[0077] Determination method: Take the musk gland organoids inoculated and cultured for 4 days in the example and the comparative example, aspirate the culture medium, fix with 10% neutral formaldehyde for 30 minutes, rinse with distilled water, stain with Oil Red O at room temperature for 20 minutes, discard the excess dye, observe and photograph under an inverted microscope, randomly record the number of stained positive cells and the total number of cells in 3 fields of view, and then calculate the adipogenic differentiation rate.

[0078] The calculation method is as follows: Adipogenic differentiation rate = (number of positively stained cells / total number of cells) × 100%

[0079] Test results: Figure 3-4 ; Figure 3 This is a morphological picture of the Musk Musk glandular organoids at 4 days of inoculation and culture in Example 2 without adipogenic induction staining (10x). Figure 4 This is a morphological image (10x) of the oil red staining effect of the musk gland organoid at 4 days after inoculation and culture in Example 2.

[0080] Depend on Figure 3-4 It can be seen that the musk gland organoids prepared in the present application were stained with Oil Red, and the secretion substances after adipogenic induction increased significantly.

[0081] The results of the adipogenic differentiation rate test are shown in Table 3.

[0082] Table 3 Performance test results of musk gland organoids in Examples 1-16 and Comparative Examples 1-6

[0083]

[0084]

[0085] Combined with Table 3, by comparing the detection results of the musk deer gland organoids in Examples 1-16 and Comparative Examples 1-6, the musk deer gland organoid cells obtained by three-dimensional culture in the present application can express CK14 and Ki67, indicating cell proliferation markers, indicating that the musk deer gland organoid cells have strong proliferation ability; they can express αSMA and SOX9 proteins, which shows that the musk deer gland organoids obtained by culture still have stem cell characteristics.

[0086] By comparing the test results in Examples 2-4 and Comparative Examples 1-3, it can be seen that the concentration of each raw material component in the culture medium has a great influence on the culture effect of musk gland organoid cells. The present application can significantly improve the culture effect of musk gland organoid cells by reasonably screening the dosage of each raw material component.

[0087] By comparing the test results in Comparative Examples 4-6, it can be seen that when a mixture of EGF and FGF10 with a weight ratio of 350:17 is used as a growth factor, or thyroid-stimulating hormone is used instead of insulin, or dexamethasone is used instead of hydrocortisone, the proliferation markers of musk gland organoid cells are fewer and the adipogenic differentiation rate is lower.

[0088] By comparing the test results in Examples 2 and 5-8, the present application controls the concentration of raw insulin in the culture medium to be lower than the concentration of hydrocortisone, which can further improve the culture effect of musk gland organoid cells.

[0089] By comparing the test results in Examples 1-2 and 9-16, the present application can further improve the culture effect of musk gland organoid cells by adding 15-20 μg / mL prostaglandins and 0.5-5 μg / mL dexamethasone as functional additives in the culture medium.

[0090] (3) Detection of the main components of musk (musk ketone, steroids, fatty acids)

[0091] The culture methods provided in Example 1, Example 2, and Comparative Example 1 were used as the detection objects, and the musk gland organoids were taken on the 4th day of culture, and the musk gland organoids were treated with 0.05 g / ml agonist aqueous solution (sourced from Beijing Yanshe Biotechnology Development Co., Ltd.) for 7 days; the main components of the musk gland organoids (musk ketone, steroid substances, fatty acids) were determined by gas chromatography-mass spectrometry.

[0092] Test results: as shown in Table 4.

[0093] Table 4 Detection of main components of musk gland organoids in Example 2

[0094]

[0095] It can be seen from the test results in Table 4 that the main components of musk - musk ketone, steroids, fatty acids and other substances - were detected in the musk gland organoids obtained by using the culture medium and culture method provided by the present application, which further shows that the technical solution of the present application has made the laboratory stage of musk deer in vitro fragrance production a success.

[0096] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A musk gland organoid culture medium, characterized in that: The culture medium uses DMEM / F12 as the basic culture medium; contains the following components at the following concentrations: 0.05-0.2 mg / mL albumin, 0.5-5 mg / mL B-27, 5-15 mM HEPES, 0.05-0.1 mg / mL glutamine, 1-5 mM N-acetyl-L-cysteine, 5-15 mM niacinamide, 300-400 ng / mL growth factor, 60-120 ng / mL Wnt3a, 0.2-1.6 μM A83-01, 200-400 μM Forskolin, 60-140 U / mL penicillin, 0.05-0.25 mg / mL streptomycin, 25-75 ng / mL hydrocortisone, and 20-50 ng / mL insulin; The growth factors are EGF and bFGF at a weight ratio of 350:(11-21).

2. The musk gland organoid culture medium according to claim 1, characterized in that The culture medium contains the following components at the following concentrations: 0.1-0.2 mg / mL albumin, 1-3 mg / mL B-27, 8-12 mM HEPES, 0.05-0.1 mg / mL glutamine, 1-5 mM N-acetyl-L-cysteine, 7-13 mM niacinamide, 330-380 ng / mL growth factor, 80-100 ng / mL Wnt3a, 0.6-1.2 μM A83-01, 280-320 μM Forskolin, 90-120 U / mL penicillin, 0.1-0.2 mg / mL streptomycin, 25-75 ng / mL hydrocortisone, and 20-50 ng / mL insulin.

3. The musk gland organoid culture medium according to claim 1, characterized in that The insulin concentration is lower than the hydrocortisone concentration.

4. The musk gland organoid culture medium according to claim 1, characterized in that The culture medium contains the following components as functional additives at the following concentrations: 8-20 μg / mL prostaglandin, 0.5-5 μg / mL dexamethasone.

5. The musk gland organoid culture medium according to claim 4, characterized in that The culture medium contains the following components as functional additives at the following concentrations: 15-20 μg / mL prostaglandin, 0.5-2.5 μg / mL dexamethasone.

6. The method for preparing the musk gland organoid culture medium according to any one of claims 1 to 5, characterized in that: The specific steps include: Weigh the corresponding weight of each raw material, fully dissolve it in 950 mL of basal culture medium DMEM / F12, adjust the pH to 6.6-7.2, add sterile deionized water to make up to 1000 mL, and then filter it with a filter membrane with a pore size of 0.22 μm to obtain the culture medium, and store it at 4°C for use.

7. Use of the forest musk gland organoid culture medium as described in any one of claims 1 to 5 in culturing forest musk gland organoids.

8. A method for culturing musk gland organoids, characterized in that: Cultivation using the culture medium described in any one of claims 1 to 5 specifically comprises the following steps: mixing the musk gland organoid cells with the finished matrix gel, and culturing them in the musk gland organoid culture medium at a rate of (0.4-4)×10 5 The cells were inoculated at a density of 1.54 cells / mL, and then the musk gland organoids were cultured at 37°C and 5% CO2 under saturated humidity conditions.

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