Arenal gland organ culture medium, culture method of adrenal gland organ and construction method of primary adrenal cortical carcinoma animal model

Abstract

The invention relates to an adrenal gland organ culture medium, a culture method of an adrenal gland organ and a construction method of a primary adrenal cortical carcinoma animal model, and belongs to the technical field of biology. The invention provides an adrenal gland organ culture medium, which is prepared by taking a DMEM (Dulbecco's Modified Eagle Medium) / F12 culture medium as a basic culture medium and adding the following components: 40x-60 B27, 1-1.5 mM of N-acetylcysteine, 40-60 ng / ml of EGF (Epidermal Growth Factor), 80-120 ng / ml of Noggin, 8-12% of R-ponding1, 8-12 [mu] M of Y27632, 160-240 nM of A83-01 and 2.4-3.6 nM of ACTH. The construction period of the tumor model is greatly shortened compared with that of a genetic engineering animal model, animal death before tumor formation cannot be caused, and the tumor formation rate reaches up to 100%. The primary immune sound mouse adrenal cortical carcinoma tumor model constructed by the invention can simulate the process of transforming normal cells into tumor cells due to genetic alteration in a human body, and can dynamically represent the tumor generation and development process; the gene level, tumor microenvironment, tumor development, physiology and pathology and other aspects are closer to the real situation of tumor occurrence and development.

Description

Technical Field

[0001] The invention relates to an adrenal organoid culture medium, an adrenal organoid culture method, and a method for constructing a primary adrenal cortical carcinoma animal model, and belongs to the field of biotechnology. Background Art

[0002] Adrenocortical carcinoma is a rare but highly aggressive adrenal tumor that usually originates from the adrenal cortex. In the past few decades, surgery and mitotane plus platinum have been the main treatments for patients with adrenocortical carcinoma, and there has been no breakthrough in the clinical treatment of adrenocortical carcinoma. It is particularly urgent to deeply understand the pathogenesis of adrenocortical carcinoma, find the key factors involved, and then develop more effective treatments. Tumor animal models play a vital role in exploring the mechanisms of tumor occurrence and development and developing new drugs. The mouse adrenocortical carcinoma model provides a powerful tool for studying the mechanisms of adrenocortical carcinoma occurrence and metastasis, anti-tumor drug screening, and immunotherapy evaluation.

[0003] At present, the commonly used mouse adrenocortical carcinoma models mainly include transplant tumor models and spontaneous tumor models. The transplant tumor model uses immunodeficient mice (such as athymic nude mice, SCID mice) as transplant recipients, and tumor cells (cell lines) or tumor tissue blocks are transplanted into immunodeficient mice to form transplant tumors. Spontaneous tumor model mice mainly include genetically engineered mouse adrenocortical carcinoma models. The reported genetically engineered mouse adrenocortical carcinoma models mainly include TP53-deficient and b-catenin-overexpressing mice. This type of mouse uses homologous recombination of mouse embryonic stem cells to knock out or knock in specific genes in the mouse genome.

[0004] However, the current mouse adrenocortical carcinoma model has many shortcomings and is insufficient in meeting the needs of basic and preclinical research. The mouse transplant tumor model has been controversial because the transplant tumor is not a tumor formed spontaneously in the mouse and cannot reflect the true process of tumor formation in the body. More importantly, the body's immune system is very important in the process of tumor formation, and immunodeficient mice lack a complete immune system. The use of the transplant tumor model can no longer meet the needs of tumor immunology treatment research. The spontaneous tumor model based on genetically engineered mice has been recognized because it is similar to the tumor occurrence process in the human body. However, the occurrence of tumors is a process of cumulative changes in multiple genes. Traditional genetically engineered mice only introduce one or two gene changes at a time. If you want to obtain multiple gene mutation mice, you need to mate and iterate multiple times, which is time-consuming. If you want to obtain a large number of tumor mice with the same genotype at the same time, you need more parent mice, which increases the research cost. At the same time, because genetically engineered mice are germline mutations, they are prone to induce tumors in multiple systems and organs, which is different from the actual occurrence of tumors. In addition, due to individual differences in mice, the cycle of spontaneous tumor formation is also quite different, making it difficult to achieve synchronization. More importantly, the technical threshold for constructing genetically engineered mice is high. Constructing a new genotype mouse model takes a long time and is costly, so general research institutions cannot carry it out routinely. Summary of the invention

[0005] The purpose of the present invention is to provide a primary adrenocortical carcinoma model of immune-competent mice that is closer to the biological characteristics of adrenocortical carcinoma and has a short preparation period.

[0006] The present invention provides an adrenal organoid culture medium, which is based on DMEM / F12 culture medium and is composed of the following components: 40x-60 B27, N-acetylcysteine ​​1-1.5mM, EGF 40-60ng / ml, Noggin 80-120ng / ml, R-sponding1 8-12%, Y27632 8-12uM, A83-01 160-240nM, ACTH 2.4-3.6nM.

[0007] Preferably, it is based on DMEM / F12 medium, plus the following components: 50x B27, N-acetylcysteine ​​1.25mM, EGF 50ng / ml, Noggin100ng / ml, R-sponding1 10%, Y27632 10uM, A83-01 200nM, ACTH 3nM.

[0008] The present invention provides a method for culturing adrenal organoids, comprising the following steps: a. Take the animal adrenal gland, wash it, and cut it into 1-2 mm pieces; b. Digest the tissue using type I collagenase and type IV collagenase at 37 degrees Celsius, using a pipette; c. Pipette once every 10 minutes until the tissue is digested into single cells; d. Filter the undigested tissue to obtain the cell suspension, centrifuge, and discard the supernatant; e. Take the precipitate and resuspend it in matrix gel. After the matrix gel solidifies, add the culture medium described in claim 1 or 2 to culture to obtain organoids.

[0009] The culture conditions in step e are as follows: culture in a cell culture incubator at 37°C and 5% CO2, replace the culture medium every 2-3 days, and perform subculture every 7-8 days.

[0010] The present invention provides a method for constructing a primary adrenocortical carcinoma animal model, which comprises the following steps: a. Injecting the cultured adrenal organoids subcutaneously into the animal; b. Performing genetic modification on animals, wherein the genetic modification is to knock out tumor suppressor genes and / or overexpress oncogenes.

[0011] Among them, the genetic modification method is to knock out TP53, CDKN2A, ZNRF3, and NF1 genes, and overexpress the MYC gene.

[0012] The genetic modification includes transferring fluorescent marker genes into the organoids.

[0013] The present invention provides use of the adrenal organoid culture medium in preparing adrenal organoids.

[0014] The present invention provides use of the primary adrenocortical carcinoma animal model constructed by the construction method in drug screening, drug toxicity test or immunotherapy test.

[0015] The adrenal organoids constructed by the present invention are digested into single cells from mature adrenal organs of mice, and grown and differentiated by adding specific culture media. The main source is adult stem cells, and the main research purpose is to construct a mouse primary tumor model. There is an essential difference in the source from the organoids induced from pluripotent stem cells. The organoids induced from pluripotent stem cells are mainly used to study specific organ functions and simulate organ development processes (see literature: Guo Yajie, Establishment and Identification of Organoid Culture System from Human Embryonic Adrenal Cells, Master's Thesis of Guangxi Medical University, May 2022). The present invention focuses on the construction of mouse organoids and primary tumor mouse models after gene editing. The adrenocortical carcinoma mouse model based on mouse adrenal organoids in the present invention has similar pathological and biological characteristics to those of adrenocortical carcinoma patients, a high tumor formation rate, and a shorter tumor formation time than that of genetic mice. The tumor tissue after tumor formation can then be cultured as an organoid, and at the same time provides a tumor research approach in mice in vivo and in vitro, which is more conducive to immunological research, gene therapy, drug screening and other experiments of adrenal tumors.

[0016] The tissue source of the present invention is the adrenal gland of mature, immune-complete mice. This type of organoid derived from adult stem cells can more specifically simulate the true physiological and pathological state of the organ, laying the foundation for the cultivation of specific organoids in patients with adrenocortical carcinoma in the future. Secondly, compared with the adrenal organoids cultivated from induced pluripotent stem cells, the adrenal organoids cultured by the present invention have a fast growth rate and simple technology, save the technical requirements for differentiation and cultivation, reduce the complexity of operation, and have better practicality and repeatability. Genetic mutations may occur in pluripotent stem cells during the differentiation process, especially in long-term in vitro culture. The tissue-derived organoids in this patent are generally more stable, with a lower risk of gene mutations, and are more helpful in maintaining the phenotype and functional characteristics of the original tissue, and are more direct and efficient in gene editing tumorigenesis and personalized medicine for patients.

[0017] Beneficial effects of the present invention: The tumor model construction cycle of the present invention is greatly shortened compared with the genetic engineering animal model, and will not cause the death of animals before tumor formation, and the tumor formation rate is as high as 100%. The primary mouse adrenocortical carcinoma tumor model of immune-competent mice constructed by the present invention can simulate the process of normal cells transforming into tumor cells due to genetic changes in the human body, and can dynamically characterize the tumor occurrence and development process, and is closer to the actual situation of tumor occurrence and development in terms of gene level, tumor microenvironment, tumor development and physiological pathology.

[0018] In summary, the method of the present invention can efficiently prepare an adrenocortical carcinoma model that is closer to adrenocortical carcinoma and meets the needs of clinical research; this model can provide a useful tool in research fields such as exploring the occurrence and development mechanism of adrenal carcinoma, finding and optimizing new possible treatment methods for adrenocortical carcinoma, etc.

[0019] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0020] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a gross image of the mouse adrenal gland and a picture of the cultured adrenal organoids; Figure 2 HE image of adrenal organoid; Figure 3 This is an immunohistochemical staining of adrenal organoid steroidogenic factor 1; Figure 4 White light and fluorescence images of gene-edited adrenal organoids; Figure 5 This is a picture of T7 endonuclease detection in gene-edited adrenal organoids; Figure 6 These are white light and fluorescence images of subcutaneous adrenal tumors in mice; Figure 7 This is a picture of mouse subcutaneous adrenal tumor organoids; Figure 8 HE image of subcutaneous adrenal tumor in mice; Fig. 9 This is an immunofluorescence staining image of steroidogenic factor 1 (green) in a mouse subcutaneous adrenal tumor (where blue represents the cell nucleus); Fig.10 This is an immunofluorescence staining image of the steroid hormone production enzyme-related gene CYP11A1 (cytochrome P450 family 11A1) in subcutaneous adrenal tumors of mice (green), and blue is the cell nucleus; Fig.11 A diagram showing the growth status of adrenal organoids under culture conditions with different ACTH concentrations. DETAILED DESCRIPTION

[0022] Some of the English abbreviations in the present invention are explained as follows: DMEM: It is a widely used culture medium that can be used for many mammalian cell cultures. It was purchased from GIBCO. DMEM / F12: F12 medium and DMEM medium are combined in a 1:1 ratio, called DMEM / F12 medium. It combines the advantages of F12 containing richer ingredients and DMEM containing higher concentrations of nutrients. Purchased from GIBCO.

[0023] Matrigel is isolated from EHS mouse tumors rich in extracellular matrix proteins. Its main components are laminin, type IV collagen, nestin, heparin sulfate glycoprotein, etc. It also contains growth factors and matrix metalloproteinases, etc. It was purchased from Corning.

[0024] B27, i.e. B27 supplement, is a commercially available product that can be used to prepare culture media. B27 supplement is provided as a 50-fold concentrated liquid (50-fold dilution when used) and contains, among other ingredients, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin. It was purchased from Life Technologies.

[0025] N-acetylcysteine: N-acetylcysteine ​​was purchased from Sigma.

[0026] EGF, epidermal growth factor, a commercially available product, was purchased from R&D Company.

[0027] Noggin, a cell growth protein component, is a commercially available product purchased from Peprotech.

[0028] R-spondin1, a human cell growth encoding protein, is a commercially available product purchased from Peprotech.

[0029] A83-01, a TGF-B inhibitor, was purchased from Tocris Bioscience.

[0030] Y-27632, a ROCK specific pathway blocker, was purchased from Abmole Bioscience.

[0031] ACTH, adrenocorticotropic hormone, was purchased from MCE.

[0032] TrypLE, a recombinant digestion enzyme used to dissociate adherent mammalian cells, was purchased from GIBCO.

[0033] Example 1 Preparation of adrenal organoid culture medium of the present invention The culture medium of the present invention is obtained by adding the following additives to the DMEM / F12 culture medium: Element content Element content B27 50x R-sponding 1 10% N-acetylcysteine 1.25mM Y27632 10uM EGF 50ng / ml A83-01 200nM Noggin 100ng / ml ACTH 3nM Example 2 Construction and verification of the mouse adrenocortical carcinoma model of the present invention 1. Use organoid culture technology to culture adrenal organoids in vitro, such as Figure 1-3 shown.

[0034] (1) Take the adrenal gland of C57BL / 6 mice, wash it with pre-cooled DPBS solution, cut it into 1-2 mm tissue pieces, and digest it with type I collagenase and type IV collagenase in a shaking incubator at 37 degrees Celsius for 20 minutes, pipetting 20-30 times with a 10 ml pipette; (2) After that, pipette and perfuse every 10 minutes until the tissue is digested into single cells; (3) Filter the digested tissue suspension with a 70 μm filter to obtain a cell suspension, centrifuge, and discard the supernatant; (4) mixing the obtained adrenal cells with Matrigel; (5) After Matrigel solidifies, add the culture medium to obtain organoids, and culture them in a cell culture incubator at 37°C and 5% CO2. Replace the culture medium every 2-3 days and perform subculturing every 7-8 days.

[0035] 2. Viral vector construction, packaging and in vitro validation Gene overexpression technology is used to overexpress oncogenes, and CRISPR / Cas9 gene knockout technology is used to knock out tumor suppressor genes, as follows: (1) Starting from the pil-Luci vector, the target oncogene is constructed into the pil-Luci vector through conventional enzyme digestion and enzyme ligation operations. The vector carries the luciferase reporter gene Luciferase; (2) Starting from the V2TC empty plasmid, the sgRNA of the target tumor suppressor gene was introduced through conventional enzyme digestion and enzyme ligation operations. The Cas9 protein was derived from the LentiCRISPRv2 plasmid; (3) The above-mentioned target oncogene is C-MYC; (4) The above target tumor suppressor genes are TP53, CDKN2A, NF1, and ZNRF3; (5) The constructed viral vector and packaging plasmid were co-transfected into 293T cells, and the DMEM / F12 basal medium (without FBS) was replaced at 10h and 24h, and the viral supernatant was collected at 36h, 48h and 72h, respectively. After filtering at 0.45 to remove cell debris, the viral liquid was used to infect the target cells in vitro, and DNA was extracted. The mutation of the target tumor suppressor gene was identified by direct sequencing or T7E1 or enzyme digestion. The gRNA with the best mutation effect was selected for subsequent experiments; (6) Mouse adrenal organoid infection and gene knockout verification: cultured mouse adrenal organoids were digested with TrypLE to single cells, resuspended with 0.5 ml of fresh virus solution, and the transfection reagent Polybrene (final working concentration 2 ug / ml) was added. The cells were placed in a 24-well plate, centrifuged at 2000 rpm for 60 min at 31 degrees Celsius, and incubated in a 37 incubator for 4 h. The virus solution was diluted with 10 ml of DPBS solution, centrifuged at 300 g for 10 min, the supernatant was removed, the cells were resuspended with Matrigel, inoculated in a 48-well plate, adrenal organoid culture medium was added, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2. DNA was extracted from adrenal organoids cultured for 5-7 days, and the mutation of the target tumor suppressor gene was identified by direct sequencing or T7E1 or restriction digestion; (7) Organoids successfully transfected with viruses Figure 3 As shown, the upper picture is non-fluorescence observation, and the lower picture is fluorescence observation; red fluorescence can be seen from the right picture, indicating that the virus was successfully transfected; (8) Subcutaneous organoid transplantation: Gene-edited adrenal organoids were taken 5-7 days after passage, prepared into adrenal organoid DPBS suspension, and counted under a microscope; (9) 6-8 week old athymic nude mice were selected as transplant recipient mice. The armpits on both sides of the shoulder blades were shaved and disinfected with povidone-iodine. The adrenal organoid suspension was injected subcutaneously into the armpits of the mice with an insulin needle to allow the edited adrenal organoids to undergo malignant transformation in vivo. (10) Monitoring and pathological identification of mice: After transplantation, the condition of the mice was observed in real time, and the growth of subcutaneous tumors was observed. Masses began to appear under the skin of the mice about 2 months after injection; (11) Take part of the adrenal tumor tissue from nude mice to continue culturing adrenal tumor organoids, and then transplant these tumor organoids into normal immune C57B / L6 mice; (12) Monitoring and pathological identification of mice: After transplantation, the status of mice was observed in real time, and the growth of subcutaneous tumors was observed. About 20 days after injection, masses began to appear under the skin of mice. Part of the subcutaneous adrenal tumor tissue was cryopreserved in liquid nitrogen, and part of the adrenal tumor tissue was taken to continue culturing adrenal tumor organoids. Part of the adrenal tumor tissue was fixed, dehydrated, embedded, sliced, and HE stained according to conventional pathological detection methods to detect the pathological characteristics of the tissue. It was determined that the adrenal cancer model formed by transplanting edited adrenal organoids was adrenocortical carcinoma.

[0036] The beneficial effects of the present invention are further described below in the form of experimental examples.

[0037] Experimental Example 1 Construction of a primary mouse adrenal cancer model with TP53 / CDKN2A / NF1 / ZNRF knockout 1. Methods: Adrenal tissues of TP53- / - cas9 C57BL / 6 mice were taken and organoid culture was performed in sequence using the method of Example 1 (such as Figure 1 As shown), passage, genetic modification, and gene knockout verification, and the genetically modified organoids were transplanted subcutaneously into athymic nude mice to observe the growth of subcutaneous tumors in mice. After 70 days of transplantation, the mice were sacrificed, and the tumor tissues were transplanted subcutaneously into immune-competent C57BL / 6 mice for a second time to observe the growth of subcutaneous tumors. After 25 days of transplantation, the mice were sacrificed, and the subcutaneous tumor tissues were taken and observed by HE staining and immunohistochemistry staining.

[0038] The genetic modification utilizes CRISPR / Cas9 gene knockout technology to knock out tumor suppressor genes NF1, ZNRF3, and CDKN2A.

[0039] 2. Results 2.1 The above method was used to culture adrenal organoids, and HE staining was performed. Immunohistochemical staining showed that steroid factor 1 was positive ( Figure 1 , 2 、3) 2.2 Gene knockout verification Adrenal organoids were infected with sgRNA (NF1, ZNRF3, CDKN2A) using lentiviral packaging technology. After infection, cells and fluorescence showed successful infection ( Figure 4 ) T7 endonuclease was used to detect the mutations of NF1, ZNRF3, and CDKN2A in genetically modified organoids. Figure 5 As shown, NF1, ZNRF3, and CDKN2A were successfully knocked out.

[0040] 2.2 Observation of the morphology of subcutaneous adrenal tumors in mice Figure 6 shown.

[0041] 2.3 Mouse adrenal tumors can continue to be cultured into organoids after digestion and can be stably passaged and expanded. Figure 7 shown.

[0042] 2.4HE, IF staining HE staining of mouse tumor sections shows that cancer cells show different degrees of atypia, with different sizes and shapes, and pathological nuclear division images can be seen ( Figure 8 ), while IF staining showed that steroidogenic factor 1 and steroidogenic enzyme-related gene CYP11A1 were positive ( Fig. 9 , Fig.10). The adrenal cancer model obtained in this experimental example belongs to the adrenal cancer model. All five athymic nude mice successfully developed adrenocortical cancer within 80 days, and all five C57BL / 6 mice successfully developed adrenocortical cancer within 30 days after secondary transplantation.

[0043] Experimental Example 3 Relationship between the amount of ACTH in the culture medium of the present invention and organoid culture 1. Take the adrenal glands of C57B / L6 mice, digest them according to the method of Experimental Example 2, and evenly seed them into 4 wells of organoids; 2. The basal culture medium is 50x B27, N-acetylcysteine ​​1.25mM, EGF 50ng / ml, Noggin100ng / ml, R-sponding1 10%, Y27632 10uM, A83-01 200nM. On this basis, ACTH is added or not, and the added ACTH is set to 3 concentrations: 3nM, 30nM, and 300nM; 3. After adding the above four culture media, culture in a cell culture incubator at 37°C and 5% CO2, and take pictures under a microscope every 24 hours to observe the formation of adrenal organoids.

[0044] like Fig.11 As shown in the figure, under the condition of culture without ACTH, the digested adrenal cells could not form organoid morphology until 48 hours, indicating that ACTH is crucial for whether primary adrenal cells can form organoids. At 24 hours, the adrenal organoids cultured under the conditions of 3nM and 300nM ACTH concentrations were similar in size, while the adrenal organoids cultured under the conditions of 30nM ACTH concentration were slightly smaller. After 48 hours, the adrenal organoids showed a larger diameter as the concentration of ACTH increased. This shows that the adrenal organoids can effectively simulate the real physiological state in which the growth of the adrenal gland is regulated by the upstream hormone ACTH.

Claims

1. An adrenal organoid culture medium, characterized in that: It is based on DMEM / F12 medium, plus the following components: 40x-60 B27, N-acetylcysteine ​​1-1.5mM, EGF 40-60ng / ml, Noggin 80-120ng / ml, R-sponding1 8-12%, Y27632 8-12uM, A83-01 160-240nM, ACTH 2.4-3.6nM.

2. The adrenal organoid culture medium according to claim 1, characterized in that: It is based on DMEM / F12 medium, plus the following components: 50x B27, N-acetylcysteine ​​1.25mM, EGF 50ng / ml, Noggin100ng / ml, R-sponding110%, Y27632 10uM, A83-01 200nM, ACTH 3nM.

3. A method for culturing adrenal organoids, characterized in that: The steps include: a. Take the animal adrenal gland, wash it, and cut it into 1-2 mm pieces; b. Digest the tissue using type I collagenase and type IV collagenase at 37 degrees Celsius, using a pipette; c. Pipette once every 10 minutes until the tissue is digested into single cells; d. Filter the undigested tissue to obtain the cell suspension, centrifuge, and discard the supernatant; e. Take the precipitate and resuspend it in matrix gel. After the matrix gel solidifies, add the culture medium described in claim 1 or 2 to culture to obtain organoids.

4. The method for culturing adrenal organoids according to claim 3, characterized in that: The culture conditions described in step e are: culture in a cell culture incubator at 37°C and 5% CO2, replace the culture medium every 2-3 days, and perform subculturing every 7-8 days.

5. A method for constructing an animal model of primary adrenocortical carcinoma, characterized in that: It includes the following steps: a. injecting the adrenal organoid cultured according to claim 3 or 4 into the subcutaneous tissue of an animal; b. Performing genetic modification on animals, wherein the genetic modification is to knock out tumor suppressor genes and / or overexpress oncogenes.

6. The method for constructing a primary adrenocortical carcinoma animal model according to claim 5, characterized in that: The genetic modification method is to knock out the TP53, CDKN2A, ZNRF3, and NF1 genes and overexpress the MYC gene.

7. The method for constructing a primary adrenocortical carcinoma animal model according to claim 5, characterized in that: The genetic modification includes transferring fluorescent marker genes into the organoids.

8. Use of the adrenal organoid culture medium according to claim 1 or 2 in preparing adrenal organoids.

9. Use of the primary adrenocortical carcinoma animal model constructed by the construction method according to any one of claims 5 to 7 in drug screening, drug toxicity testing or immunotherapy testing.

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