CN113897336A - Preparation method and application of WRN conditioned medium - Google Patents
Preparation method and application of WRN conditioned medium Download PDFInfo
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Abstract
The invention provides a preparation method of a WRN conditioned medium, which comprises the following steps: respectively constructing expression plasmids of three genes of Wnt3a, R-spondin and Noggin, transfecting the three plasmids into cells, and adding antibiotics for screening to obtain a WRN cell line stably expressing and secreting the Wnt3a, R-spondin and Noggin genes; preparing WRN conditioned medium by using the WRN cell line stably expressing Wnt3a, R-spondin and Noggin protein. The invention also provides application of the WRN conditioned medium. According to the preparation method and the application of the WRN conditioned medium, the WRN conditioned medium enables primary tumor cells to quickly form organoids, enough cells are obtained within an effective time, the culture speed and the success rate of the organoids are improved, the cost generated in the culture process is reduced, the tumor organoid modeling efficiency is improved, and the organoid cell activity is improved.
Description
Technical Field
The invention relates to the technical field of culture medium preparation, in particular to a preparation method and application of a WRN conditioned medium.
Background
Stomach cancer is one of the most common digestive tract malignant tumors seriously threatening human health in China and even worldwide, and has high morbidity and mortality. Statistics of relevance indicate that the mortality rate of gastric cancer is in malignant tumor 2 nd place in whole asia. In recent years, the first successful breeding of 3D organoids (PDOs) derived from patient tumor tissues opens a new era of tumor individualized treatment, and is expected to be a novel platform for screening anticancer drugs and precise tumor therapy. These individualized tumor PDOs, which retain the characteristics and function of tumor cells in patients. PDOs, in the narrow sense, refers to protozoan cytoplasm differentiated to form a three-dimensional structure composed of aggregated cells resembling some organs of higher animals, and the spatial organization structure is similar to that of organs. The PDOs of tumor tissue mentioned herein is an organoid cultured from tissue derived from a tumor patient, a minute tumor grown in a culture dish. Compared with the traditional 2D culture system, the most important characteristic of the technical innovation is that the tissues of the patient can be directly used for culturing the organoids, and meanwhile, the organoids can well replicate some key characteristics of the primary tumor and keep the pathological morphology and the biological mechanism of the tissues of the patient; and can simulate the behavior of the original tumor in the early in vitro experiment, and truly reflect the biological characteristics of the tumor in the aspects of genetic development, invasion, metastasis and the like.
However, the preparation method of the PDOs conditioned medium has high cost in the culture process and low organoid cell viability.
Disclosure of Invention
The invention aims to provide a preparation method and application of a WRN conditioned medium, and aims to solve the problem of low organoid cell viability prepared by the existing preparation method of PDOs conditioned medium.
The invention provides a preparation method of a WRN conditioned medium, which comprises the following steps: respectively constructing expression plasmids of three genes of Wnt3a, R-spondin and Noggin, transfecting the three plasmids into cells, and adding antibiotics for screening to obtain a WRN cell line stably expressing and secreting the Wnt3a, R-spondin and Noggin genes; preparing WRN conditioned medium by using the WRN cell line stably expressing Wnt3a, R-spondin and Noggin protein.
According to the preparation method and the application of the WRN conditioned medium, the WRN conditioned medium enables primary tumor cells to quickly form organoids, enough cells are obtained within an effective time, the culture speed and the success rate of the organoids are improved, the cost generated in the culture process is reduced, the tumor organoid modeling efficiency is improved, and the organoid cell activity is improved.
Further, the method for constructing the expression plasmids of the three genes of Wnt3a, R-spondin and Noggin comprises the following steps:
cloning three genes of Wnt3a, R-spondin and Noggin respectively, and performing gene sequencing and sequence comparison;
carrying out double enzyme digestion on Wnt3a, R-spondin, Noggin gene sequences and vector genes, and inserting the Wnt3a, R-spondin and Noggin gene sequences into a vector to obtain the expression vectors of Wnt3a, R-spondin and Noggin.
Furthermore, the expression vector has a resistance gene sequence of G418 or hygromycin or puromycin, and subsequent experiments can screen positive expression cells of Wnt3a, R-spondin and Noggin by using G418, hygromycin or puromycin.
Further, the positive expression cells include L cells, Hela cells, 293T cells, CHO cells and mammalian cell lines.
Further, the method for preparing WRN conditioned medium by using the WRN cell line stably expressing Wnt3a, R-spondin and Noggin protein comprises the following steps: unfreezing the frozen WRN cell line in a water bath at 37 ℃, immediately transferring the WRN cell line to a preheated DMEM medium containing 10% fetal calf serum after ice is melted, transferring the WRN cell line to a cell culture bottle of 150cm2, and placing the cell culture bottle in a cell culture box for culture; and when the cell density exceeds 90%, replacing a new DMEM medium containing 10% fetal calf serum, starting to harvest culture supernatants after culturing for 24 hours, continuously harvesting for three days, and combining the harvested culture supernatants to obtain the WRN conditioned medium.
The invention also provides an application of the WRN conditioned medium, wherein the WRN conditioned medium obtained by any one of the above is used for inducing and culturing tumor cells derived from a patient into a tumor organoid for subsequent tumor drug sensitivity screening or tumor pathogenesis research.
Further, the tumor cell derived from the patient includes any one selected from the group consisting of lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancy, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, cancer of the anal region, cancer of the testis, cancer of the fallopian tube, cancer of the endometrium, cancer of the vagina, hodgkin's disease, non-hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, or cancer of the urethra.
Further, the method for inducing and culturing tumor cells derived from a patient into a neoplastic tumor organoid by using the WRN conditioned medium comprises: sampling: obtaining a clinical tumor tissue specimen, carrying out primary treatment and preservation on the tumor tissue specimen, then carrying out refrigeration at 4 ℃ and transporting to a tissue cell culture laboratory; enzymolysis separation: washing a tumor tissue specimen by adopting a 10mL DMEM medium, shearing the tumor tissue specimen, adding 2mL of digestive juice containing pancreatin, II-type collagenase and DNA enzyme, placing the digestive juice on a rotary oscillator for digesting for 2 hours at a constant temperature of 37 ℃, and then centrifuging and removing supernatant to obtain cells to be cultured; cell inoculation: freezing and thawing matrix gel (Matrigel) and uniformly mixing the matrix gel into a homogenate shape, gently mixing cells to be cultured and the Matrigel matrix in a 1.5ml centrifuge tube to enable the cells to be cultured to be suspended in the Matrigel matrix, transferring the cell mixture into a precooled cell culture plate, then placing the cell mixture in an incubator at 37 ℃ for 30min, and gelling and solidifying the matrix into a jelly shape; organoid culture: adding WRN conditioned medium into the cell culture plate, placing the cell culture plate in a constant temperature and humidity incubator for static culture, replacing the culture medium every 3-4 days, carrying out passage every 1-2 weeks, and freezing and storing the cells when the cells reach a certain amount.
Drawings
FIG. 1 is a flowchart of a method for preparing a WRN conditioned medium according to a first embodiment of the present invention;
FIG. 2 is an enlarged structural view of organoids of a tumor cultured in PDO medium;
FIG. 3 is an enlarged schematic structural view of organoids of tumors cultured in WRN conditioned medium;
FIG. 4 is a flow chart of a procedure for conditioning a medium for WRN according to a second embodiment of the present invention.
The following detailed description will further illustrate the invention in conjunction with the above-described figures.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Several embodiments of the invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only.
Unless defined otherwise, all 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Referring to FIG. 1, the present invention provides a method for preparing a WRN conditioned medium, including steps S01 through S02.
Step S01, expression plasmids of three genes of Wnt3a, R-spondin and Noggin are respectively constructed, the three plasmids are transfected into cells, antibiotics are added for screening, and WRN cell lines which stably express and secrete the Wnt3a, the R-spondin and the Noggin genes are obtained.
Step S02, preparing WRN conditioned medium using the WRN cell line stably expressing Wnt3a, R-spondin, Noggin-secreting proteins. Wherein Wnt3a belongs to Wntl protein, the function of Wnt in cell is realized by regulating the function of each component protein of Wnt classical signal channel, R-spondin has the function of enhancing Wnt/beta-catenin signal, and Noggin protein is an inhibitor secreted by body of bone morphogenetic genetic protein (BMP) playing a key role in development.
Specifically, in this embodiment, the method for constructing expression plasmids of three genes, Wnt3a, R-spondin and Noggin, includes: cloning three genes of Wnt3a, R-spondin and Noggin respectively, and performing gene sequencing and sequence comparison; carrying out double enzyme digestion on Wnt3a, R-spondin, Noggin gene sequences and vector genes, and inserting the Wnt3a, R-spondin and Noggin gene sequences into a vector to obtain the expression vectors of Wnt3a, R-spondin and Noggin.
Specifically, in the embodiment, the expression vector has a resistance gene sequence of G418 or hygromycin or puromycin, and subsequent experiments can screen positive expression cells of Wnt3a, R-spondin and Noggin by using G418, hygromycin or puromycin.
Specifically, in this embodiment, the positive expression cells can be L cells, Hela cells, 293T cells, CHO cells, and mammalian cell lines.
Specifically, in this embodiment, the method for preparing WRN conditioned medium using the WRN cell line stably expressing Wnt3a, R-spondin, Noggin protein secretion comprises: unfreezing the frozen WRN cell line in a water bath at 37 ℃, immediately transferring the WRN cell line to a preheated DMEM medium containing 10% fetal calf serum after ice is melted, transferring the WRN cell line to a cell culture bottle of 150cm2, and placing the cell culture bottle in a cell culture box for culture; and when the cell density exceeds 90%, replacing a new DMEM medium containing 10% fetal calf serum, starting to harvest culture supernatants after culturing for 24 hours, continuously harvesting for three days, and combining the harvested culture supernatants to obtain the WRN conditioned medium.
Referring to fig. 4, a WRN conditioned medium according to a second embodiment of the present invention is used to induce and culture patient-derived tumor cells into a tumor organoid for subsequent tumor drug sensitivity screening or tumor pathogenesis research.
Specifically, in this embodiment, the tumor cells derived from the patient include any one selected from the group consisting of cells of lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, cancer of the anal region, cancer of the testis, cancer of the fallopian tube, cancer of the endometrium, cancer of the vagina, hodgkin's disease, non-hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, or cancer of the urethra.
Specifically, in this embodiment, the method for inducing and culturing tumor cells derived from a patient into a neoplastic tumor organoid by using the WRN-conditioned medium comprises: sampling: obtaining a clinical tumor tissue specimen, carrying out primary treatment and preservation on the tumor tissue specimen, then carrying out refrigeration at 4 ℃ and transporting to a tissue cell culture laboratory; enzymolysis separation: washing a tumor tissue specimen by adopting a 10mL DMEM medium, shearing the tumor tissue specimen, adding 2mL of digestive juice containing pancreatin, II-type collagenase and DNA enzyme, placing the digestive juice on a rotary oscillator for digesting for 2 hours at a constant temperature of 37 ℃, and then centrifuging and removing supernatant to obtain cells to be cultured; cell inoculation: freezing and thawing matrix gel (Matrigel) and uniformly mixing the matrix gel into a homogenate shape, gently mixing cells to be cultured and the Matrigel matrix in a 1.5ml centrifuge tube to enable the cells to be cultured to be suspended in the Matrigel matrix, transferring the cell mixture into a precooled cell culture plate, then placing the cell mixture in an incubator at 37 ℃ for 30min, and gelling and solidifying the matrix into a jelly shape; organoid culture: adding WRN conditioned medium into the cell culture plate, placing the cell culture plate in a constant temperature and humidity incubator for static culture, replacing the culture medium every 3-4 days, carrying out passage every 1-2 weeks, and freezing and storing the cells when the cells reach a certain amount.
Referring to fig. 2 and fig. 3, it can be seen by comparison that, in the preparation method and application of the WRN conditioned medium, the WRN conditioned medium enables primary tumor cells to rapidly form organoids, and sufficient cells are obtained within an effective time, so that the organoids culture speed and success rate are improved, the cost generated in the culture process is reduced, the tumor organoids modeling efficiency is improved, and the organoids cell viability is improved.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A method for preparing a WRN conditioned medium, comprising:
respectively constructing expression plasmids of three genes of Wnt3a, R-spondin and Noggin, transfecting the three plasmids into cells, and adding antibiotics for screening to obtain a WRN cell line stably expressing and secreting the Wnt3a, R-spondin and Noggin genes;
preparing WRN conditioned medium by using the WRN cell line stably expressing Wnt3a, R-spondin and Noggin protein.
2. The method for preparing WRN conditioned medium according to claim 1, wherein the method for constructing expression plasmids for three genes Wnt3a, R-spondin and Noggin comprises:
cloning three genes of Wnt3a, R-spondin and Noggin respectively, and performing gene sequencing and sequence comparison;
carrying out double enzyme digestion on Wnt3a, R-spondin, Noggin gene sequences and vector genes, and inserting the Wnt3a, R-spondin and Noggin gene sequences into a vector to obtain the expression vectors of Wnt3a, R-spondin and Noggin.
3. The method for preparing WRN conditioned medium according to claim 2, wherein the expression vector has the resistance gene sequence of G418 or hygromycin or puromycin, and subsequent experiments can screen positive expression cells of Wnt3a, R-spondin and Noggin using G418, hygromycin or puromycin.
4. A method of making a WRN conditioned medium according to claim 3, wherein said positively expressing cells comprise L cells, Hela cells, 293T cells, CHO cells, and mammalian cell lines.
5. The method for preparing conditioned medium according to claim 1, wherein said method for preparing WRN conditioned medium using said WRN cell line stably expressing Wnt3a, R-spondin, and Noggin-secreting proteins comprises: unfreezing the frozen WRN cell line in a water bath at 37 ℃, immediately transferring the WRN cell line to a preheated DMEM medium containing 10% fetal calf serum after ice is melted, transferring the WRN cell line to a cell culture bottle of 150cm2, and placing the cell culture bottle in a cell culture box for culture; and when the cell density exceeds 90%, replacing a new DMEM medium containing 10% fetal calf serum, starting to harvest culture supernatants after culturing for 24 hours, continuously harvesting for three days, and combining the harvested culture supernatants to obtain the WRN conditioned medium.
6. Use of WRN conditioned medium obtained according to any of claims 1-5 for the induced culture of patient-derived tumor cells into neoplastic tumor organoids for subsequent tumor drug sensitivity screening or tumor pathogenesis studies.
7. Use of WRN conditioned medium according to claim 6, wherein the patient derived tumor cells comprise any one of cells from lung, ovarian, colon, rectal, melanoma, kidney, bladder, breast, liver, lymphoma, hematologic malignancies, head and neck, glioma, stomach, nasopharyngeal, laryngeal, cervical, uterine body, osteosarcoma, bone, pancreatic, skin, prostate, cutaneous or intraocular malignant melanoma, uterine, anal region, testicular, fallopian tube, endometrial, vaginal, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal, small intestine, endocrine, thyroid, parathyroid, adrenal, soft tissue sarcoma or urethral cancer.
8. Use of WRN conditioned medium according to claim 6, in a method for inducing culture of patient-derived tumor cells into neoplastic tumor organoids comprising:
sampling: obtaining a clinical tumor tissue specimen, carrying out primary treatment and preservation on the tumor tissue specimen, then carrying out refrigeration at 4 ℃ and transporting to a tissue cell culture laboratory;
enzymolysis separation: washing a tumor tissue specimen by adopting a 10mL DMEM medium, shearing the tumor tissue specimen, adding 2mL of digestive juice containing pancreatin, II-type collagenase and DNA enzyme, placing the digestive juice on a rotary oscillator for digesting for 2 hours at a constant temperature of 37 ℃, and then centrifuging and removing supernatant to obtain cells to be cultured;
cell inoculation: freezing and thawing matrix gel (Matrigel) and uniformly mixing the matrix gel into a homogenate shape, gently mixing cells to be cultured and the Matrigel matrix in a 1.5ml centrifuge tube to enable the cells to be cultured to be suspended in the Matrigel matrix, transferring the cell mixture into a precooled cell culture plate, then placing the cell mixture in an incubator at 37 ℃ for 30min, and gelling and solidifying the matrix into a jelly shape;
organoid culture: adding WRN conditioned medium into the cell culture plate, placing the cell culture plate in a constant temperature and humidity incubator for static culture, replacing the culture medium every 3-4 days, carrying out passage every 1-2 weeks, and freezing and storing the cells when the cells reach a certain amount.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851577A (en) * | 2022-12-23 | 2023-03-28 | 北京市创伤骨科研究所 | Method for constructing osteosarcoma organoid model based on single cell |
| CN116836934A (en) * | 2023-08-31 | 2023-10-03 | 北京大橡科技有限公司 | Osteosarcoma organoid culture solution, culture reagent combination and culture method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019021990A1 (en) * | 2017-07-28 | 2019-01-31 | 国立大学法人大阪大学 | Enterocyte-like cells |
| JP2019103399A (en) * | 2017-12-08 | 2019-06-27 | 京ダイアグノスティクス株式会社 | Method of producing cancer spheroid |
| CN110408595A (en) * | 2019-08-23 | 2019-11-05 | 厦门博创盛世生物技术有限公司 | A kind of cultural method of the tumour 3D organoid for gastric cancer test |
-
2020
- 2020-06-22 CN CN202010576001.6A patent/CN113897336A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019021990A1 (en) * | 2017-07-28 | 2019-01-31 | 国立大学法人大阪大学 | Enterocyte-like cells |
| JP2019103399A (en) * | 2017-12-08 | 2019-06-27 | 京ダイアグノスティクス株式会社 | Method of producing cancer spheroid |
| CN110408595A (en) * | 2019-08-23 | 2019-11-05 | 厦门博创盛世生物技术有限公司 | A kind of cultural method of the tumour 3D organoid for gastric cancer test |
Non-Patent Citations (4)
| Title |
|---|
| ELOÏSE MUSSARD ET AL.: "Reconstitution of intestinal stem cell niche in vitro with pharmacological inhibitors or L-WRN conditioned medium differentially regulates epithelial proliferation, differentiation and barrier function in rabbit caecum organoids", 《BIORXIV》, pages 6 * |
| HIROYUKI MIYOSHI ET AL.: "In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture", 《NATURE PROTOCOLS》, vol. 8, no. 12, pages 2472 * |
| 宋东娟 等: "小肠类器官培养技术的建立和优化", 《胃肠病学》, vol. 21, no. 2, pages 76 * |
| 宋东娟;童锦禄;冉志华;郑青;: "小肠类器官培养技术的建立和优化", 胃肠病学, no. 02 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851577A (en) * | 2022-12-23 | 2023-03-28 | 北京市创伤骨科研究所 | Method for constructing osteosarcoma organoid model based on single cell |
| CN116836934A (en) * | 2023-08-31 | 2023-10-03 | 北京大橡科技有限公司 | Osteosarcoma organoid culture solution, culture reagent combination and culture method |
| CN116836934B (en) * | 2023-08-31 | 2023-11-24 | 北京大橡科技有限公司 | Osteosarcoma organoid culture solution, culture reagent combination and culture method |
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