Smooth myosarcoma organoid culture medium and construction and culture method thereof

Abstract

The invention discloses a smooth muscle sarcoma organoid culture medium and a construction and culture method.The culture medium comprises bFGF, Insulin, FGF-10, ydrocortisone, SB202190, PGE2, Y-27632 and the like, isolated smooth muscle sarcoma tissue can be efficiently constructed into a smooth muscle sarcoma organoid, meanwhile, matrigel is used for simulating an extracellular mechanism to provide support for forming a three-dimensional structure for the organoid, continuous culture is conducted, and the smooth muscle sarcoma organoid culture medium is obtained. The obtained smooth muscle sarcoma organ has dryness and heterogeneity consistent with those of an original tumor tissue, and a new platform is provided for establishment of a smooth muscle sarcoma disease research model and in-vitro drug research and development.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor organoid culture, and particularly relates to a leiomyosarcoma organoid culture medium, and also relates to a method for constructing and culturing leiomyosarcoma organoids. Background Art

[0002] Tumor organoids are three-dimensional organoids derived from patient tissues and cultured in vitro. Such organoids derived from the patient itself can not only maintain the heterogeneity of the tumor and the heterogeneity among patients, but also maximize the restoration of characteristics such as the primary tumor subtype and genomic changes, providing a fast and excellent technical platform for fields such as tumor pathogenesis research, drug screening, personalized precision medicine, and regenerative medicine.

[0003] Leiomyosarcoma is a malignant tumor originating from mesenchyme and derived from smooth muscle throughout the body. It is very rare, mostly occurring in the elderly, usually appearing in the abdominal cavity, the inner wall of the uterus, the blood vessel wall, and very rarely in the trunk and extremities of the body. Its five-year average survival rate is only 41%. Usually, there are no obvious symptoms, only a local mass is found, it grows relatively fast and has a relatively large volume. Each subtype of leiomyosarcoma may have complex abnormal chromosomes, and the clinical manifestations and patient outcomes also vary greatly. Surgical resection of the tumor focus is the main treatment method for early-stage leiomyosarcoma, but often the tumor is relatively large and difficult to completely resect, and other treatments are needed in combination. Radiotherapy can be applied before or after surgery, which can reduce recurrence and relieve symptoms, but it does not significantly help in prolonging the survival period; chemotherapy is performed on patients who cannot be completely surgically resected to relieve symptoms and prolong the survival period. The efficacy of chemotherapy is related to the tumor type and varies from person to person.

[0004] Patient-derived tumor organs retain the genetic and phenotypic characteristics of the original tumor and maintain their heterogeneity, thus being widely used in many research fields. Establishing leiomyosarcoma organoids from clinical cancer patients as a potential biomarker for predicting cancer patient treatment is of great significance for leiomyosarcoma patients. Leiomyosarcoma organoids derived from leiomyosarcoma patients can be used as an important model system for patient drug testing surrogates, predicting clinical responses, preclinical studies, etc. However, at present, there are few reports on the culture protocol for leiomyosarcoma organoids. Based on this, there is an urgent need to establish a method for constructing leiomyosarcoma organoids. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a leiomyosarcoma organoid culture medium; the second purpose of the present invention is to provide a method for constructing and culturing leiomyosarcoma organoids using the said leiomyosarcoma organoid culture medium.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] 1. A smooth muscle sarcoma organoid culture medium, comprising a basal component, a specific factor component, and an additive component;

[0008] The basal component of the smooth muscle sarcoma organoid culture medium provided by the present invention includes: Advanced DMEM, Ham's F-10 Nutrient Mix, fetal bovine serum, horse serum, GlutaMAX, penicillin-streptomycin, sodium pyruvate, HEPES.

[0009] The specific factor component of the smooth muscle sarcoma organoid culture medium provided by this method includes: bFGF, Insulin, and FGF-10.

[0010] bFGF is an important inducer of morphogenesis and differentiation, showing effective angiogenesis in vivo and in vitro, stimulating the growth of smooth muscle cells, and promoting wound healing and tissue regeneration.

[0011] Insulin is the only hormone that simultaneously promotes glycogen, fat, and protein synthesis. Used as an additive in cell culture, it can accelerate the utilization of glucose by muscle cells and maintain cell viability.

[0012] FGF-10 is an important member of the fibroblast growth factor family, with a specific mitogenic effect. In the mechanism of cancer cell type III occurrence and metastasis, it is considered to be consistent with the promotion of type I epithelial-mesenchymal transition by FGF-10 during gastrulation in embryonic development.

[0013] The additive components of the smooth muscle sarcoma organoid culture medium provided by the present invention include: hydrocortisone, SB202190, PGE2, Y-27632.

[0014] In some embodiments of the present invention, the culture medium contains the following components in the following amounts: 5-10% (V / V) fetal bovine serum, 5-10% (V / V) horse serum, 2-10 mM GlutaMAX, 0.5-2% (V / V) penicillin-streptomycin, 0.5-2% (V / V) sodium pyruvate, 5-20 mM HEPES, 1-5 ng / mL bFGF, 1-10 μg / mL Insulin, 5-20 ng / mL FGF-10, 5-20 μg / mL hydrocortisone, 1-15 μM SB202190, 0.5-1.5 μM PGE2, 5-20 μM Y-27632. Dissolve the above components in a mixed culture medium formed by mixing Advanced DMEM and Ham's F-10 Nutrient Mix at a volume ratio of 1:1 to obtain.

[0015] 2. A method for constructing and culturing leiomyosarcoma organoids using the said leiomyosarcoma organoid medium, comprising the following steps:

[0016] (1) Take the tumor sample resected surgically from a leiomyosarcoma patient, transfer it into a tissue preservation solution with a volume 5 times that of the sample, and transport it in a cold chain at 0 - 8°C and transfer the sample to the laboratory in a timely manner;

[0017] (2) Take out the leiomyosarcoma sample, immerse it in a tissue washing solution with a volume 10 times that of the sample, incubate it with shaking at 200 rpm and a constant temperature of 25°C for 30 min, and replace the tissue washing solution every 5 min during this period;

[0018] (3) Collect the washed sample, cut the sample into small tissue pieces with side lengths less than 0.5 mm, and resuspend the sample using the tissue preservation solution;

[0019] (4) Filter the sample suspension through a 40 - mesh cell sieve, apply appropriate pressure to assist the sample to pass through the sieve;

[0020] (5) Collect the filtered sample suspension, centrifuge it at 4°C, 400×g for 5 min, collect the cell pellet, add the leiomyosarcoma organoid medium, mix evenly with Matrigel after forming a cell suspension, and spread the mixture evenly on a cell culture plate;

[0021] (6) Let it stand in an incubator at 37°C for 30 min, and after the Matrigel has fully solidified, supplement the leiomyosarcoma organoid medium into the well plate;

[0022] (7) Culture it overnight in an incubator at 37°C, and replace it with the leiomyosarcoma organoid medium without Y - 27632 the next day;

[0023] (8) Continuously culture it in an incubator at 37°C for 10 - 20 days, and replace the medium every 3 days during this period.

[0024] The method for constructing and culturing leiomyosarcoma organoids provided by the present invention relates to a tissue preservation solution, which comprises components with the following volume fractions: 1 - 2% penicillin - streptomycin - amphotericin B, 1 - 2% GlutaMAX, 48 - 49% Advanced DMEM, 48 - 49% Ham's F - 10 Nutrient Mix.

[0025] The method for constructing and culturing leiomyosarcoma organoids provided by the present invention relates to a tissue washing solution, which comprises components with the following contents: 2% (V / V) penicillin - streptomycin - amphotericin B, 0.1 - 0.5 mg / mL hyaluronidase, 0.05 - 0.5 mg / mL DNaseⅠ, 1×DPBS.

[0026] Hyaluronic acid is a large-molecular-weight polysaccharide with a negative charge and an important component of the extracellular matrix (ECM). It is widely present in connective tissues, epithelial tissues, and nerve tissues and participates in many biological processes. Research has found that hyaluronic acid is widely present in the tumor microenvironment, and the presence of hyaluronic acid affects subsequent sample processing. In this sample washing solution, there is 0.1 - 0.5 mg / mL hyaluronidase, which reduces the large number of bubbles generated by hyaluronic acid during the washing process. At the same time, it can make the ECM skeleton become loose, improving the recovery rate of leiomyosarcoma samples and the efficiency of organoid construction.

[0027] The method for constructing and culturing leiomyosarcoma organoids provided by the present invention involves a leiomyosarcoma organoid culture medium without Y-27632, which is composed of: 5 - 10% (V / V) fetal bovine serum, 5 - 10% (V / V) horse serum, 2 - 10 mM GlutaMAX, 0.5 - 2% (V / V) penicillin-streptomycin, 0.5 - 2% (V / V) sodium pyruvate, 5 - 20 mM HEPES, 1 - 5 ng / mL bFGF, 1 - 10 μg / mL Insulin, 5 - 20 ng / mL FGF-10, 5 - 20 μg / mL hydrocortisone, 1 - 15 μM SB202190, 0.5 - 1.5 μM PGE2. Dissolve the above components in a mixed culture medium formed by Advanced DMEM and Ham's F-10 Nutrient Mix at a volume ratio of 1:1 to obtain it.

[0028] The beneficial effects of the present invention are as follows: The present invention provides a culture medium for leiomyosarcoma organoids and a method for construction and culture, which can efficiently construct in vitro leiomyosarcoma tissues into leiomyosarcoma organoids. According to the occurrence mechanism of leiomyosarcoma, a culture medium for leiomyosarcoma organoids suitable for the growth of leiomyosarcoma organoids is prepared. Using Matrigel to simulate the extracellular mechanism provides support for the formation of a three-dimensional structure of organoids. Through continuous culture, the obtained leiomyosarcoma organoids have the same stemness and heterogeneity as the original tumor tissue. The aim is to overcome technical bottlenecks, extend the survival cycle of patients, improve the quality of life of patients, and also provide a new platform for establishing a research model for leiomyosarcoma diseases and in vitro drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0030] Figure 1 The culture result obtained in Example 1;

[0031] Figure 2 The culture result obtained in Example 2;

[0032] Figure 3The culture result obtained as a comparative example;

[0033] Figure 4 It is the result of HE staining of leiomyosarcoma organoids in Example 3. Detailed implementation manners

[0034] The present invention will be 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 be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0035] All kinds of reagents and raw materials used in the present invention are commercially available products or products that can be prepared by well-known methods.

[0036] Example 1

[0037] A leiomyosarcoma tissue preservation solution, the tissue preservation solution contains the following components by volume fraction: 2% penicillin-streptomycin-amphotericin B, 1% GlutaMAX, 48.5% Advanced DMEM, 48.5% Ham's F-10 Nutrient Mix.

[0038] A leiomyosarcoma tissue washing solution, the tissue washing solution contains the following components: 2% (V / V) penicillin-streptomycin-amphotericin B, 0.1 mg / mL hyaluronidase, 0.1 mg / mL DNase I, 1×DPBS.

[0039] A leiomyosarcoma organoid culture medium, the culture medium contains the following components: 5% (V / V) fetal bovine serum, 5% (V / V) horse serum, 5 mM GlutaMAX, 1% (V / V) penicillin-streptomycin double antibody, 1% (V / V) sodium pyruvate, 10 mM HEPES, 4 ng / mL bFGF, 8 μg / mL Insulin, 10 ng / mL FGF-10, 10 μg / mL hydrocortisone, 5 μM SB202190, 1 μM PGE2, 10 μM Y-27632, and the above components are dissolved in a mixed culture medium formed by Advanced DMEM and Ham's F-10 Nutrient Mix according to a volume ratio of 1:1.

[0040] A smooth muscle sarcoma medium without Y-27632, the medium contains the following components: 5% (V / V) fetal bovine serum, 5% (V / V) horse serum, 5 mM GlutaMAX, 1% (V / V) penicillin-streptomycin double antibody, 1% (V / V) sodium pyruvate, 10 mM HEPES, 4 ng / mL bFGF, 8 μg / mL Insulin, 10 ng / mL FGF-10, 10 μg / mL hydrocortisone, 5 μM SB202190, 1 μM PGE2, and dissolve the above components in a mixed medium formed by Advanced DMEM and Ham's F-10 Nutrient Mix according to a volume ratio of 1:1.

[0041] Use the above-prepared reagents and medium to complete the construction of smooth muscle sarcoma organoids. The specific steps are as follows:

[0042] (1) Take a tumor tissue sample surgically resected from a smooth muscle sarcoma patient, transfer it into a tissue preservation solution with a volume 5 times that of the sample, and transport it in a cold chain at 0-8°C and transfer the sample to the laboratory in a timely manner;

[0043] (2) Take out the smooth muscle sarcoma sample, immerse it in a tissue washing solution with a volume 10 times that of the sample, shake and incubate it on a constant temperature shaker at 200 rpm and 25°C for 30 min, and change the tissue washing solution every 5 min during this period;

[0044] (3) Collect the washed sample, use a sterile scalpel and sterile surgical scissors to cut the sample into small tissue pieces with a side length less than 0.5 mm, and resuspend the sample with the tissue preservation solution;

[0045] (4) Filter the sample suspension through a 40-mesh cell sieve, apply appropriate pressure to assist the sample to pass through the sieve;

[0046] (5) Collect the above sample suspension, centrifuge it at 4°C and 400×g for 5 min, discard the supernatant, collect the cell pellet, add the smooth muscle sarcoma organoid medium, mix evenly with Matrigel after forming a cell suspension, and use a wide-mouth pipette tip to evenly spread the mixture on a cell culture plate;

[0047] (6) Let it stand in a 37°C incubator for 30 min. After the Matrigel has fully solidified, supplement the smooth muscle sarcoma organoid medium into the well plate;

[0048] (7) Culture it in a 37°C incubator overnight, and replace it with a smooth muscle sarcoma organoid medium without Y-27632 the next day;

[0049] (8) Continuously culture it in a 37°C incubator for 10-20 days, and change the medium every 3 days.

[0050] The culture result after 20 days is as Figure 1As shown in the middle right figure, a relatively large number of overall shapes and organoid structures with smooth edges were formed.

[0051] Example 2

[0052] A smooth muscle sarcoma tissue preservation solution, the tissue preservation solution contains the following components by volume fraction: 2% penicillin-streptomycin-amphotericin B, 2% GlutaMAX, 48% Advanced DMEM, 48% Ham's F-10 Nutrient Mix.

[0053] A smooth muscle sarcoma tissue washing solution, the tissue washing solution contains the following components: 2% (V / V) penicillin-streptomycin-amphotericin B, 0.3 mg / mL hyaluronidase, 0.5 mg / mL DNaseⅠ, 1×DPBS.

[0054] A smooth muscle sarcoma organoid culture medium, the culture medium contains the following components: 2% (V / V) fetal bovine serum, 2% (V / V) horse serum, 5 mM GlutaMAX, 1% (V / V) penicillin-streptomycin double antibody, 1% (V / V) sodium pyruvate, 10 mM HEPES, 2 ng / mL bFGF, 5 μg / mL Insulin, 2 ng / mL FGF-10, 2 μg / mL hydrocortisone, 10 μM SB202190, 0.2 μM PGE2, 10 μM Y-27632, and the above components are dissolved in a mixed culture medium formed by mixing Advanced DMEM and Ham's F-10 Nutrient Mix at a volume ratio of 1:1.

[0055] A smooth muscle sarcoma culture medium without Y-27632, the culture medium contains the following components: 2% (V / V) fetal bovine serum, 2% (V / V) horse serum, 5 mM GlutaMAX, 1% (V / V) penicillin-streptomycin double antibody, 1% (V / V) sodium pyruvate, 10 mM HEPES, 2 ng / mL bFGF, 5 μg / mL Insulin, 2 ng / mL FGF-10, 2 μg / mL hydrocortisone, 10 μM SB202190, 0.2 μM PGE2, and the above components are dissolved in a mixed culture medium formed by mixing Advanced DMEM and Ham's F-10 Nutrient Mix at a volume ratio of 1:1.

[0056] Using the above-prepared reagents and culture medium, the culture was carried out according to the construction method of Example 1. The culture results after 20 days are as Figure 2 shown in the middle right figure, only some of the original tissue fragments formed organoid structures with smooth overall shapes and edges.

[0057] Example 3

[0058] Identification of leiomyosarcoma organoids by HE staining. HE staining was performed on the leiomyosarcoma organoids obtained in Example 1. The steps were as follows: After culturing the organoids for 20 days, they were treated with organoid recovery solution for 30 min to dissolve Matrigel. The organoids separated from Matrigel were fixed with 4% paraformaldehyde for 12 h, dehydrated through an alcohol gradient and cleared with xylene, and then embedded in paraffin. Cross-sections and longitudinal sections were made respectively. At the same time, the original leiomyosarcoma tissue was also fixed, dehydrated, cleared, and embedded in paraffin. After sectioning, H&E staining was performed on the organoids and the tissue, and their morphological structures were identified.

[0059] The original tissue and organoids after staining were as Figure 4 shown. The results showed that the cells in the original leiomyosarcoma tissue and the organoids were similar in terms of cell nucleus, cytoplasm, and nuclear-cytoplasmic ratio, and were also similar in tissue morphology, both showing an irregularly arranged cavity-like structure among cells. This similarity indicates that the constructed leiomyosarcoma organoids can better retain the genetic characteristics and biological behaviors of the original tissue.

[0060] Control

[0061] A preservation solution for leiomyosarcoma tissue. The preservation solution contains the following components: 2% penicillin-streptomycin-amphotericin B, 2% GlutaMAX, 48% Advanced DMEM, 48% Ham's F-10 Nutrient Mix.

[0062] A washing solution for leiomyosarcoma tissue. The washing solution contains the following components: 2% penicillin-streptomycin-amphotericin B, 0.5 mg / mL hyaluronidase, 0.5 mg / mL DNaseⅠ, 1×DPBS.

[0063] A culture medium for leiomyosarcoma organoids. The culture medium contains the following components: 10% fetal bovine serum, 5% horse serum, 5 mM GlutaMAX, 1% penicillin-streptomycin double antibody, 1% sodium pyruvate, 10 mM HEPES, 10 μg / mL Insulin, 20 μg / mL hydrocortisone, 15 μM SB202190, 20 μM Y-27632. The above components were dissolved in a mixed culture medium formed by Advanced DMEM and Ham's F-10 Nutrient Mix at a volume ratio of 1:1.

[0064] A smooth muscle sarcoma culture medium without Y-27632, the culture medium comprises the following components: 10% fetal bovine serum, 5% horse serum, 5 mM GlutaMAX, 1% penicillin-streptomycin double antibody, 1% sodium pyruvate, 10 mM HEPES, 10 μg / mL Insulin, 20 μg / mL hydrocortisone, 15 μM SB202190, and the above components are dissolved in a mixed culture medium formed by Advanced DMEM and Ham's F-10 Nutrient Mix according to a volume ratio of 1:1. The reagents and culture medium prepared in the comparative example are used for culturing with reference to the construction method of Example 1. The culture result after 20 days is as Figure 3 shown in the right figure in the middle above, no obvious round organoid structure is formed, and most tissue fragments tend to turn black and have adhesion phenomena.

[0065] It can be known from the comparison of the culture results of the examples and the comparative examples that the number of organoids obtained in Example 1 is the largest, the morphology is the smoothest, and there are obvious differences in the morphology compared with the original tissue fragments, indicating that the self-assembly effect of the organoids is the best under this culture condition; only some of the original tissue fragments in Example 2 self-assembled into organoid structures, and there are obvious differences in the number and morphology of the organoids compared with Example 1; while there is almost no self-assembly phenomenon in the original tissue fragments in the comparative example, and most tissue fragments tend to turn black and have adhesion phenomena, indicating that this culture scheme is a failure. In summary, the culture effect of Example 1 is the best.

[0066] The above-mentioned examples are only preferred examples given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A smooth muscle sarcoma organoid culture medium, characterized in that, The culture medium contains a basal component, a specific factor component and an additive component; the basal component includes: Advanced DMEM, Ham's F-10 Nutrient Mix, fetal bovine serum, horse serum, GlutaMAX, penicillin-streptomycin, sodium pyruvate, HEPES; the specific factor component includes: bFGF, Insulin, FGF-10; the additive component contains: hydrocortisone, SB202190, PGE2, Y-27632.

2. The smooth muscle sarcoma organoid culture medium according to claim 1, characterized in that, The culture medium contains components in the following contents: 5-10% (V / V) fetal bovine serum, 5-10% (V / V) horse serum, 2-10 mM GlutaMAX, 0.5-2% (V / V) penicillin-streptomycin, 0.5-2% (V / V) sodium pyruvate, 5-20 mM HEPES, 1-5 ng / mL bFGF, 1-10 μg / mL Insulin, 5-20 ng / mL FGF-10, 5-20 μg / mL hydrocortisone, 1-15 μM SB202190, 0.5-1.5 μM PGE2, 5-20 μM Y-27632. Dissolve the above components in a mixed culture medium formed by mixing Advanced DMEM and Ham's F-10 Nutrient Mix at a volume ratio of 1:1 to obtain the culture medium.

3. A method for constructing and culturing leiomyosarcoma organoids using the leiomyosarcoma organoid culture medium described in claim 2, characterized in that, It includes the following steps: (1) Take the tumor sample resected from a patient with leiomyosarcoma, transfer it to a tissue preservation solution with a volume 5 times that of the sample, and transport it in a cold chain at 0-8°C and transfer the sample to the laboratory in a timely manner; (2) Take out the leiomyosarcoma sample, immerse it in a tissue washing solution with a volume 10 times that of the sample, shake and incubate it on a constant temperature shaker at 25°C and 200 rpm for 30 min, and replace the tissue washing solution every 5 min during this period; (3) Collect the washed sample, cut the sample into small tissue pieces with a side length less than 0.5 mm, and resuspend the sample with the tissue preservation solution; (4) Filter the sample suspension through a 40-mesh cell sieve, apply appropriate pressure to assist the sample to pass through the sieve; (5) Collect the filtered sample suspension, centrifuge it at 4°C and 400×g for 5 min, collect the cell precipitate, add the leiomyosarcoma organoid culture medium, mix it evenly with the Matrigel after forming a cell suspension, and spread the mixture evenly on a cell culture plate; (6) Let it stand in an incubator at 37°C for 30 min. After the Matrigel has fully solidified, supplement the leiomyosarcoma organoid culture medium into the well plate; (7) Culture it in an incubator at 37°C overnight, and replace it with the leiomyosarcoma organoid culture medium without Y-27632 the next day; (8) Continuously culture it in an incubator at 37°C for 10-20 days, and replace the culture medium every 3 days during this period.

4. The method according to claim 3, wherein The tissue preservation solution contains components in the following volume fractions: 1-2% penicillin-streptomycin-amphotericin B, 1-2% GlutaMAX, 48-49% Advanced DMEM, 48-49% Ham's F-10 Nutrient Mix.

5. The method according to claim 3, characterized in that, The tissue washing solution contains components in the following contents: 2% (V / V) penicillin-streptomycin-amphotericin B, 0.1 - 0.5 mg / mL hyaluronidase, 0.05 - 0.5 mg / mL DNase I, 1×DPBS.

6. The method according to claim 3, characterized in that The smooth muscle sarcoma organoid medium without Y-27632 is: 5 - 10% (V / V) fetal bovine serum, 5 - 10% (V / V) horse serum, 2 - 10 mM GlutaMAX, 0.5 - 2% (V / V) penicillin-streptomycin, 0.5 - 2% (V / V) sodium pyruvate, 5 - 20 mM HEPES, 1 - 5 ng / mL bFGF, 1 - 10 μg / mL Insulin, 5 - 20 ng / mL FGF-10, 5 - 20 μg / mL hydrocortisone, 1 - 15 μM SB202190, 0.5 - 1.5 μM PGE2. Dissolve the above components in a mixed medium formed by Advanced DMEM and Ham's F-10 Nutrient Mix at a volume ratio of 1:1 to obtain it.

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