Culture medium for culturing liposarcoma organs, construction method and culture method

Abstract

The invention discloses a culture medium for culturing liposarcoma organs, and a construction and culture method thereof, which can efficiently convert a liposarcoma tissue sample cut by an operation into the liposarcoma organs, and prepare the liposarcoma organ culture medium suitable for specific growth of the liposarcoma organs according to a signal channel of liposarcoma generation. The Matrigel simulated extracellular matrix is used for providing 3D support and continuous culture for a sample, so that the sample always keeps dryness and heterogeneity consistent with those of an original tumor, and compared with a traditional liposarcoma model, the model construction time is shortened, and the construction efficiency is improved.

Description

Technical Field

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

[0002] Liposarcoma is a type of malignant tumor composed of fat cells with varying degrees of differentiation and heterogeneity. It accounts for about 20% of soft tissue sarcomas and is one of the most common soft tissue malignant tumors in adults. It is often found in the limbs and retroperitoneum, and is more common in 40-60 years old, with a higher incidence in men than in women. Liposarcoma has the characteristics of high recurrence rate and low treatment efficiency. Surgical treatment is the first choice for liposarcoma, but there is still a risk of in situ recurrence and metastasis after surgery. Currently, chemotherapy is an effective means of postoperative adjuvant therapy, but some patients are not sensitive to systemic chemotherapy. Therefore, the treatment of liposarcoma still needs to be further optimized. In recent years, immunotherapy such as immune checkpoint inhibitors, oncolytic viruses, adoptive cell therapy and cancer vaccines have been widely studied and applied in other tumor fields, but the high heterogeneity between different liposarcoma patients has different responses to immunotherapy. Therefore, the personalized establishment of a disease model consistent with the histopathology, genetic material and phenotype of the maternal tumor can not only realize personalized drug regimen screening, but also lay the foundation for the early conquest of liposarcoma.

[0003] As with other cancers, sarcoma cell lines and patient-derived xenograft models have been developed and used to characterize these tumors and identify therapeutic targets, but these models have inherent limitations: low establishment efficiency, inability to replicate in vivo tumor heterogeneity, genomic instability, long culture cycles, etc., resulting in extremely low clinical translation rates.

[0004] In 2009, Hans Clevers et al. + Small intestinal organoids were cultured from small intestinal stem cells, ushering in a new era of organoid culture. The concept of a systematic organoid is that stem cells or organ progenitor cells can undergo cell differentiation and lineage-directed processes similar to those in vivo in vitro culture, and self-organize to form a 3D structure with the spatial structure and specific functions of human organ parts. In 2011, Sato et al. established tumor organoid models from colon adenoma, adenocarcinoma and Barrett's esophagus, opening a new era of personalized tumor treatment. Researchers have found that tumor organoids can not only match patients, reproduce the pathological characteristics, gene copy number variation and mutation map of the original tumor, but also have high culture efficiency, only requiring very small biopsy tissues, and the construction efficiency can reach 30% or even greater than 90% in a short time (4 to 6 weeks), which is expected to provide cancer patients with diagnosis and treatment plans within the time window. However, at present, there are few culture programs for liposarcoma organoids. Based on this, it is urgent to establish a method for constructing liposarcoma organoids. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a culture medium for culturing liposarcoma organoids; a second object of the present invention is to provide a method for constructing and culturing liposarcoma organoids.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A culture medium for culturing liposarcoma organoids, comprising basic components, additive components and specific factor components of the culture medium; the basic components include: DMEM / F12, FBS, P / S, GlutaMAX, HEPES; the additive components include: Hydrocortisone, SB202190, Rspondin1, EGF, N-Acetylcysteine, Nicotinamide; the specific factor components include: PGE2, Insulin, FGF-10, A 83-01.

[0007] In some embodiments of the present invention, the concentration range of each component in the culture medium is: 3-15% FBS, 0.5-2× P / S, 1-5 mM GlutaMAX, 5-20 mM HEPES, 5-50 nM Hydrocortisone, 1-15 µM SB202190, 0.3-1.5 µg / mL Rspondin1, 1-15 ng / mL EGF, 1-5 mM N-Acetylcysteine, 1-15 mM Nicotinamide, 500-1500 nM PGE2, 1-10 µg / mL Insulin, 5-20 ng / mL FGF-10, and 5-40 nM A 83-01 are added to DMEM / F12.

[0008] In some embodiments of the present invention, the additive component further includes Y-27632.

[0009] In some embodiments of the invention, the concentration of Y-27632 is 5-30 µM.

[0010] A method for constructing and culturing liposarcoma organoids comprises the following steps: (1) After the liposarcoma surgical specimen is removed from the body, it is washed once with a sample cleaning and preservation solution of 5 times the sample volume, and then transferred to a new sample cleaning and preservation solution of 10 times the sample volume, transported in a cold chain at 0-8°C, and quickly transferred to the laboratory; (2) Sample cleaning: Take out the liposarcoma sample and immerse it in a sample cleaning and preservation solution with a volume 10 times that of the sample. Incubate it in a constant temperature shaker at 200 rpm and 25°C for 30 min. During this period, the sample cleaning and preservation solution should be replaced every 10 min. (3) After cleaning, collect the sample and cut it into 1 mm pieces using a sterile scalpel. 3 For minced meat pieces, resuspend the sample in 1× DPBS and place in a 15 mL centrifuge tube; (4) Centrifuge at 300 rpm for 3 min, discard the supernatant, collect the sample precipitate, add the medium containing Y-27632 to resuspend it to form a cell suspension, mix it with Matrigel to form a mix, and use a wide-mouth pipette tip to evenly spread the mix on the cell culture plate; (5) Incubate at 37°C for 30 min. After Matrigel is fully solidified, add liposarcoma organoid culture medium into the well plate. (6) Cultivate in a 37°C incubator overnight, and replace with the culture medium without Y-27632 the next day; (7) Culture in a 37°C incubator for 10 to 40 days, changing the medium every 3 days.

[0011] In some embodiments of the present invention, the sample washing and preservation solution is formulated as follows: 1-2× penicillin-streptomycin-amphotericin B and 5-10 µM Y-27632 are added to 1× DPBS, prepared immediately before use, and temporarily stored or transported at 0-8°C.

[0012] The beneficial effects of the present invention are as follows: the present invention provides a culture medium, construction and culture method for liposarcoma organoids, which is intended to overcome the above-mentioned technical obstacles, assist precision medicine, prolong patient survival time, and improve patient quality of life; at the same time, it also provides a new in vitro research platform for subsequent disease research and related drug development. The culture medium, construction and culture method for liposarcoma organoids provided by the present invention can efficiently convert surgically removed liposarcoma tissue samples into liposarcoma organoids. According to the signaling pathways of liposarcoma occurrence, a liposarcoma organoid culture medium suitable for the specific growth of liposarcoma organoids is prepared, and Matrigel is used to simulate the extracellular matrix to provide 3D support for the sample, and continuous culture is carried out so that it always retains the stemness and heterogeneity consistent with the original tumor. Compared with the traditional liposarcoma model, the model construction time is shortened and the construction efficiency is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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: Figure 1 This is a morphological picture of the organoid cultured in Example 1 after 20 days.

[0014] Figure 2 These are the HE staining results of tissues and organoids in Example 1.

[0015] Figure 3 This is a morphological picture of the organoid cultured in Example 2 after 20 days.

[0016] Figure 4 This is a morphological picture of the organoid of Comparative Example 1 after 20 days of culture.

[0017] Figure 5 This is the morphological picture of the organoids of Comparative Example 2 after 20 days of culture. DETAILED DESCRIPTION

[0018] 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.

[0019] The various reagents and raw materials used in the present invention are all commodities that can be purchased from the market or products that can be prepared by known methods. The specific technical scheme is as follows: The liposarcoma organoid culture medium provided by the present invention comprises specific factor components, additive components and basic components of the culture medium; wherein the specific factor components include: PGE2, Insulin, FGF-10, A 83-01.

[0020] PGE2 has multiple biological functions, is synthesized in almost all tissues, and participates in multiple physiological processes. It is an important lipid mediator in the body. It exerts its biological effects by binding to four G protein-coupled receptors (PGE2 receptors) and participates in the regulation of fat metabolism. The effective concentration range of PGE2 described in the present invention is 500-1500 nM.

[0021] Insulin is the only hormone in the body known to reduce blood sugar, and is also the only hormone that promotes the synthesis of glycogen, fat, and protein at the same time. When used as an additive in cell culture, insulin can accelerate the utilization of glucose and inhibit the production of glucose, inhibit the decomposition of fat, promote the synthesis and storage of fat, and maintain cell activity; the effective concentration range of the insulin described in the present invention is 1-10 µg / mL.

[0022] FGF-10 is one of the important members of the fibroblast growth factor family and has a specific effect on promoting mitosis. In addition, FGF-10 is consistent with the occurrence and metastasis mechanism of type III cancer cells, and it is believed that during gastrulation of embryonic development, FGF-10 can promote the process of type I epithelial-mesenchymal transformation; the effective concentration range of FGF-10 in the present invention is 5-20 ng / mL.

[0023] A83-01 is an effective inhibitor that blocks Smad2 phosphorylation and inhibits TGF-β-induced epithelial-mesenchymal transition. The TGF-β signaling pathway plays a key regulatory role in multiple cell functions such as cell proliferation, apoptosis and fibrosis. A83-01 can effectively block the growth inhibition induced by TGF-β, and at the same time inhibit Smad2 phosphorylation, thereby maintaining the self-renewal and proliferation of tumor stem cells. Therefore, A83-01 plays an important role in promoting cell proliferation, preventing cell differentiation, maintaining stem cell stemness, inhibiting cell apoptosis and aging; the effective concentration range of A83-01 described in the present invention is 5-40 nM.

[0024] The liposarcoma culture medium provided by the present invention further comprises the basic components of the culture medium, including: DMEM / F12, 3-15% FBS, 0.5-2× P / S, 1-5 mM GlutaMAX, 5-20 mM HEPES; The liposarcoma culture medium provided by the present invention further comprises culture medium additive components, including: 5-50 nM Hydrocortisone, 1-15 µM SB202190, 0.3-1.5 µg / mL Rspondin1, 1-15 ng / mL EGF, 5-30 µM Y-27632, 1-5 mM N-Acetylcysteine, 1-15 mM Nicotinamide; Prepare liposarcoma organoid culture medium: add 3-15% FBS, 0.5-2× P / S, 1-5mM GlutaMAX, 5-20 mM HEPES, 5-50 nM Hydrocortisone, 1-15 µM SB202190, 0.3-1.5 µg / mL Rspondin1, 1-15 ng / mL EGF, 5-30 µM Y-27632, 1-5 mM N-Acetylcysteine, 1-15 mMNicotinamide, 500-1500 nM PGE2, 1-10 µg / mL Insulin, 5-20 ng / mL FGF-10, and 5-40 nM A83-01 to DMEM / F12.

[0025] Prepare liposarcoma organoid culture medium without Y-27632: add 3-15% FBS, 0.5-2×P / S, 1-5 mM GlutaMAX, 5-20 mM HEPES, 5-50 nM Hydrocortisone, 1-15 µM SB202190, 0.3-1.5 µg / mL Rspondin1, 1-15 ng / mL EGF, 1-5 mM N-Acetylcysteine, 1-15mM Nicotinamide, 500-1500 nM PGE2, 1-10 µg / mL Insulin, 5-20 ng / mL FGF-10, and 5-40 nM MA 83-01 to DMEM / F12.

[0026] Example 1 A liposarcoma organoid culture medium, comprising the following components: 5% FBS, 1×P / S, 2 mMGlutaMAX, 10 mM HEPES, 20 nM Hydrocortisone, 5 µM SB202190, 1 µg / mL Rspondin1, 5 ng / mL EGF, 10 µM Y-27632, 1 mM N-Acetylcysteine, 10 mM Nicotinamide, 900 nM PGE2, 5 µg / mL Insulin, 10 ng / mL FGF-10, 20 nM A 83-01, the above components are dissolved in DMEM / F12; A Y-27632-free liposarcoma organoid culture medium, the culture medium comprising the following components: 5% FBS, 1×P / S, 2 mM GlutaMAX, 10 mM HEPES, 20 nM Hydrocortisone, 5 µM SB202190, 1 µg / mL Rspondin1, 5 ng / mL EGF, 1 mM N-Acetylcysteine, 10 mM Nicotinamide, 900 nM PGE2, 5µg / mL Insulin, 10 ng / mL FGF-10, 20 nM A 83-01, the above components are dissolved in DMEM / F12; A sample cleaning and preservation solution comprises the following components: 2× penicillin-streptomycin-amphotericin B, 10 μM Y-27632, the above components are dissolved in 1× DPBS, prepared before use, and temporarily stored or transported at 0-8°C.

[0027] Use the above culture medium, sample washing and storage solution to complete the construction of liposarcoma organoids. The specific steps are as follows: (1) After the liposarcoma surgical specimen is removed from the body, it is washed once with a sample cleaning and preservation solution of 5 times the sample volume, and then transferred to a new sample cleaning and preservation solution of 10 times the sample volume, transported in a cold chain at 0-8℃, and quickly transferred to the laboratory; (2) Sample cleaning: Take out the liposarcoma sample and immerse it in a sample cleaning and preservation solution with a volume 10 times that of the sample. Incubate it in a constant temperature shaker at 200 rpm and 25°C for 30 min. During this period, the sample cleaning and preservation solution should be replaced every 10 min. (3) After cleaning, collect the sample and cut it into 1 mm pieces using a sterile scalpel. 3 For minced meat pieces, resuspend the sample in 1× DPBS and place in a 15 mL centrifuge tube; (4) Centrifuge at 300 rpm for 3 min, discard the supernatant, collect the sample precipitate, add liposarcoma organoid culture medium to resuspend to form a cell suspension, mix it thoroughly with Matrigel to form a mix, and use a wide-mouth pipette tip to evenly spread the mix on the cell culture plate; (5) Incubate at 37°C for 30 min. After Matrigel is fully solidified, add liposarcoma organoid culture medium into the well plate. (6) Culture in a 37°C incubator overnight and replace with liposarcoma organoid culture medium without Y-27632 the next day; (7) Cultivate in a 37°C incubator for 10 to 40 days, changing the medium every 3 days; The morphology of organoids after 20 days of culture is shown in Figure 1 , the cell morphology was intact and the organoid structure was clear under bright field; tissues and organoids were taken for HE staining, and the staining results are shown in Figure 2 , the organoids are consistent with the original tissue morphology and structure.

[0028] Example 2 A liposarcoma organoid culture medium, comprising the following components: 10% FBS, 2× P / S, 5 mMGlutaMAX, 20 mM HEPES, 30 nM Hydrocortisone, 10 µM SB202190, 1.5 µg / mL Rspondin1, 10 ng / mL EGF, 10 µM Y-27632, 2 mM N-Acetylcysteine, 15 mM Nicotinamide, 1000 nMPGE2, 10 µg / mL Insulin, 5 ng / mL FGF-10, 40 nM A 83-01, the above components are dissolved in DMEM / F12; A Y-27632-free liposarcoma organoid culture medium, the culture medium comprising the following components: 10% FBS, 2×P / S, 5 mM GlutaMAX, 20 mM HEPES, 30 nM Hydrocortisone, 10 µM SB202190, 1.5 µg / mL Rspondin1, 10 ng / mL EGF, 2 mM N-Acetylcysteine, 15 mM Nicotinamide, 1000 nM PGE2, 10 µg / mL Insulin, 5 ng / mL FGF-10, 40 nM A 83-01, the above components are dissolved in DMEM / F12; A sample cleaning and preservation solution, comprising the following components: 1× penicillin-streptomycin-amphotericin B, 8 μM Y-27632, the above components are dissolved in 1× DPBS, prepared before use, and temporarily stored or transported at 0-8°C; The above-prepared sample washing and preservation solution and culture medium are used to complete the construction of liposarcoma organoids. The specific steps are as follows: (1) After the liposarcoma surgical specimen is removed from the body, it is washed once with a sample cleaning and preservation solution of 5 times the sample volume, and then transferred to a new sample cleaning and preservation solution of 10 times the sample volume, transported in a cold chain at 0-8℃, and quickly transferred to the laboratory; (2) Sample cleaning: Take out the liposarcoma sample and immerse it in a sample cleaning and preservation solution with a volume 10 times that of the sample. Incubate it in a constant temperature shaker at 200 rpm and 25°C for 30 min. During this period, the sample cleaning and preservation solution should be replaced every 10 min. (3) After cleaning, collect the sample and cut it into 1 mm pieces using a sterile scalpel. 3 For minced meat pieces, resuspend the sample in 1× DPBS and place in a 15 mL centrifuge tube; (4) Centrifuge at 300 rpm for 3 min, discard the supernatant, collect the sample precipitate, add liposarcoma organoid culture medium to resuspend to form a cell suspension, mix it thoroughly with Matrigel to form a mix, and use a wide-mouth pipette tip to evenly spread the mix on the cell culture plate; (5) Incubate at 37°C for 30 min. After Matrigel is fully solidified, add liposarcoma organoid culture medium into the well plate. (6) Culture in a 37°C incubator overnight and replace with liposarcoma organoid culture medium without Y-27632 the next day; (7) Cultivate in a 37°C incubator for 10 to 40 days, changing the medium every 3 days; The morphology of organoids after 20 days of culture is shown in Figure 3, the cell morphology was observed to be intact and the organoid structure was clear under bright field.

[0029] Comparative Example 1 A liposarcoma organoid culture medium, comprising the following components: 10% FBS, 2× P / S, 5 mMGlutaMAX, 20 mM HEPES, 30 nM Hydrocortisone, 10 µM SB202190, 1.5 µg / mL Rspondin1, 10 ng / mL EGF, 10 µM Y-27632, 2 mM N-Acetylcysteine, 15 mM Nicotinamide, 1000 nMPGE2, 10 µg / mL Insulin, the above components are dissolved in DMEM / F12; A Y-27632-free liposarcoma organoid culture medium, the culture medium comprising the following components: 10% FBS, 2×P / S, 5 mM GlutaMAX, 20 mM HEPES, 30 nM Hydrocortisone, 10 µM SB202190, 1.5 µg / mL Rspondin1, 10 ng / mL EGF, 2 mM N-Acetylcysteine, 15 mM Nicotinamide, 1000 nM PGE2, 10 µg / mL Insulin, the above components are dissolved in DMEM / F12; A sample cleaning and preservation solution, comprising the following components: 1× penicillin-streptomycin-amphotericin B, 8 μM Y-27632, the above components are dissolved in 1× DPBS, prepared before use, and temporarily stored or transported at 0-8°C; The above-prepared sample washing and preservation solution and culture medium are used to complete the construction of liposarcoma organoids. The specific steps are as follows: (1) After the liposarcoma surgical specimen is removed from the body, it is washed once with a sample cleaning and preservation solution of 5 times the sample volume, and then transferred to a new sample cleaning and preservation solution of 10 times the sample volume, transported in a cold chain at 0-8℃, and quickly transferred to the laboratory; (2) Sample cleaning: Take out the liposarcoma sample and immerse it in a sample cleaning and preservation solution with a volume 10 times that of the sample. Incubate it in a constant temperature shaker at 200 rpm and 25°C for 30 min. During this period, the sample cleaning and preservation solution should be replaced every 10 min. (3) After cleaning, collect the sample and cut it into 1 mm pieces using a sterile scalpel. 3 The minced meat pieces were collected into 15 mL centrifuge tubes and digested with 0.25% trypsin at 5 times the sample volume for 5 min, and digestion was terminated with FBS; (4) Centrifuge at 300 rpm for 3 min, discard the supernatant, collect the sample precipitate, add liposarcoma organoid culture medium to resuspend to form a cell suspension, mix it with Matrigel to form a mix, and evenly drip 30-40 μL / drop into the well plate; (5) Incubate at 37°C for 30 min. After Matrigel is fully solidified, add liposarcoma organoid culture medium into the well plate. (6) Culture in a 37°C incubator overnight and replace with liposarcoma organoid culture medium without Y-27632 the next day; (7) Cultivate in a 37°C incubator for 10 to 40 days, changing the medium every 3 days; The morphology of organoids after 20 days of culture is shown in Figure 4 , liposarcoma cells ruptured and died, and no organoid structure was formed, indicating that FGF-10 and A 83-01 play a key role in the formation of liposarcoma organoids; Comparative Example 2 A liposarcoma organoid culture medium, comprising the following components: 10% FBS, 2× P / S, 5 mM GlutaMAX, 20 mM HEPES, 30 nM Hydrocortisone, 10 µM SB202190, 1.5 µg / mL Rspondin1, 10 ng / mL EGF, 10 µM Y-27632, 2 mM N-Acetylcysteine, 15 mM Nicotinamide, 10 ng / mL FGF-10, 20 nM A 83-01, the above components are dissolved in DMEM / F12; A Y-27632-free liposarcoma organoid culture medium, the culture medium comprising the following components: 10% FBS, 2×P / S, 5 mM GlutaMAX, 20 mM HEPES, 30 nM Hydrocortisone, 10 µM SB202190, 1.5 µg / mL Rspondin1, 10 ng / mL EGF, 2 mM N-Acetylcysteine, 15 mM Nicotinamide, 10 ng / mL FGF-10, 20 nM A 83-01, the above components are dissolved in DMEM / F12; A sample cleaning and preservation solution, comprising the following components: 1× penicillin-streptomycin-amphotericin B, 8 μM Y-27632, the above components are dissolved in 1× DPBS, prepared before use, and temporarily stored or transported at 0-8°C; The above-prepared sample washing and preservation solution and culture medium are used to complete the construction of liposarcoma organoids. The specific steps are as follows: (1) After the liposarcoma surgical specimen is removed from the body, it is washed once with a sample cleaning and preservation solution of 5 times the sample volume, and then transferred to a new sample cleaning and preservation solution of 10 times the sample volume, transported in a cold chain at 0-8℃, and quickly transferred to the laboratory; (2) Sample cleaning: Take out the liposarcoma sample and immerse it in a sample cleaning and preservation solution with a volume 10 times that of the sample. Incubate it in a constant temperature shaker at 200 rpm and 25°C for 30 min. During this period, the sample cleaning and preservation solution should be replaced every 10 min. (3) After cleaning, collect the sample and cut it into 1 mm pieces using a sterile scalpel. 3 The minced meat pieces were collected into 15 mL centrifuge tubes and digested with 0.25% trypsin at 5 times the sample volume for 5 min, and digestion was terminated with FBS; (4) Centrifuge at 300 rpm for 3 min, discard the supernatant, collect the sample precipitate, add liposarcoma organoid culture medium to resuspend to form a cell suspension, mix it with Matrigel to form a mix, and evenly drip 30-40 μL / drop into the well plate; (5) Incubate at 37°C for 30 min. After Matrigel is fully solidified, add liposarcoma organoid culture medium into the well plate. (6) Culture in a 37°C incubator overnight and replace with liposarcoma organoid culture medium without Y-27632 the next day; (7) Cultivate in a 37°C incubator for 10 to 40 days, changing the medium every 3 days; The morphology of organoids after 20 days of culture is shown in Figure 5 , liposarcoma cells ruptured and died, and no organoid structure was formed, indicating that PGE2 and Insulin play a key role in the formation of liposarcoma organoids.

[0030] 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 culturing liposarcoma organoids, characterized in that: Contains basic components, additive components and specific factor components of the culture medium; The basic components include: DMEM / F12, FBS, P / S, GlutaMAX, HEPES; the additive components include: Hydrocortisone, SB202190, Rspondin1, EGF, N-Acetylcysteine, Nicotinamide; the specific factor components include: PGE2, Insulin, FGF-10, A 83-01.

2. The culture medium according to claim 1, characterized in that: The concentration range of each component in the culture medium is: adding 3-15% FBS, 0.5-2× P / S, 1-5 mM GlutaMAX, 5-20 mM HEPES, 5-50 nM Hydrocortisone, 1-15 µM SB202190, 0.3-1.5 µg / mL Rspondin1, 1-15 ng / mL EGF, 1-5 mM N-Acetylcysteine, 1-15 mM Nicotinamide, 500-1500 nM PGE2, 1-10 µg / mL Insulin, 5-20 ng / mL FGF-10, and 5-40 nM A 83-01 to DMEM / F12.

3. The culture medium according to claim 2, characterized in that: The additive composition also includes Y-27632.

4. The culture medium according to claim 3, characterized in that: The concentration of Y-27632 is 5-30 µM.

5. A method for constructing and culturing liposarcoma organoids, characterized in that: The following steps are included: (1) After the liposarcoma surgical specimen is removed from the body, it is washed once with a sample cleaning and preservation solution of 5 times the sample volume, and then transferred to a new sample cleaning and preservation solution of 10 times the sample volume, transported in a cold chain at 0-8°C, and quickly transferred to the laboratory; (2) Sample cleaning: Take out the liposarcoma sample and immerse it in a sample cleaning and preservation solution with a volume 10 times that of the sample. Incubate it in a constant temperature shaker at 200 rpm and 25°C for 30 min. During this period, the sample cleaning and preservation solution is replaced every 10 min. (3) After cleaning, collect the sample and cut it into 1 mm pieces using a sterile scalpel. 3 For minced meat pieces, resuspend the sample in 1× DPBS and place in a 15 mL centrifuge tube; (4) Centrifuge at 300 rpm for 3 min, discard the supernatant, collect the sample precipitate, add the culture medium described in claim 4 to resuspend it to form a cell suspension, mix it with Matrigel to form a mix, and use a wide-mouth pipette tip to evenly spread the mix on the cell culture plate; (5) Place in a 37°C incubator for 30 min. After Matrigel is fully solidified, add liposarcoma organoid culture medium into the well plate. (6) Cultivate in a 37°C incubator overnight and replace with the culture medium described in claim 2 the next day; (7) Culture in a 37°C incubator for 10 to 40 days, changing the medium every 3 days.

6. The method according to claim 5, characterized in that: The sample washing and preservation solution is formulated as follows: add 1-2× penicillin-streptomycin-amphotericin B and 5-10 μM Y-27632 to 1×DPBS, prepare before use, and store or transport at 0-8°C.

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