A culture medium and a culture method for hepatocellular carcinoma organoids
A specialized culture medium and method for liver cancer organoids using RPMI1640 medium and specific additives address the low success rate issue, enhancing cultivation success and maintaining genetic and structural integrity for effective drug sensitivity testing.
Patent Information
- Application Number
- CN202310052968.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-03
- Publication Date
- 2025-07-15
- Estimated Expiration
- 2043-02-03
AI Technical Summary
In the prior art, the success rate of hepatocellular carcinoma organoid culture is extremely low, which is difficult to reflect the genetic heterogeneity between different patients, and there is a lack of sufficient liver cancer organoid models for preclinical research and drug development.
The culture medium consisting of RPMI1640 basal culture medium and specific additive factors, including streptomycin, human insulin, human transferrin, Primocin, sodium selenite, fetal bovine serum, etc., was optimized for the culture conditions of hepatocellular carcinoma organoids in combination with specific culture steps and passage methods.
It improves the success rate of hepatocellular carcinoma organoid culture, maintains the molecular characteristics and morphological structure of liver cancer tissue, reduces the risk of microbial contamination, and provides an efficient experimental system for liver cancer mechanism research and drug development.
Smart Images
Figure CN115948337B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell carcinoma organoids, and specifically, to a culture medium and a culture method for hepatocellular carcinoma organoids. Background Art
[0002] Primary liver cancer is the third leading cause of cancer death globally, and hepatocellular carcinoma (HCC) accounts for approximately 90% of it. Cancer preclinical models are crucial for understanding tumor biological mechanisms and translational therapies. Organoids are 3D multicellular tissue constructs cultured in vitro that can mimic the corresponding in vivo tissues. The patient-derived tumor organoid (PDO) model is a major technological breakthrough in cancer biology research, overcoming the limitations of cell lines and mouse xenograft models and integrating the advantages of both models. The tumor organoid model has significantly improved the success rate of culturing cancer cells in vitro, retaining both the advantages of easy operation and high throughput of tumor cell lines and the ability of in vivo models to grow three-dimensionally and have a microenvironment, and being able to better mimic the characteristics of tumors, which has become a hot topic in cancer research. Based on the established culture system, the success rate of colorectal cancer, breast cancer, and pancreatic cancer organoids can reach 70% or even higher, and a certain number of tumor organoid biobanks have been established.
[0003] However, due to tissue specificity and culture condition limitations, the success rate of liver cancer, especially HCC organoids, is extremely low (5%-30%), making it very difficult to establish a large-scale liver cancer organoid model and difficult to reflect the genetic heterogeneity of liver cancer among different patients; and due to the limited quantity, there is a lack of comparison of molecular typing characteristics between liver cancer organoids and liver cancer tissues. Therefore, it is highly necessary to establish a certain number of liver cancer organoid biobanks to characterize the heterogeneity among liver cancers and provide a powerful experimental system and preclinical model for liver cancer mechanism research and drug development.
[0004] Chinese Patent Document CN111394314A discloses a culture medium and a culture method for colorectal cancer organoids. The culture medium includes a basal medium Advanced DMEM / F12, specific additives, and sterile water; wherein, the mass ratio of the basal medium Advanced DMEM / F12 to the sterile water is 99:1; the specific additives include B27 without vitamin A, N-acetylcysteine, EGF, Noggin, R-spondin 1, Wnt3a, CHIR99021, thiazovivin, Gastrin I, penicillin-streptomycin mixture, and Primocin. Using this culture medium to culture colorectal cancer organoids can maintain the morphological structure and genetic characteristics of the primary tissue, effectively reduce the risk of microbial contamination in colorectal cancer culture, and improve the success rate and survival rate of colorectal cancer organoid culture. At present, there is no report on a culture medium and a culture method for hepatocellular carcinoma organoids, especially the specific experimental procedures, operation steps, culture conditions, that is, the culture medium formula has not been reported much. Summary of the Invention
[0005] In view of the deficiencies in the prior art, the first object of the present invention is to provide a culture medium for hepatocellular carcinoma organoids.
[0006] The second object of the present invention is to provide a culture method for hepatocellular carcinoma organoids.
[0007] To achieve the above first object, the technical solution adopted by the present invention is:
[0008] A culture medium for hepatocellular carcinoma organoids, the culture medium includes: RPMI1640 basal medium and specific additives; the composition of the specific additives is: penicillin-streptomycin 100 μg / mL; human insulin 0.5 - 5 μM; human epidermal growth factor 50 ng / mL; human transferrin 20 - 100 nM; Primocin 100 μg / mL; sodium selenite 10 - 50 nM; sodium pyruvate 0.2 - 5 mM; fetal bovine serum 1% - 10%, v / v; A83 - 01 5 μM; Y-27632 10 μM; the concentration of each component of the specific additives is based on its concentration in the RPMI1640 basal medium.
[0009] Preferably, the human insulin in the culture medium is 2.5 μM, the human transferrin is 60 nM, and the sodium selenite is 40 nM.
[0010] To achieve the above second object, the technical solution adopted by the present invention is:
[0011] A culture method for hepatocellular carcinoma organoids, including the following steps:
[0012] (1) The obtained hepatocellular carcinoma tissue is pretreated, and the pretreated hepatocellular carcinoma tissue is thoroughly washed with a washing solution containing PBS phosphate buffer and penicillin-streptomycin.
[0013] (2) Wash the above hepatocellular carcinoma tissue, precipitate, and discard about the supernatant; repeat the washing once and discard the supernatant.
[0014] (3) Add 10 mL of collagenase digestion solution and digest in a 37 °C incubator for 30 - 180 minutes; the collagenase digestion solution contains PBS phosphate buffer, penicillin-streptomycin, and collagenase.
[0015] (4) Stop digestion when 50% - 100% of single cells are present in the suspension, filter through a 100 - micron pore size filter membrane, centrifuge at a gradient speed, and wash the cells.
[0016] (5) Resuspend the cells with the medium for hepatocellular carcinoma organoids, count, and resuspend the cells in the mixture of the medium for hepatocellular carcinoma organoids and Matrigel. The volume ratio of the medium for hepatocellular carcinoma organoids to Matrigel in the mixture of the medium for hepatocellular carcinoma organoids and Matrigel is 1:1 - 4.
[0017] (6) Replace the medium for hepatocellular carcinoma organoids every 3 - 5 days and passage once every 1 - 3 weeks.
[0018] (7) Resuspend the cells with the medium for hepatocellular carcinoma organoids, count, and resuspend the cells in the mixture of the medium for hepatocellular carcinoma organoids and Matrigel for culture to obtain the product.
[0019] Preferably, in step (1), the hepatocellular carcinoma tissue includes hepatocellular carcinoma, mixed hepatocellular carcinoma, and hepatoblastoma.
[0020] Preferably, in step (1), the concentration of penicillin-streptomycin is 50 - 400 μg / mL.
[0021] Preferably, in step (3), based on the collagenase digestion solution, the concentration of penicillin-streptomycin is 50 - 400 μg / mL, and the collagenase is type IV collagenase with a content of 1 - 10 mg / mL.
[0022] Preferably, in step (5), 2000 - 10000 cells are resuspended in 50 μL of the mixture of the medium for hepatocellular carcinoma organoids and Matrigel.
[0023] Preferably, in step (6), the passage ratio is 1:2 - 5.
[0024] Preferably, in step (7), 2000 - 10000 cells are resuspended in 50 μL of the culture medium - Matrigel mixture of the hepatocellular carcinoma organoids for culture.
[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0026] 1. The culture medium of the present invention contains unique components for culturing hepatocellular carcinoma organoids, and can culture organoids derived from multiple types of hepatocellular carcinoma tissues such as hepatocellular carcinoma, combined hepatocellular - cholangiocarcinoma, and hepatoblastoma.
[0027] 2. Human insulin and human transferrin, which can promote the gene expression of hepatocellular carcinoma, are added to the components of the culture medium of the present invention, enabling the established hepatocellular carcinoma organoids to better maintain the molecular characteristics of hepatocellular carcinoma tissues.
[0028] 3. A trace amount of fetal bovine serum is contained in the culture medium of the present invention, which can promote the proliferation and structural maintenance of hepatocellular carcinoma organoids and improve the success rate of culturing hepatocellular carcinoma organoids.
[0029] 4. The culture medium of the present invention is suitable for culturing hepatocellular carcinoma organoids, and the cultured hepatocellular carcinoma organoids can maintain the gene expression and morphological structure of the primary tissue.
[0030] 5. The present invention can effectively reduce the risk of microbial and mycoplasma contamination in hepatocellular carcinoma culture and improve the success rate of culturing hepatocellular carcinoma organoids. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 It is a functional analysis diagram of the components of the culture medium of hepatocellular carcinoma organoids in the examples.
[0032] Figure 2 For the examples: (A) There is no contamination of normal bile duct cells; (B) All the established hepatocellular carcinoma organoids can be cultured for a long time.
[0033] Figure 3 It is the HE and immunohistochemical staining results of hepatocellular carcinoma - specific markers of a strain of HCC organoids and the source tissue in the examples.
[0034] Figure 4 For the examples, in two strains of HCC organoids, β - Catenin and GS are both membrane - localized, and KI67 is nuclear - localized.
[0035] Figure 5 For the examples: (A) The organoids are more sensitive to the chemotherapeutic drug Doxorubicin; (B) The organoids show high sensitivity to sorafenib. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0036] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
[0037] The culture medium for hepatocellular carcinoma organoids used in the following examples includes RPMI1640 basal medium and specific additives; the composition of the specific additives is as follows: penicillin-streptomycin 100 μg / mL; human insulin 0.5 - 5 μM; human epidermal growth factor 50 ng / mL; human transferrin 20 - 100 nM; Primocin 100 μg / mL; sodium selenite 10 - 50 nM; sodium pyruvate 0.2 - 5 mM; fetal bovine serum 1% - 10%, v / v; A83 - 01 5 μM; Y - 27632 10 μM; the concentrations of each component of the specific additives are based on their concentrations in the RPMI1640 basal medium.
[0038] Preferably, in the above medium, human insulin is 2.5 μM, human transferrin is 60 nM, sodium selenite is 40 nM, and other components and contents remain unchanged.
[0039] Example 1
[0040] 1. Culture method of hepatocellular carcinoma organoids
[0041] 1.1 Trim the edge part of the obtained hepatocellular carcinoma tissue, thoroughly wash the tissue with the washing solution, and then cut the hepatocellular carcinoma tissue into small pieces of 0.5 - 1 mm 3 in size with scissors. The washing solution contains PBS phosphate buffer and penicillin-streptomycin, and the concentration of penicillin-streptomycin is 50 - 400 μg / mL.
[0042] 1.2 Transfer the tissue pieces to a 15 mL centrifuge tube, add 10 mL of ice-cold washing solution, and gently pipette to wash the tissue pieces; wait for the tissue pieces to precipitate, and discard about 7.5 mL of the supernatant (which contains blood cells and fat); repeat the washing once and discard the supernatant.
[0043] 1.3 Add 50 - 200 μL of collagenase digestion solution, continue to cut the tissue with the collagenase digestion solution, add 10 mL of collagenase digestion solution and transfer the cut tissue to the centrifuge tube, and enzymatically digest in a 37 °C incubator for 30 - 180 min. Microscopically examine whether the number of single cells is sufficient. If it is less, continue digestion. Among them, the digestion solution contains PBS phosphate buffer, penicillin-streptomycin, and collagenase. Based on the digestion solution, the concentration of penicillin-streptomycin is 50 - 400 μg / mL, and the content of collagenase is 1 - 10 mg / mL.
[0044] 1.4 Stop digestion when 50%-100% of the cells in the suspension are single cells. Add 10 mL of washing solution to stop digestion, filter through a 100-μm pore size filter membrane, centrifuge at 1000 rpm, 800 rpm, and 600 rpm respectively, and resuspend and wash the centrifugal precipitate with the washing solution each time.
[0045] 1.5 After the last centrifugation, discard the supernatant, resuspend the cells with the medium for hepatocellular carcinoma organoids, count, resuspend 2000-10000 cells in 50 μL of the mixture of the medium for hepatocellular carcinoma organoids and Matrigel, drop it into the culture plate, and add the medium for hepatocellular carcinoma organoids for culture after solidification at 37°C. The volume ratio of the medium for hepatocellular carcinoma organoids to Matrigel in the mixture of the medium for hepatocellular carcinoma organoids and Matrigel is 1:1-4.
[0046] 1.6 Subsequently, change the medium for hepatocellular carcinoma organoids every 3-5 days, passage once every 1-3 weeks, and the passage ratio is 1:2-5. Aspirate the medium for hepatocellular carcinoma organoids, add ice-cold phosphate buffer to disperse the organoids, transfer them to a 15-mL centrifuge tube, centrifuge for 5 min at 1000 rpm, completely discard the supernatant, add 2 mL of preheated TrypLE to resuspend the organoids, pipette several times with a pipette tip, digest at 37°C for 1-5 min, observe the digestion situation under bright field in the middle, add 8 mL of ice-cold phosphate buffer to terminate digestion, and centrifuge.
[0047] 1.7 Discard the supernatant, resuspend the cells with the medium for hepatocellular carcinoma organoids, count, resuspend 2000-10000 cells in 50 μL of the mixture of the medium for hepatocellular carcinoma organoids and Matrigel, drop it into the culture plate, and add the medium for hepatocellular carcinoma organoids for culture after solidification at 37°C.
[0048] 2. Components of the medium for hepatocellular carcinoma organoids
[0049] RPMI1640 basal medium (purchased from Gibco); penicillin-streptomycin 100 μg / mL; human insulin 0.5-5 mM; human epidermal growth factor 50 ng / mL; human transferrin 20-100 nM; Primocin (Primocin - primary cell antibiotic, purchased from InvivoGen) 100 μg / mL; sodium selenite 10-50 nM; sodium pyruvate 0.2-5 mM; fetal bovine serum 1%-10%, v / v; A83-01 (inhibitor of TGF-β type I receptor (ALK5-TD), purchased from Sigma) 5 μM; Y-27632 (specific inhibitor of ROCK, purchased from Sigma) 10 μM.
[0050] It was found through experiments that in the medium of the above hepatocellular carcinoma organoids, removing human insulin would reduce the expression of liver cancer marker genes; removing human transferrin would reduce the expression of liver cancer marker genes; removing fetal bovine serum would affect organoid proliferation and reduce the success rate; removing Y-27632 would reduce the success rate of organoids.
[0051] 3. Experimental Results
[0052] 3.1 Functional Analysis of Medium Components
[0053] Based on the above medium of hepatocellular carcinoma organoids, removing human insulin or human transferrin would reduce the expression of liver cancer-related genes. For the functional analysis of medium components, see Figure 1 .
[0054] 3.2 Morphology of Organoids Cultured by the Culture Method of Hepatocellular Carcinoma Organoids
[0055] The main reasons for the failure of liver cancer organoids are the inability of in vitro tumor cells to expand and the "contamination" caused by the proliferation advantage of normal bile duct cells. In this example, by optimizing the medium components, applying the above culture method and medium of hepatocellular carcinoma organoids, the morphology during the culture of two cases of hepatocellular carcinoma HCC organoids was demonstrated. The growth was in the form of solid spheres with a three-dimensional structure at different passages, and there was no contamination by normal bile duct cells ( Figure 2 A), and the established liver cancer organoids could be cultured for a long time ( Figure 2 B).
[0056] 3.2 Immunohistochemical Staining of Organoids Cultured by the Culture Method of Hepatocellular Carcinoma Organoids
[0057] To verify whether the tissue structure and marker molecule expression of the constructed liver cancer organoids are consistent with those of the source liver cancer tissue, HE staining and immunohistochemical staining of liver cancer-specific molecular markers GPC3, AFP, and proliferation marker KI67 were performed on liver cancer organoids and the source cancer tissues ( Figure 3 ). The staining results of HCC organoids showed that the constructed HCC organoids were composed of typical epithelial-like cell arrangements and had a liver tissue structure, which was similar to the structure of the non-necrotic and non-fibrotic regions of the source liver cancer tissue. The strong positive expression of the proliferation marker KI67 in the organoids indicated a fast proliferation rate, and the membrane protein GPC3 and secreted protein AFP were expressed.
[0058] 3.3 Immunofluorescence Staining of Organoids Cultured by the Culture Method of Hepatocellular Carcinoma Organoids
[0059] The co-localization of β-Catenin and GS was detected by immunofluorescence staining. The results showed that β-Catenin and GS were both membrane-localized, and KI67 was nuclear-localized in two strains of HCC organoids ( Figure 4 ).
[0060] 3.4 Culturing organoids using the culturing method of hepatocellular carcinoma organoids for drug screening
[0061] To evaluate the drug sensitivity of liver cancer organoids and establish a high-throughput drug screening model, the drug IC50 values of a strain of liver cancer organoids to the liver cancer-targeted drug Sorafenib and the chemotherapeutic drug Doxorubicin were first detected. The IC50 of the established organoids to Doxorbucin was 1.031 mM, and the IC50 to Sorafenib was 2.189 mM. The organoids were more sensitive to the chemotherapeutic drug Doxorubicin( Figure 5 A).
[0062] The established organoids were used to screen the drug sensitivity of small molecule compounds. It was found that liver cancer organoids were more sensitive to chemotherapeutic drugs and multi-kinase inhibitors, and there were significant differences in the sensitivity to different drugs. By comparing the sensitivities of organoids to the liver cancer clinical drugs Sorafenib, Lenvatinib, Regorafenib, and the chemotherapeutic drug Doxorubicin, it was found that different from the high sensitivity of 2D cell lines, about 50% of the organoids had low sensitivity to Doxorubicin, suggesting that it might be caused by the significant differences in the three-dimensional structure and drug metabolism of organoids and 2D cells. The organoids showed high sensitivity to Sorafenib( Figure 5 B).
[0063] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.
Claims
1. A culture medium for hepatocellular carcinoma organoids, characterized in that, The culture medium comprises RPMI 1640 basal medium and specific addition factors; wherein: The composition of the specific addition factors is as follows: penicillin-streptomycin 100 μg / mL; human insulin 0.5 - 5 μM; human epidermal growth factor 50 ng / mL; human transferrin 20 - 100 nM; Primocin 100 μg / mL; sodium selenite 10 - 50 nM; sodium pyruvate 0.2 - 5 mM; fetal bovine serum 1% - 10%, v / v; A83-01 5 μM; Y-27632 10 μM; The concentration of each component of the specific addition factors is based on its concentration in the RPMI 1640 basal medium.
2. The culture medium for hepatocellular carcinoma organoids according to claim 1, characterized in that, In the culture medium, the human insulin is 2.5 μM, the human transferrin is 60 nM, and the sodium selenite is 40 nM.
3. A method for culturing hepatocellular carcinoma organoids, characterized in that, It includes the following steps: (1) The obtained hepatocellular carcinoma tissue is pretreated and thoroughly washed with a washing solution; the washing solution contains PBS phosphate buffer and penicillin-streptomycin, the hepatocellular carcinoma tissue includes hepatocellular carcinoma, mixed hepatocellular carcinoma, and hepatoblastoma, and the concentration of penicillin-streptomycin is 50 - 400 μg / mL; (2) Wash the above hepatocellular carcinoma tissue, centrifuge, and discard the supernatant; repeat the washing once and discard the supernatant; (3) Add 10 mL of collagenase digestion solution and digest in a 37 °C incubator for 30 - 180 minutes; the collagenase digestion solution contains PBS phosphate buffer, penicillin-streptomycin, and collagenase; based on the collagenase digestion solution, the concentration of penicillin-streptomycin is 50 - 400 μg / mL, and the collagenase is type IV collagenase with a content of 1 - 10 mg / mL; (4) Stop digestion when 50% - 100% of single cells are present in the suspension, filter through a 100 - micron pore size filter membrane, centrifuge at a gradient speed, and wash the cells; (5) Resuspend the cells with the culture medium for hepatocellular carcinoma organoids described in claim 1 or 2, count, and resuspend the cells in a culture medium - Matrigel mixture for hepatocellular carcinoma organoids, where the volume ratio of the culture medium for hepatocellular carcinoma organoids to Matrigel in the culture medium - Matrigel mixture for hepatocellular carcinoma organoids is 1:1 - 4, and resuspend 2000 - 10000 cells in 50 μL of the culture medium - Matrigel mixture for hepatocellular carcinoma organoids; (6) Replace the culture medium for hepatocellular carcinoma organoids every 3 - 5 days and passage once every 1 - 3 weeks, and the passage ratio is 1:2 - 5; (7) Resuspend the cells with the culture medium for hepatocellular carcinoma organoids, count, and resuspend the cells in the culture medium - Matrigel mixture for hepatocellular carcinoma organoids for culture, and resuspend 2000 - 10000 cells in 50 μL of the culture medium - Matrigel mixture for hepatocellular carcinoma organoids for culture.