CN117701505A - Sarcoma tissue organoid medium, method for constructing sarcoma tissue organoid and sarcoma tissue organoid library, and method for constructing animal disease model - Google Patents
Sarcoma tissue organoid medium, method for constructing sarcoma tissue organoid and sarcoma tissue organoid library, and method for constructing animal disease model Download PDFInfo
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- CN117701505A CN117701505A CN202410167784.0A CN202410167784A CN117701505A CN 117701505 A CN117701505 A CN 117701505A CN 202410167784 A CN202410167784 A CN 202410167784A CN 117701505 A CN117701505 A CN 117701505A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a sarcoma tissue organoid culture medium, a method for constructing a sarcoma tissue organoid and a sarcoma tissue organoid library and a method for constructing an animal disease model, and relates to the field of organoid culture. The culture medium comprises: a basal medium and a specific additive factor, the specific additive factor comprising: nicotinamide, B27 additive, N-acetylcysteine, EGF protein, FGF2 protein, FGF7 protein, and estradiol. The culture medium is used for culturing the sarcoid tissue organoids, has high organoid growth speed and multiple passage times, has high biological consistency with the animal sarcoid tissue derived from organoid culture cells, has high research value and has wide application prospect.
Description
Technical Field
The invention relates to the field of organoid culture, in particular to a sarcomas tissue organoid culture medium, a method for constructing sarcomas tissue organoids and a sarcomas tissue organoid library and a method for constructing an animal disease model.
Background
The main treatment means for sarcoma diseases such as carcinoma (Carcinosarcomas), malignant soft tissue sarcoma (Malignant soft tissue tumors (soft tissue Sarcomas, STS)), endometrial sarcoma (Sarcabas), malignant endometrial stromal tumor (Leucomatous), and leiomyosarcoma (Uterine leiomyosarcoma, u-LMS) are surgery, with poor prognosis and low overall survival rate. Patient survival has not improved significantly over the past decades due to the lack of more effective treatments.
The sarcoma organoid constructed by using the tumor stem cells derived from the tumor tissue of the patient can simulate the clinical treatment result of the patient in vitro, thereby being beneficial to realizing personalized accurate treatment, improving prognosis and increasing total survival rate. Therefore, the sarcoma organoid can provide a research model for the occurrence, development and disease treatment of various sarcomas, and can be used for the clinical effect evaluation and pre-clinical research and development of personalized accurate treatment schemes.
Thus, there is a great need in the art for a rapid and efficient in vitro establishment of a sarcoid model, particularly a medium suitable for sarcoid culture.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides the sarcoma tissue organoid culture medium, the method for constructing the sarcoma tissue organoid and the sarcoma tissue organoid library and the method for constructing the animal disease model, and the culture medium is used for culturing the sarcoma tissue organoid, and has the advantages of high organoid growth speed, multiple passage times, high consistence with animal sarcoma tissue organisms derived from organoid culture cells, high research value and wide application prospect.
In a first aspect of the invention, the invention provides a sarcoid organoid medium. According to an embodiment of the invention, the medium comprises: a basal medium and a specific additive factor, the specific additive factor comprising: nicotinamide, B27 additive, N-acetylcysteine, EGF protein, FGF2 protein, FGF7 protein, and estradiol. The culture medium provided by the embodiment of the invention is used for culturing the sarcoid tissue organoid, the organoid has the advantages of high growth rate and multiple passage times, and meanwhile, the organoid obtained by using the culture medium has high biological consistency with the animal sarcoid tissue derived from organoid culture cells, high research value and wide application prospect.
According to an embodiment of the invention, the concentration of estradiol is 10 to 50 ng/mL, preferably 25 ng/mL. The inventor obtains the above preferred estradiol concentration through a plurality of experiments, thereby further improving the growth rate of the sarcoid tissue organoid and improving the biological consistency of the obtained organoid and the animal sarcoid tissue.
According to an embodiment of the invention, the specific addition factor further comprises: fetal bovine serum, R-Spondin protein, and Wnt3a protein. The inventors have further studied and found that partial sarcomas, such as interstitial sarcoma and leiomyosarcoma, are better in growth rate in the presence of fetal bovine serum, R-Spondin protein and Wnt3a protein.
According to an embodiment of the invention, the concentration of fetal bovine serum is 5-15% by volume based on the total volume of the organoid medium; preferably 10% by volume. The inventors have determined this preferred concentration through a number of optimization experiments.
According to an embodiment of the invention, the concentration of the R-Spondin protein is 100 ng/mL-500 ng/mL based on the total volume of the organoid medium; preferably 500 ng/mL. The inventors have determined this preferred concentration through a number of optimization experiments.
According to an embodiment of the invention, the concentration of Wnt3a protein is 10 ng-50 ng/mL based on the total volume of the organoid medium; preferably 50 ng/mL. The inventors have determined this preferred concentration through a number of optimization experiments.
According to an embodiment of the invention, the concentration of nicotinamide is 1-10 mM based on the total volume of the organoid medium; preferably 10 mM. The inventors have determined this preferred concentration through a number of optimization experiments. When nicotinamide in the culture medium is within this concentration range, organoid cell expansion and long-term subculture can be achieved.
According to an embodiment of the invention, the concentration of the B27 additive is 1-3% by volume based on the total volume of the organoid medium; preferably 1% by volume. The inventors have determined this preferred concentration through a number of optimization experiments. Thereby, the organoid growth rate is further increased.
According to an embodiment of the present invention, the concentration of N-acetylcysteine is 0.5 to 2.5. 2.5 mM based on the total volume of the organoid medium; preferably 1.25, mM. The inventors have determined this preferred concentration through a number of optimization experiments. When N-acetylcysteine in the culture medium is in the concentration range, the cell expansion and long-term subculture of organoids can be realized.
According to an embodiment of the present invention, the concentration of the EGF protein is 10-100 ng/mL based on the total volume of the organoid medium; preferably 50 ng/mL. The inventors have determined this preferred concentration through a number of optimization experiments. When EGF protein in the culture medium is within this concentration range, organoid cell expansion and long-term subculture can be achieved.
According to an embodiment of the present invention, the concentration of FGF2 protein is 10-100 ng/mL based on the total volume of the organoid medium; preferably 100 ng/mL. The inventors have determined this preferred concentration through a number of optimization experiments. When FGF2 protein in the medium is in this concentration range, organoid cell expansion and long-term subculture can be achieved.
According to an embodiment of the present invention, the concentration of FGF7 protein is 10-100 ng/mL based on the total volume of the organoid medium; preferably 100 ng/mL. The inventors have determined this preferred concentration through a number of optimization experiments. When FGF7 protein in the medium is in this concentration range, organoid cell expansion and long-term subculture can be achieved.
According to an embodiment of the invention, the basal medium is a DMEM/F12 reduced serum medium. When the basal medium is a DMEM/F12 serum-reduced medium, organoid cell expansion and long-term subculture can be realized.
According to an embodiment of the present invention, the concentration of the DMEM/F12 reduced serum medium is 82 to 99% by volume based on the total volume of the organoid medium; preferably 89 to 99% by volume. The inventors have determined this preferred concentration through a number of optimization experiments. When the concentration of DMEM/F12 reduced serum medium in the medium is within this range, the organoid growth rate is faster.
According to an embodiment of the invention, the basal medium further comprises: hydrogen ion buffer, penicillin and streptomycin.
According to an embodiment of the invention, the hydrogen ion buffer is 4-hydroxyethylpiperazine ethanesulfonic acid.
According to an embodiment of the present invention, the concentration of the hydrogen ion buffer is 5 to 15 mM based on the volume of the DMEM/F12 reduced serum medium; preferably 10 mM. When the concentration of the 4-hydroxyethyl piperazine ethane sulfonic acid in the culture medium is within the concentration range, the cell expansion and long-term subculture of organoids can be realized.
According to the embodiment of the invention, the concentration of penicillin is 20-70U/mL and the concentration of streptomycin is 0.02-0.07 mg/mL based on the volume of the DMEM/F12 serum-reduced medium; preferably 0.05 mg/mL.
According to an embodiment of the invention, the organoid medium comprises: a basal medium and a specific additive factor, the specific additive factor comprising: nicotinamide, B27 additive, N-acetylcysteine, EGF protein, FGF2 protein, FGF7 protein, and estradiol; the basal medium comprises: DMEM/F12 reduced serum medium, hydrogen ion buffer, penicillin and streptomycin; the organoid medium comprises the following concentrations of components, based on the total volume of the organoid medium: 89-99% by volume of DMEM/F12 serum-reduced medium, 1-10% by volume of nicotinamide, 1-2% by volume of B27 additive, 0.5-2.5 mM of N-acetylcysteine, 10-100 ng/mL of EGF protein, 10-100 ng/mL of FGF2 protein, 10-100 ng/mL of FGF7 protein, and 10-50 ng/mL of estradiol; the organoid medium further comprises the following concentrations of components, based on the volume of the DMEM/F12 reduced serum medium: 5 to 15 of mM of hydrogen ion buffer, 20 to 70U/mL of penicillin and 0.02 to 0.07 mg/mL of streptomycin. The culture medium provided by the embodiment of the invention is used for culturing the sarcoid tissue organoids, has the advantages of high organoid growth rate and high passage times, can be used for quickly and effectively constructing the sarcoid tissue organoids, organoid libraries and animal disease models in vitro, and meanwhile, the organoids obtained by using the culture medium have high consistence with animal sarcoid tissue organisms derived from organoid cultured cells, high research value and wide application prospect.
According to an embodiment of the invention, the specific addition factor further comprises: fetal bovine serum, R-Spondin protein, and Wnt3a protein. The organoid medium according to the embodiments of the present invention enables the organoid culture of a variety of sarcoma tissues, and in particular enables the organoid culture of interstitial sarcoma and leiomyosarcoma tissues.
According to an embodiment of the invention, the organoid medium further comprises specific additive factors in the following concentrations, based on the total volume of the organoid medium: 5-15% by volume of fetal bovine serum, 100 ng-500 ng/mL of R-Spondin protein, 10 ng-50 ng/mL of Wnt3a protein. The inventor determines the preferred concentration through a plurality of optimization experiments, thereby further improving the growth rate of the organoid and improving the biological consistency of the organoid and the animal sarcoma tissue from which the organoid cultured cells are derived.
According to an embodiment of the invention, the organoid medium comprises the following concentrations of components, based on the total volume of the organoid medium: 99% by volume of DMEM/F12 reduced serum medium, 10 mM of nicotinamide, 1% by volume of B27 additive, 1.25 mM of N-acetylcysteine, 50 ng/mL of EGF protein, 100 ng/mL of FGF2 protein, 100 ng/mL of FGF7 protein, 25 ng/mL of estradiol. The inventor determines the preferred concentration through a large number of optimization experiments, thereby realizing the rapid and effective in vitro construction of the sarcoid tissue organoid and improving the biological consistency of the organoid and the animal sarcoid tissue derived from the organoid culture cells.
According to an embodiment of the invention, the organoid medium further comprises the following concentrations of components, based on the volume of the DMEM/F12 reduced serum medium: 10 mM hydrogen buffer, 50U/mL penicillin, 0.05 mg/mL streptomycin. When the concentration of the 4-hydroxyethyl piperazine ethane sulfonic acid in the culture medium is within the concentration range, the cell expansion and long-term subculture of organoids can be realized.
According to an embodiment of the invention, the organoid medium comprises the following concentrations of components, based on the total volume of the organoid medium: 89% by volume of DMEM/F12 reduced serum medium, 10 mM of nicotinamide, 1% by volume of B27 additive, 1.25. 1.25 mM of N-acetylcysteine, 50 ng/mL of EGF protein, 100 ng/mL of FGF2 protein, 100 ng/mL of FGF7 protein, 25 ng/mL of estradiol, 10% by volume of fetal bovine serum, 500 ng/mL of R-Spondin protein, 50 ng/mL of Wnt3a protein. The inventor determines the preferred concentration through a large number of optimization experiments, thereby realizing the rapid and effective in vitro construction of the sarcomas tissue organoids (especially the interstitial sarcomas and the leiomyosarcoma tissue organoids) and improving the biological consistency of the organoids and the animal sarcomas tissue derived from the organoids cultured cells.
In a second aspect of the invention, the invention provides a method of constructing a sarcomatous tissue organoid. According to an embodiment of the invention, the method comprises: culturing sarcoma tissue cells in the culture medium to obtain the sarcoma tissue organoid. The sarcoma organoid obtained by the method according to the embodiment of the invention can be used as a research model for occurrence, development and disease treatment of various sarcomas, and can be used for clinical effect evaluation and pre-clinical research and development of personalized accurate treatment schemes.
Those skilled in the art will appreciate that the features and advantages described above for the medium apply equally to the method and are not described in detail herein.
According to an embodiment of the invention, the sarcoma tissue cells are made into a single cell suspension prior to the culturing process.
According to an embodiment of the invention, before the culture treatment, the single-cell suspension is subjected to a resuspension treatment by using matrigel to obtain matrigel containing cells, wherein the concentration of the matrigel is 2.5-5 mg/mL. The inventors have unexpectedly achieved the preferred concentration ranges herein through extensive optimization experiments, thereby further increasing the organoid growth rate and the clonogenic rate.
According to an embodiment of the invention, the cell density in the matrigel containing cells is 100,000 ～ 500,000 cells/mL. Thereby, the growth rate and the cloning efficiency of the organoids are further improved.
According to an embodiment of the invention, the sarcoma tissue organoid is at least one of a carcinomatous tissue organoid, an osteosarcoma organoid, a bone and soft tissue sarcoma organoid, a synovial sarcoma organoid, a rhabdomyosarcoma organoid, a human malignant soft tissue sarcoma organoid, an endometrial sarcoma organoid, a mesenchymal sarcoma organoid and a leiomyosarcoma organoid.
In a third aspect of the invention, the invention provides a method of constructing a model of an animal disease. According to an embodiment of the invention, the method comprises: obtaining a sarcoma tissue organoid according to the method, and transplanting the sarcoma tissue organoid into the animal body to obtain the animal disease model. The animal disease model obtained by the method of the embodiment of the invention can more accurately simulate the occurrence and development processes of various sarcoma diseases in human bodies, is beneficial to research on disease mechanism and development of related medicaments and formulation of treatment schemes.
Those skilled in the art will appreciate that the features and advantages described above for the medium apply equally to the method and are not described in detail herein.
In a fourth aspect of the invention, the invention provides a method of constructing a sarcomas tissue organoid library. According to an embodiment of the invention, the method comprises: obtaining a sarcoma tissue organoid according to the method, subculturing the sarcoma tissue organoid to obtain the subcultured sarcoma tissue organoid, and performing freezing treatment on the subcultured sarcoma tissue organoid to obtain the sarcoma tissue organoid library.
Those skilled in the art will appreciate that the features and advantages described above for the medium apply equally to the method and are not described in detail herein.
Compared with the prior art, the invention has the beneficial effects that:
1. the culture medium of the invention is widely suitable for the culture of sarcomas tissue organoids, and has fast organoids growth and multiple passage times.
2. The organoid obtained by using the culture medium has high tissue biological consistency with animal sarcoma derived from organoid cultured cells, can be used as a research model for the occurrence, development and disease treatment of various sarcomas, has high research value, can be used for evaluating the clinical effect of a personalized accurate treatment scheme and researching and developing drugs before clinic, and has wide application value.
3. The culture medium is suitable for quickly and effectively constructing sarcoma tissue organoids, organoid libraries and animal disease models in vitro, and is particularly beneficial to evaluating the sensitivity of various sarcomas and establishing personalized treatment schemes.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a diagram showing the growth state of human carcinomatous sarcoma on day 6 of primary culture in example 3 according to the present invention, wherein the scale is: 100. μm;
FIG. 2 is a graph showing the growth state of human carcinomatous sarcoma on day 5 of subculture in example 3 of the present invention, wherein the scale is: 100. μm;
fig. 3 is a growth state diagram of human interstitial sarcoma organoid in example 4 of the present invention, wherein organoids were in matrigel when photographed, and the scale is: 100. μm;
fig. 4 is a diagram showing the growth state of human smooth muscle primary sarcoma organoid in example 4 of the present invention, wherein organoids were in matrigel when photographed, and the scale is: 100. μm;
FIG. 5 is a graph showing the growth state of human smooth muscle metastasis sarcoma organoids in example 4 of the present invention, wherein the organoids were in matrigel when photographed, and the scale is: 100. μm;
FIG. 6 is a graph showing the H & E staining results of human stromal sarcoma in example 5 of the present invention, wherein (A) is a graph showing the H & E staining results of human stromal sarcoma tissue (Tumor tissue) sample; (B) H & E staining results for human stromal sarcoma Organoid (Organoid) samples after passage of example 4;
FIG. 7 is a graph showing immunofluorescence staining results of human stromal sarcoma in example 5 of the present invention, wherein (A) to (F) are graphs showing immunofluorescence staining results of Primary tissue (Primary tissue) of human stromal tumor for organoid culture; (G) (L) is a graph showing immunofluorescence staining results of human stromal sarcoma Organoid (Organoid) after passage of example 4; wherein, (a) is a DNA immunofluorescence staining result map of a Primary tissue (Primary tissue) sample of human stromal tumor, (B) is an Estrogen Receptor (ER) immunofluorescence staining result map of a Primary tissue (Primary tissue) sample of human stromal tumor for culture of organoids, (C) is a DNA immunofluorescence staining result map of a Primary tissue (Primary tissue) sample of human stromal tumor, (D) is a Progestin Receptor (PR) immunofluorescence staining result map of a Primary tissue (Primary tissue) sample of human stromal tumor for culture of organoids, (E) is a DNA immunofluorescence staining result map of a Primary tissue (Primary tissue) sample of human stromal tumor for culture of organoids, (F) is a middle filament protein (Vimentin) of a Primary tissue (Primary tissue) sample of human stromal tumor for culture of organoids, wherein (a) is a control of (B), (C) is a control of (D), and (E) is a control of (F); (G) A DNA immunofluorescence staining profile for a human stromal sarcoma Organoid (Organoid) sample, (H) an Estrogen Receptor (ER) immunofluorescence staining profile for a human stromal sarcoma Organoid (Organoid) sample, (I) a DNA immunofluorescence staining profile for a human stromal sarcoma Organoid (Organoid) sample, (J) a Progestin Receptor (PR) immunofluorescence staining profile for a human stromal sarcoma Organoid (Organoid) sample, (K) a DNA immunofluorescence staining profile for a human stromal sarcoma Organoid (Organoid) sample, (L) an intermediate silk protein (Vimentin) immunofluorescence staining profile for a human stromal sarcoma Organoid (Organoid) sample, wherein (G) is a control of (H), (I) is a control of (J), and (K) is a control of (L).
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
Terms and definitions
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
In this context, the term "FBS" is an abbreviation for "Fetal Bovine Serum" and is equivalent to "fetal bovine serum".
As used herein, the term "DMEM/F12 minus serum medium" is Advanced DMEM/F12.
In this context, the reagent "green streptomycin diabody" is a penicillin streptomycin mixture of a 100-fold concentration mother liquor. The recommended penicillin working concentration in cell culture is 100U/mL, streptomycin working concentration is 0.1 mg/mL, and penicillin-streptomycin mixed solution of 100 times concentration mother solution is diluted 100 times.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The major reagent manufacturers and numbers used are shown in Table 1, and the reagents not contained in Table 1 and the equipment not specifying the manufacturer are conventional products commercially available.
Table 1 major reagent manufacturers and product numbers
| Reagent(s) | Merchant | Goods number |
| Green Streptomycin double antibody (Penicillin-Streptomycin) | Life Technologies | 15140-122 |
| DMEM/F12 serum-reduced medium (Advanced DMEM/F12) | Life Technologies | 12634-010 |
| 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) | Life Technologies | 15630-056 |
| Nicotinamide (Nicotinamide) | Sigma-Aldrich | N0636 |
| N-acetylcysteine (NAC) | Sigma-Aldrich | A9165 |
| GlutaMaxTM | Life Technologies | 35050-068 |
| FBS（Fetal bovine serum） | FBS four seasons green | 11011-8611 |
| Tissue preservation solution | bioGenous | K601005 |
| Erythrocyte lysate | bioGenous | E238010 |
| B27 additive | bioGenous | S223151 |
Example 1: human carcinomatous sarcoma tissue organoid culture medium
In this example, human carcinomatous tissue organoid medium was formulated with reference to the formulations of tables 2 and 3.
TABLE 2 human carcinomatous sarcoma tissue organoid Medium composition Table
TABLE 3 basal medium composition Table containing trophic factors
| Component (A) | Final concentration |
| DMEM/F12 serum-reduced medium | / |
| 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) | 10 mM |
| Green Streptomycin double antibody (Penicillin-Streptomycin) | 0.5× |
| L-glutamine derivative (GlutaMax) | 5 mM |
Remarks: "0.5X" means that the double antibody to penicillin was diluted 200 times, whereby the final penicillin concentration was 100U/mL and the final streptomycin concentration was 0.1 mg/mL.
Example 2: tissue organoid culture medium for human interstitial sarcoma and leiomyosarcoma
In this example, human interstitial sarcoma/leiomyosarcoma tissue organoid medium was formulated as per the formulations of tables 3, 4.
TABLE 4 human interstitial sarcoma/leiomyosarcoma tissue organoid Medium composition Table
Example 3: human carcinomatosis tissue organoid culture and construction of organoid library
In this example, the human carcinomatous tissue organoid of example 1 was used to perform primary culture of human carcinomatous tissue organoids to obtain human carcinomatous tissue organoids, then subcultured, selected organoids with good growth state, frozen, and stored in liquid nitrogen for a long period of time to construct a human carcinomatous tissue organoid library.
The primary culture operation steps are as follows:
(1) Transferring human carcinoma sarcoma tissue sample in tissue preservation solution to laboratory after 1 XDPBS cleaning and tissue preservation solution soaking, cleaning sarcoma tissue 3 times with 1 XDPBS, and shearing tissue with scissors; the crushed tissue was again washed with 1 XDPBS, and then added with a tissue digestion solution (Table 5), and subjected to enzymolysis in a shaker at 37℃and 100 rpm for 0.5 to 1 hour, to obtain a human carcinomatous sarcoma tissue digestion suspension.
Human carcinoma sarcoma tissue samples were obtained from hospital surgical resection specimens as follows: fresh human sarcoma tissue surgical excision specimens were immersed in tissue preservation solution and transferred from the hospital to the laboratory for organoid culture-related experiments. Before relevant experiments such as specimen sampling, organoid culture and the like are carried out, informed consent of the patient is obtained, and the patient voluntarily participates in the experiment and signs the informed consent.
Table 5 tissue digestate formulation
| Adding reagent | Working concentration |
| Advanced DMEM/F12 | 1× |
| Small molecule compound Y-27632 | 10 μM |
| Collagen hydrolase (Collagenase I) | 400 U/mL |
| Deoxyribonuclease (DNase I) | 10 U/mL |
(2) Blowing the tissue digestion suspension by using a pipetting gun until the tissue is dispersed to obtain fully digested tissue digestion suspension; adding fetal bovine serum with the final concentration of 10% into the digested tissue digestion suspension, allowing the tissue digestion suspension to pass through a 100 μm cell filter screen to obtain single cell suspension, and respectively washing the cells with a basal medium (Advanced DMEM/F12) for 2 times (centrifuging 300 g for 3 min after mixing uniformly, discarding the supernatant); if the cells obtained contain more erythrocytes, the erythrocytes can be lysed by adding a erythrocyte lysate (hypotonic ammonium chloride solution) to the pellet before washing.
(3) Counting a small amount of washed cell suspension, adding matrigel according to the cell density of 100,000-500,000 cells/mL at low temperature and the concentration of 2.5-5 mg/mL, and uniformly mixing on ice to obtain matrigel containing cells; the mixed matrigel containing cells was placed on ice and seeded in 24-well plates (or 10. Mu.L/well in 48-well plates) at 30. Mu.L/well.
(4) The 24-well plate (or 48-well plate) inoculated with the cells was placed in an incubator at 37℃and incubated upside down for 30 minutesCuring the matrigel; after the matrigel was solidified, human carcinomatous tissue organoid medium of example 1 (500. Mu.L/well for 24 well plates and 250. Mu.L/well for 48 well plates) was slowly added along the well walls of the cell culture well plates, and cells were cultured under standard cell culture conditions (cell incubator, 37 ℃,5% CO 2 ) P0 generation (primary) culture was performed. The culture medium is replaced every 3 to 4 days, the culture is continued, the growth condition of human carcinomatous sarcoma tissue organoid is recorded by photographing, and the culture is carried out for about 15 days for passage.
The subculture operation steps are as follows:
(1) Sucking off the original organoid culture medium in a 24-well plate (or a 48-well plate), adding precooled 1 XDPBS according to the amount of 1mL per well, and standing on ice for 5 min; blowing matrigel in the 24 pore plate by using a 1mL gun head to make the matrigel fall off; the shed organoid-containing matrigel was transferred to a 15 mL centrifuge tube, the well plate was washed with pre-chilled 1 XPBS, and the wash solution was collected and pooled into the 15 mL centrifuge tube described above.
(2) Inserting a 15 mL centrifuge tube into ice, standing for 15 min to soften matrigel and obtain organoid suspension; the organoid suspension in the 15 mL centrifuge tube was pipetted 15 times with a 10 mL pipette to separate the organoid from the matrigel; the organoid suspension in 15 mL centrifuge tubes was centrifuged at 250 g at 4℃for 5 min, the supernatant removed and 1 XDPBS pre-chilled in 1mL was added to resuspend the organoid.
(3) Centrifuging at 4deg.C under 250 g for 5 min, removing supernatant, and standing on ice for 5 min; the refluxed liquid (residual 1×dpbs) and matrigel were sucked off; adding new matrigel to re-suspend organoids according to the amount of 8000-10000 cells/10 mu L matrigel, and adding matrigel according to the concentration of 2.5-5 mg/mL; the cells were seeded at 30. Mu.L/well in 24-well plates (or 10. Mu.L/well in 48-well plates).
(4) Placing a 24-well plate (48-well plate) inoculated with cells in a 37 ℃ incubator for inversion incubation for 30 minutes, and solidifying matrigel; after the matrigel was solidified, human carcinomatous tissue organoid medium of example 1 (24 well plate 500. Mu.L/well, 48 well plate 250. Mu.L/well) was slowly added along the well wall of the cell culture well plate, and cells were cultured under standard cell culture conditions (cell incubator，37℃，5% CO 2 ). Changing the culture medium every 3-4 days, culturing for about 6-12 days, and performing freezing preservation; the photo was taken to record the growth of human carcinomatous tissue organoid.
The human carcinomatous organoid growth status of the primary culture for 6 days is shown in FIG. 1.
The growth status of human carcinomatous sarcoid organoid after 5 days of subculture is shown in FIG. 2.
The results show that: (1) The organoid medium of example 1 was used to obtain human carcinomatous sarcoid organoids with intact morphology by primary culture for 6 days. (2) The organoid medium of example 1 was used for subculture for 5 days to obtain the human carcinomatous sarcoma organoid with complete morphology.
Example 4: human interstitial sarcoma, human leiomyosarcoma tissue organoid culture and construction of organoid library
In this example, human interstitial sarcoma, human leiomyosarcoma and osteosarcoma tissue organoids were primary cultured using the organoid medium of example 2 by the method of example 3 to obtain human interstitial sarcoma and human leiomyosarcoma tissue organoids, then subcultured, organoids with good growth state were selected, and after freezing, liquid nitrogen was stored for a long period of time, thereby constructing a human interstitial sarcoma and human leiomyosarcoma tissue organoid library.
Wherein, human interstitial sarcoma, human leiomyosarcoma (primary smooth muscle sarcoma and smooth muscle metastasis sarcoma) tissue samples were obtained from hospital surgical excision specimens in the following manner: fresh human stromal sarcoma, leiomyosarcoma and osteosarcoma tissue surgical resection specimens were immersed in tissue preservation solution and transferred from the hospital to the laboratory for organoid culture-related experiments. Before relevant experiments such as specimen sampling, organoid culture and the like are carried out, informed consent of the patient is obtained, and the patient voluntarily participates in the experiment and signs the informed consent.
The growth status of human interstitial sarcoma organoids after 5 days of primary culture is shown in fig. 3.
The primary human smooth muscle primary sarcoma organoid growth status of primary culture for 5 days is shown in fig. 4.
The growth status of human smooth muscle metastasis sarcoma organoids in primary culture for 8 days is shown in fig. 5.
Example 5: human carcinoma sarcoma, human interstitial sarcoma, human leiomyosarcoma organoid histology and pathological identification
In this example, human carcinoma sarcoma, human interstitial sarcoma, human leiomyosarcoma organoids obtained in examples 3 and 4 were identified from the histological and pathological aspects by means of H & E staining and immunofluorescence staining to examine the consistency of the organoids obtained with the tissue of origin.
Specifically, the passaged organoids were H & E stained as follows:
(1) Organoids were recovered at low temperature and fixed with 4% paraformaldehyde for 30 min at room temperature; washing organoids three times with 1×dpbs, wrapping organoids with 3% low melting agarose, and standing on ice for 30 min until it solidifies; taking out solidified agarose blocks containing organoid precipitate, dehydrating in gradient ethanol for 1 hr, respectively 70%, 80%, 90%, 95% ethanol, and 100% ethanol for 30 min twice, and placing on low-speed horizontal shaker.
(2) The dehydrated agarose blocks were transferred to tissue embedding cassettes (the agarose blocks were immersed in absolute ethanol before xylene treatment), and the tissue embedding cassettes were transferred to xylene for transparentization twice for 5 min each.
(3) Immediately transferring the tissue embedding box after the transparentizing treatment into a melted wax cylinder, immersing the wax for 2 hours at 60 ℃, replacing the wax cylinder, and immersing the tissue embedding box in 2 h; transferring the tissue embedding box after the wax dipping is completed to a melted paraffin preservation box, taking out the die, firstly dripping 20% -30% paraffin, then placing the agarose block in the middle of the metal die, then dripping the paraffin for embedding, and disassembling the tissue embedding box and buckling at the top.
(4) Placing the embedded agarose blocks in a cooling table, taking care of removing the die after complete solidification, and slicing; sequentially placing the slices into xylene for 10 min-xylene (replaced by new xylene) for 10 min-absolute ethanol for 5 min-absolute ethanol (replaced by new absolute ethanol) for 5 min-95% alcohol for 5 min-90% alcohol for 5 min-80% alcohol for 5 min-70% alcohol for 5 min-distilled water washing; putting the washed slices into Harris hematoxylin for dyeing for 3-8 min, washing for 30 sec, differentiating 1% hydrochloric acid alcohol for several seconds, washing for 30 sec with tap water, returning blue with 0.6% ammonia water, and washing for 30 sec with running water; and then putting the slice into eosin dye solution for dyeing for 1-3 min.
(5) Sequentially placing the dyed slices into 95% alcohol (replaced by new 95% alcohol) for 5 min-absolute alcohol (replaced by new absolute alcohol) for 5 min-xylene (replaced by new xylene) for 5 min for dehydration and transparency, taking out the slices from the xylene, slightly airing, and sealing the neutral resin slices; microscopic examination, image acquisition and analysis.
As a control, H & E staining was performed on human carcinomatosis, human stromal sarcoma, and human leiomyosarcoma tissue samples, as described above.
Immunofluorescence staining is carried out on the organoids after passage, and the specific steps are as follows:
(1) Dewaxing the passaged organoid paraffin section in xylene for 5 min, dewaxing by fresh xylene, dewaxing by using xylene for 3 times, dewaxing by using absolute ethyl alcohol for 5 min, twice by 90% ethyl alcohol for 5 min, twice by using 70% ethyl alcohol for 5 min, once by using distilled water for 5 min, twice by using 10 mM citric acid buffer solution for antigen retrieval, heating for 12 min at 95 ℃, and slowly cooling to room temperature within about 30 min.
(2) Adding immunostaining blocking solution (1×PBS containing 5% BSA and 0.25% Triton X-100) into the organoparaffin section after antigen retrieval, blocking for 60 min, sucking off the blocking solution, immediately adding 1% primary antibody diluted with primary antibody diluent (ER, PR, vimentin), and slowly shaking at 4deg.C for incubation overnight; recovering primary antibody, adding immunostaining washing liquid (1 XDPBS contains Tween 20 with final concentration of 0.1%), slowly shaking on a side shaking table for 5 min, adding new washing liquid after sucking the washing liquid, washing for 5 min, and washing for 3 times.
(3) Adding the fluorescent-labeled secondary antibody diluted by the immunofluorescent-stained secondary antibody diluent according to a proper proportion into the washed organoparaffin section, slowly shaking at room temperature in a lateral shaking table in a dark place for incubation for one hour, recovering the secondary antibody, adding the immunostaining washing solution, slowly shaking and washing for 5 min on the lateral shaking table, and adding new washing solution after the washing solution is completely absorbed, washing for 5 min and washing for 3 times.
(4) Adding DAPI-containing anti-fluorescence quencher dropwise into the washed organoid paraffin section, adding a cover glass, performing microscopic examination under a confocal microscope, and performing image acquisition analysis.
As a control, immunofluorescent staining was performed on human carcinomatosis, human stromal sarcoma, and human leiomyosarcoma tissue samples, as described above.
Wherein, primary antibody (0.5%), primary antibody diluent (1 XPBS+5% BSA+0.02% Tween-20), secondary antibody (0.5%), secondary antibody diluent (1 XPBS+0.5% BSA+0.02% Tween-20), the dilution method and working concentration are carried out by referring to the product instruction book.
Exemplary results of H & E staining and immunofluorescence staining are shown in fig. 6, 7.
The results show that: the organoid media of examples 1 and 2 were used to obtain human carcinomatosis, human stromal sarcoma, human leiomyosarcoma organoids, (1) the cell morphology and nuclear morphology were similar to human carcinomatosis, stromal sarcoma, leiomyosarcoma cells; (2) The pathological characteristics of the kit are high in consistency with human-derived carcinoma sarcoma, interstitial sarcoma and leiomyosarcoma tissue samples.
The results show that the human carcinomatosis, human interstitial sarcoma and human leiomyosarcoma organoids obtained by using the organoid culture medium can highly simulate the molecular pathological characteristics of sarcoma tissues, can be used as a sarcoma disease model, and provides a more accurate organoid model for the occurrence, development and treatment of diseases and drug development.
Example 6: human carcinoma sarcoma, human malignant soft tissue sarcoma, endometrial sarcoma, interstitial sarcoma, leiomyosarcoma, osteosarcoma organoid culture
In this example, the inventors used the organoid medium of example 1 or 2, refer to the method of example 3, and perform primary culture of human carcinomatous sarcoma (example 1), human malignant soft tissue sarcoma (example 2), endometrial sarcoma (example 2), interstitial sarcoma (example 2), leiomyosarcoma (example 2), osteosarcoma (example 2) organoids to obtain the corresponding organoids, and subculture of organoids to count the number of organoids formed and the organoid diameter size, unless otherwise specified.
The difference from the method of example 3 is that this example further performs screening optimization on the concentration of matrigel. The specific screening concentrations were as follows: 0 to 2.5 mg/mL, 2.5 to 5 mg/mL, 7.5 to 9.5 mg/mL, 9.5 to 11.5 mg/mL, 11.5 to 13.5 mg/mL.
The examination results are shown in Table 6.
The results show that: when the matrigel concentration is 2.5-5 mg/mL, the growth rate and the clone formation rate of the sarcoma tissue organoid are obviously increased.
TABLE 6 Effect of matrigel at different concentrations on growth of sarcomas tissue organoids
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (11)
1. A sarcoma tissue organoid medium comprising: a basal medium and a specific additive factor, the specific additive factor comprising: nicotinamide, B27 additive, N-acetylcysteine, EGF protein, FGF2 protein, FGF7 protein, and estradiol.
2. The medium of claim 1, wherein the concentration of estradiol is 10-50 ng/mL.
3. The medium of claim 1, wherein the specific addition factor further comprises: fetal bovine serum, R-Spondin protein, and Wnt3a protein;
the concentration of the fetal bovine serum is 5-10% by volume based on the total volume of the organoid medium, the concentration of the R-Spondin protein is 100 ng-500 ng/mL, and the concentration of the Wnt3a protein is 10 ng-50 ng/mL.
4. The medium of claim 1, wherein the concentration of nicotinamide is 1-10 mM, the concentration of B27 additive is 1-3 vol%, the concentration of N-acetylcysteine is 0.5-2.5 mM, the concentration of EGF protein is 10-100 ng/mL, the concentration of FGF2 protein is 10-100 ng/mL, and the concentration of FGF7 protein is 10-100 ng/mL, based on the total volume of the organoid medium.
5. The culture medium of claim 1, wherein the basal medium is DMEM/F12 reduced serum medium;
the concentration of the DMEM/F12 reduced serum medium is 82-99 vol.% based on the total volume of the organoid medium.
6. The culture medium of claim 5, wherein the organoid medium further comprises the following concentrations of components, based on the volume of DMEM/F12 reduced serum medium: 5-15 of mM of hydrogen ion buffer, 20-70U/mL of penicillin and 0.02-0.07 mg/mL of streptomycin, wherein the hydrogen ion buffer is 4-hydroxyethyl piperazine ethane sulfonic acid.
7. The culture medium of claim 1, wherein the basal medium is DMEM/F12 reduced serum medium; the organoid medium comprises the following concentrations of components, based on the total volume of the organoid medium:
99% by volume of DMEM/F12 reduced serum medium, nicotinamide 10 mM, B27 additive 1% by volume, N-acetylcysteine 1.25 mM, EGF protein 50 ng/mL, FGF2 protein 100 ng/mL, FGF7 protein 100 ng/mL, estradiol 25 ng/mL;
the organoid medium further comprises the following concentrations of components, based on the volume of the DMEM/F12 reduced serum medium: 10 mM hydrogen buffer, 50U/mL penicillin, 0.05 mg/mL streptomycin, 4-hydroxyethylpiperazine ethanesulfonic acid.
8. A culture medium according to claim 3, wherein the basal medium is DMEM/F12 reduced serum medium; the organoid medium comprises the following concentrations of components, based on the total volume of the organoid medium:
89% by volume of DMEM/F12 reduced serum medium, 10 mM of nicotinamide, 1% by volume of B27 additive, 1.25. 1.25 mM of N-acetylcysteine, 50 ng/mL of EGF protein, 100 ng/mL of FGF2 protein, 100 ng/mL of FGF7 protein, 25 ng/mL of estradiol, 10% by volume of fetal bovine serum, 500 ng/mL of R-Spondin protein, 50 ng/mL of Wnt3a protein;
the organoid medium further comprises the following concentrations of components, based on the volume of the DMEM/F12 reduced serum medium: 10 mM hydrogen buffer, 50U/mL penicillin, 0.05 mg/mL streptomycin, 4-hydroxyethylpiperazine ethanesulfonic acid.
9. A method of constructing a sarcoma tissue organoid comprising:
culturing sarcoma tissue cells in the culture medium according to any one of claims 1-8 to obtain the sarcoma tissue organoid;
preparing the sarcoma tissue cells into a single cell suspension before culturing;
before culturing, re-suspending the single cell suspension with matrigel to obtain matrigel containing cells, wherein the matrigel concentration is 2.5-5 mg/mL;
in the matrigel containing cells, the cell density was 100,000 ～ 500,000 cells/mL.
10. A method of constructing an animal disease model comprising:
the method of claim 9, wherein the sarcoma tissue organoid is obtained,
and transplanting the sarcoma tissue organoid into the animal body to obtain the animal disease model.
11. A method of constructing a sarcomas tissue organoid library comprising:
the method of claim 9, wherein the sarcoma tissue organoid is obtained,
subculturing the sarcoma tissue organoid to obtain the sarcoma tissue organoid after subculture,
and carrying out freezing treatment on the sarcoma tissue organoids after subculture to obtain the sarcoma tissue organoid library.
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