Culture medium and culture method for dedifferentiating liposarcoma organs

Abstract

The invention discloses a culture medium and a culture method for dedifferentiating liposarcoma organs, the culture medium takes DMEM / F12 as a basic culture medium, and the following components with final concentration are added: 1-5 mM of N-acetylcysteine, 1-20 mM of nicotinamide, 10-30 ng / ml of fibroblast growth factor 2, 1 * B27, 10 [mu] M of Y-27632, 500-1000 ng / ml of R-spondin1, 500-1000 nM of prostaglandin E2, 5-10 [mu] M of SB202190, 1 * HEPES, 1 * GlutaMAX, 1 * P / S, 10-50 nM of hydrocortisone and 5-10% of fetal calf serum; the components of the culture medium interact with one another, in-vitro growth of the dedifferentiated liposarcoma can be promoted under the overall action effect, the dedifferentiated liposarcoma organoid is obtained, and the obtained organoid can be stably cultured and amplified in vitro for a long time.

Description

Technical Field

[0001] The present invention relates to the field of organoid culture, in particular to a culture medium for dedifferentiated liposarcoma organoids, and also to a method for culturing dedifferentiated liposarcoma organoids using the culture medium. Background Art

[0002] Dedifferentiated liposarcoma is a highly malignant subtype of liposarcoma, which is highly invasive and prone to recurrence. Its five-year survival rate is about 30-40%, especially in recurrent or metastatic cases, where the prognosis is poor (Schwab JH, Boland PJ, Antonescu CR, et al. A comparison of 3 retrospective cohorts of patients with primary, recurrent, and dedifferentiated liposarcoma. Cancer 2009; 115: 5716-5727.). Organoids can more realistically simulate the biological characteristics and microenvironment of tumors and are an important model for studying the molecular mechanisms of tumors in vitro. And different tumor patients have different responses to treatment. Organoid technology can screen multiple drugs in vitro and test the efficacy of different drug combinations, providing a platform for personalized treatment and new drug development (Li Y, Zhang Q, Sun X, et al. The establishment and drug response profiles of patient-derived organoids of liposarcoma. Clin Cancer Res. 2021; 27 (13): 3714-3724.). In the prior art, the construction of tumor organoids mainly extracts cells from patient tumor tissues, and cultures them in three dimensions through specific culture media and conditions to simulate the microenvironment of tumors in vivo. For example, breast cancer organoids were constructed by combining specific culture media and growth factors, which can simulate the tumor microenvironment in vivo and maintain the survival of tumor cells in vitro and their tumor characteristics (CN117987371A).

[0003] At present, tumor organoid culture technology has been applied to the study of various solid tumors and has made certain progress, but there are still technical challenges in the construction of dedifferentiated liposarcoma organoids. Traditional organoid culture methods are difficult to provide sufficient microenvironment and nutritional support for dedifferentiated liposarcoma, making it difficult for organoid models to expand long-term or maintain structural integrity. At present, dedifferentiated liposarcoma cell lines are the main in vitro research model of dedifferentiated liposarcoma. Although they have certain molecular and biological characteristics of dedifferentiated liposarcoma, 2D culture cannot fully reflect the biological behavior of tumors in vivo. Therefore, it is necessary to construct dedifferentiated liposarcoma organoids to study the mechanism of tumor progression and drug response evaluation. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide a culture medium for dedifferentiated liposarcoma organoids, and a second object of the present invention is to provide a culture method for dedifferentiated liposarcoma organoids using the culture medium.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The culture medium for dedifferentiated liposarcoma organoids is based on DMEM / F12 as the basic medium, and the following components are added at the following final concentrations: 1-5mM N-acetylcysteine, 1-20mM nicotinamide, 10-30ng / ml fibroblast growth factor 2, 1×B27, 10μM Y-27632, 500-1000ng / ml R-spondin1, 500-1000nM prostaglandin E2, 5-10μM SB202190, 1×HEPES, 1×GlutaMAX, 1×P / S, 10-50nM hydrocortisone, and 5-10% fetal bovine serum.

[0007] Preferably, the culture medium is based on DMEM / F12, and the following components are added at the following final concentrations: 1 mM N-acetylcysteine, 10 mM nicotinamide, 15 ng / ml fibroblast growth factor 2, 1×B27, 10 μM Y-27632, 800 ng / ml R-spondin1, 900 nM prostaglandin E2, 5 μM SB202190, 1×HEPES, 1×GlutaMAX, 1×P / S, 20 nM hydrocortisone, and 5% fetal bovine serum.

[0008] 2. A method for culturing liposarcoma organoids using the culture medium to differentiate the liposarcoma organoids comprises the following steps:

[0009] (1) Dedifferentiated liposarcoma tissue was washed and cut, and digested with 0.5 U / ml collagenase at 37°C for 30-50 min to obtain dedifferentiated liposarcoma cell clumps;

[0010] (2) Dilute Matrigel to a volume percentage of 20-70% with DMEM / F12 medium, resuspend the cell mass to a density of 100-200 cells / ml, seed the cell mass suspension with gel droplets, incubate in a 37°C incubator for 30-50 min to solidify the gel droplets, add the dedifferentiated liposarcoma organoid culture medium described in claim 1 or 2, and incubate at 37°C, 5% CO 2 Maintain the culture in an incubator;

[0011] (3) Replace the culture medium with fresh medium every 2 days for 15 to 25 days.

[0012] Preferably, in step (1), 0.5 U / ml collagenase is used and shaken at 37° C. for 30 min.

[0013] Preferably, in step (2), Matrigel is diluted with DMEM / F12 medium to a concentration of 20%, the cell aggregates are resuspended to a density of 1000 cells / ml, and 100 μl of the cell aggregate suspension is added to the gel droplets and incubated in a 37°C incubator for 50 min to solidify the gel droplets.

[0014] Preferably, in step (1), 0.5 U / ml collagenase is used and shaken at 37° C. for 30 min.

[0015] Preferably, in step (2), Matrigel is diluted with DMEM / F12 medium to a concentration of 20%, the cell aggregates are resuspended to a density of 1000 cells / ml, and 100 μl of the cell aggregate suspension is added to the gel droplets and incubated in a 37°C incubator for 50 min to solidify the gel droplets.

[0016] The beneficial effects of the present invention are as follows: the present invention provides a culture medium for dedifferentiated liposarcoma organoids, which can maintain long-term in vitro culture of dedifferentiated liposarcoma cells, promote cell proliferation and self-assembly, and the added specific components, such as fibroblast growth factor 2 and R-spondin1, can promote cell proliferation and maintain organoid growth; B27, GlutaMAX, and fetal bovine serum provide vitamins and nutrients required for organoid growth; nicotinamide and hydrocortisone regulate cell metabolism; N-acetylcysteine, Y-27632, and SB202190 regulate cell proliferation and differentiation and resist cell apoptosis; when dedifferentiated liposarcoma organoids are cultured, prostaglandin E2 plays a key role in cell growth and regeneration. The dedifferentiated liposarcoma organoid culture method of the present invention is conducive to the construction and growth of organoids, and the method of digesting cell clumps is conducive to the in vitro proliferation of dedifferentiated liposarcoma cells. The use of the culture medium of the present invention at the conventionally used 70% Matrigel concentration can maintain the growth and proliferation of organoids, but the low concentration of 20% Matrigel can further promote the self-assembly of cells and the growth of organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0018] Figure 1 The dedifferentiated liposarcoma organoid obtained in Example 2;

[0019] Figure 2 The dedifferentiated liposarcoma organoid obtained in Example 3;

[0020] Figure 3 Staining for dedifferentiated liposarcoma tissue and dedifferentiated liposarcoma organoids obtained in Example 3;

[0021] Figure 4 This is the dedifferentiated liposarcoma organoid obtained in the comparative example. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0023] Example 1

[0024] Dedifferentiated liposarcoma organoid culture medium, the culture medium comprising the following components at final concentrations: 1 mM N-acetylcysteine, 10 mM nicotinamide, 15 ng / ml fibroblast growth factor 2, 1×B27, 10 μM Y-27632, 800 ng / ml R-spondin1, 900 nM prostaglandin E2, 5 μM SB202190, 1×HEPES, 1×GlutaMAX, 1×P / S, 20 nM hydrocortisone, 5% fetal bovine serum, and the basal culture medium is DMEM / F12.

[0025] The method for preparing the dedifferentiated liposarcoma organoid culture medium is to add each additional component into the basic culture medium and mix them evenly.

[0026] Example 2

[0027] The method for culturing dedifferentiated liposarcoma organoids comprises the following steps:

[0028] (1) The obtained dedifferentiated liposarcoma tissue was washed and cut, and digested with 0.5 U / ml collagenase at 37°C for 30 min to obtain dedifferentiated liposarcoma cell clumps;

[0029] (2) Matrigel was diluted with DMEM / F12 medium to a volume percentage of 70%, and the cell mass was resuspended to a density of 1000 cells / ml. The cell mass suspension was seeded with 100 μl of gel droplets and incubated in a 37°C incubator for 30 min to solidify the gel droplets. The dedifferentiated liposarcoma organoid culture medium of Example 1 was added and incubated at 37°C, 5% CO 2 Maintain the culture in an incubator;

[0030] (3) Replace the culture medium with fresh medium every 2 days (Day19).

[0031] The culture results are as follows Figure 1 As shown, the organoid structure formed has a denser texture than the primary tissue, but the overall shape and edges are less smooth.

[0032] (4) Take photos at regular intervals, measure the total area of ​​the organoids, and calculate the average value as shown in Table 1.

[0033] Table 1

[0034] Group Example 2 Example 3 Comparative Example <![CDATA[Organoid area (μm 2 )]]> 4.30E5 4.67E5 2.26E5

[0035] Example 3

[0036] The method for culturing dedifferentiated liposarcoma organoids comprises the following steps:

[0037] (1) The obtained dedifferentiated liposarcoma tissue was washed and cut, and digested with 0.5 U / ml collagenase at 37°C for 30 min to obtain dedifferentiated liposarcoma cell clumps;

[0038] (2) Matrigel was diluted to a concentration of 20% with DMEM / F12 medium, and the cell mass was resuspended to a density of 1000 cells / ml. The cell mass suspension was seeded with 100 μl of gel droplets and incubated in a 37°C incubator for 50 min to solidify the gel droplets. The dedifferentiated liposarcoma organoid culture medium of Example 2 was added and incubated at 37°C, 5% CO 2 Maintain the culture in an incubator;

[0039] (3) Replace the culture medium with fresh medium every 2 days (Day 22).

[0040] The culture results are as follows Figure 2 As shown, an organoid structure is formed with a denser texture than the primary tissue and a smoother overall shape and edges.

[0041] (4) Take photos at regular intervals, measure the total area of ​​the organoids, and calculate the average value as shown in Table 1.

[0042] Example 4

[0043] Identification of dedifferentiated liposarcoma organoids. The dedifferentiated liposarcoma organoids obtained in Example 3 were identified as follows: After 20 days of organoid culture, the organoids were treated with organoid recovery solution for 30 minutes to dissolve Matrigel, and the organoids separated from Matrigel were fixed with 4% paraformaldehyde for 12 hours, dehydrated with alcohol gradient and transparent with xylene, and then embedded in paraffin, and cross-sectioned and longitudinally cut, respectively. At the same time, the dedifferentiated liposarcoma tissue was also fixed, dehydrated, transparent, and paraffin-embedded. After sectioning, the organoids and tissues were simultaneously stained with H&E and Oil Red O to identify their morphological structures.

[0044] The image of the stained organoid is as follows Figure 3 The results showed that after H&E staining, the morphological structure of dedifferentiated liposarcoma organoids was the same as that of dedifferentiated liposarcoma tissue. After Oil Red O staining, there was no obvious lipid staining and adipocytes in both dedifferentiated liposarcoma organoids and dedifferentiated liposarcoma tissue, indicating that the organoids cultured in vitro can maintain a dedifferentiated state.

[0045] Comparative Example 1

[0046] The dedifferentiated liposarcoma organoid culture medium is different from that of Example 1 in that the culture medium components do not contain prostaglandin E2, and the rest are the same. The culture results obtained by using the culture medium of the comparative example and the culture method of Example 3 are as follows: Figure 4 shown.

[0047] From the comparison of the organoid morphology obtained in the embodiment and the comparative example, it can be seen that the culture medium components in Example 1 interact with each other, and under the overall effect, they can promote the in vitro growth of dedifferentiated liposarcoma, which is beneficial to obtain dedifferentiated liposarcoma organoids. Under three-dimensional culture conditions, the organoids obtained in Example 2 can grow and proliferate stably. The results of Example 3 show that in addition to enabling the stable growth and proliferation of organoids, low-concentration Matrigel is more conducive to the self-assembly of organoids. The edges of the organoids tend to be smooth and grow into a round shape, and a spherical structure is formed after maintained culture. The results in the comparative example show that after continuous culture, the tissue morphology did not change significantly, and no obvious organoid-like structure was formed. In summary, the culture effect of Example 3 is better.

[0048] The identification results of Example 4 found that after H&E staining, the dedifferentiated liposarcoma organoids had the same morphological structure as the dedifferentiated liposarcoma tissue, and after Oil Red O staining, the dedifferentiated liposarcoma organoids and the dedifferentiated liposarcoma tissue had no obvious lipid staining and adipocytes, indicating that the organoids cultured in vitro can maintain a dedifferentiated state.

[0049] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A culture medium for dedifferentiated liposarcoma organoids, characterized in that The culture medium is based on DMEM / F12, and the following components are added at final concentrations as follows: 1-5 mM N-acetylcysteine, 1-20 mM nicotinamide, 10-30 ng / ml fibroblast growth factor 2, 1×B27, 10 μM Y-27632, 500-1000 ng / ml R-spondin1, 500-1000 nM prostaglandin E2, 5-10 μM SB202190, 1×HEPES, 1×GlutaMAX, 1×P / S, 10-50 nM hydrocortisone, and 5-10% fetal bovine serum.

2. The culture medium for dedifferentiated liposarcoma organoids according to claim 1, characterized in that: The culture medium is based on DMEM / F12, and the following components are added at the following final concentrations: 1 mM N-acetylcysteine, 10 mM nicotinamide, 15 ng / ml fibroblast growth factor 2, 1×B27, 10 μM Y-27632, 800 ng / ml R-spondin1, 900 nM prostaglandin E2, 5 μM SB202190, 1×HEPES, 1×GlutaMAX, 1×P / S, 20 nM hydrocortisone, and 5% fetal bovine serum.

3. A method for culturing liposarcoma organoids using the culture medium of claim 1 or 2, characterized in that: The steps include: (1) Dedifferentiated liposarcoma tissue was washed and cut, and digested with 0.5 U / ml collagenase at 37°C for 30-50 min to obtain dedifferentiated liposarcoma cell clumps; (2) Diluting Matrigel to a volume percentage of 20-70% with DMEM / F12 medium, resuspending the cell mass to a density of 100-200 cells / ml, seeding the cell mass suspension with gel droplets, incubating in a 37°C incubator for 30-50 min to solidify the gel droplets, adding the dedifferentiated liposarcoma organoid culture medium described in claim 1 or 2, and maintaining the culture in a 37°C, 5% CO2 incubator; (3) Replace the culture medium with fresh medium every 2 days for 15 to 25 days.

4. The method for culturing dedifferentiated liposarcoma organoids according to claim 3, characterized in that: In step (1), 0.5 U / ml collagenase was used and shaken at 37°C for 30 min.

5. The method for culturing dedifferentiated liposarcoma organoids according to claim 3, characterized in that: In step (2), Matrigel was diluted to a concentration of 20% with DMEM / F12 medium, and then the cell aggregates were resuspended to a density of 1000 cells / ml. The cell aggregate suspension was seeded with 100 μl of gel droplets and incubated in a 37°C incubator for 50 min to allow the gel droplets to solidify.

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