CN111411084A - Culture medium and culture method for constructing liver tumor stent-free organoid - Google Patents
Culture medium and culture method for constructing liver tumor stent-free organoid Download PDFInfo
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Abstract
The invention discloses a culture medium and a culture method for constructing a liver tumor stent-free organoid. The culture medium contains the following components: advanced DMEM/F12, FBS, diabody, N-2, Noggin, B-27, EGF, FGF-10, Y-27632, A83-01, SB202190, R-Spondin, N-acetylcysteine, Nicotinamide, Wnt3A, HGF and PrimocinTM. The culture method comprises the steps of adding the primary tumor cells obtained by separation into the special culture medium for resuspension, counting, inoculating into an ultra-low adsorption U-shaped pore plate, and carrying out centrifugal culture until organoid is formed. The culture medium and the culture method can realize the in-vitro proliferation of the primary liver cancer cells, can quickly construct the liver tumor stent-free organoid, and can keep the long-term stable growth of the liver tumor organoid. The tumor organoids formed by the technology of the invention reserve the heterogeneity of tumor tissues of patients, and are suitable for drug sensitivity detection and tumor occurrence and development related research of in vitro liver cancer chemotherapeutic drugs.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a culture medium and a culture method for constructing a stent-free liver tumor organoid.
Background
Hepatocellular carcinoma ranks second in the world's cancer mortality ranking, with high metastasis rates, low prognosis rates, difficult to treat, and the like. The malignant invasion and metastasis of the tumor lead to the recurrence of the tumor of the patient, and the treatment effect is poor and the prognosis effect is poor. Once a tumor cell is transformed into a malignant cell, its invasive and migratory capabilities also become abnormally strong. Tumor cells are not limited to the migration of single cells. In many tumor types, the usually observed invasive unit is a cell population, the behavior of which determines malignant function. Two-dimensional (2D) cell culture has been widely used for biological studies, but cells grown at two-dimensional levels do not accurately mimic the in vivo conditions. Compared with 2D culture, three-dimensional (3D) cell culture can simulate the living environment of cells in vivo by applying reconstructed extracellular matrix (such as Matrigel, collagen I, hydrogel and the like), constructing a bracket or by means of a certain carrier, provide a microenvironment which is closer to the living condition in vivo for the cells, be more favorable for developing the research of invasion and migration of tumor cells to surrounding tissues and establish a drug sensitive screening model which is closer to the real environment in vivo. However, the current tumor organoid 3D culture technology has certain defects (Science 364,2019, 952-: the forms of tumor models of different organs are not easy to distinguish and lack representativeness; the culture period is long, and the culture price is relatively high; organoid culture is mostly limited in matrix materials such as matrigel, hydrogel and the like, the mode limits gas exchange and substance metabolism between the organoid and the outside, and the exchange limitation seriously influences the absorption of nutrients required by the organoid and the elimination of metabolic waste. Aiming at the defects, the patent technology provides a culture method of a liver tumor stent-free 3D organoid, and the culture method is applied to drug sensitivity screening of anti-tumor drugs. The culture system can realize the rapid external proliferation of primary liver tumor cells, improve the modeling and culture success rate of the 3D organs of the liver tumors, and greatly reduce the culture cost and the culture period.
Disclosure of Invention
The invention aims to provide a culture medium and a culture method for constructing a liver tumor stent-free organoid, so as to solve the problems in the background technology.
To achieve inThe culture medium for constructing the liver tumor stent-free organoid is characterized by comprising serum substitutes N2 and B27, Noggin, double antibody, epidermal growth factor, fibroblast growth factor, selective ROCK1 inhibitor, A L K5 inhibitor, p38 MAPK inhibitor, N-acetylcysteine, R-Spondin protein, nicotinamide, Wnt3A cytokine, hepatocyte growth factor, PrimocinTMFetal bovine serum and basal medium Advance DMEM/F12.
As a further embodiment of the present invention, the final concentrations of the serum substitutes N2 and B27 are 1% and 2% (volume percentage content), respectively, the final concentration of Noggin is 100ng/m L, the final concentration of the double antibody is 1-2% (volume percentage content), the final concentration of Epidermal Growth Factor (EGF) is 50-100ng/m L, the final concentration of fibroblast growth factor (FGF-10) is 10-20ng/m L, the final concentration of the selective ROCK1 inhibitor (Y-27632) is 10-20 μmol/L, the final concentration of the A L K5 inhibitor (A83-01) is 0.5-1.0 μmol/L, the final concentration of the p38 MAPK inhibitor (SB202190) is 5-15 μmol/L, the final concentration of N-acetylcysteine is 0.5-1.5 mmol/L, the final concentration of the R-Sponn protein is 200-200 ng/200 mg, the final concentration of the Wnt cell growth factor (Wnt) is 100-100 ng/m 638-100 mg/L, the final concentration of the cell growth factor (HGF-100 mg/3) is 100 mg/m 638) and the final concentration of the cell growth factor (EGF) is 10-100 mg/LTMThe final concentration of the compound is 100-200 mu g/m L, the final concentration of fetal calf serum is 5 percent (volume percentage content), and the balance is Advanced DMEM/F12.
TABLE 1 complete culture medium composition special for liver cancer organoid
As a further embodiment of the invention, the dual antibody (penicillin/streptomycin mixture) is added with a GlutaMax component, which is beneficial to the growth of organoid 3D cell balls.
A method for culturing the organs without scaffold for liver tumor includes such steps as inoculating the primary liver tumor cells to an ultra-low adsorption U-shaped 96-well plate, adding said culture medium, centrifugal treating at 1000rpm for 3min to promote the aggregation of primary liver tumor cells, and culturing.
As a further scheme of the invention, the inoculation number of the primary liver tumor cells is in the range of 1000-12000 cells/hole.
As a further embodiment of the present invention, the number of primary cells seeded after purification ranged from 2000-6000 cells/well.
As a further embodiment of the present invention, after the cells are seeded and the well plate is centrifuged, a coagulated cell pellet morphology can be formed within 24 hours, and at this time, in order to maintain the stability of the primarily formed cell pellets, the culture medium is changed every 2 days for 4 days before the seeding and every day for 4 days after the seeding to maintain the supply of nutrients.
The basic medium used was Advanced DMEM/F12 medium. The primary cell antibiotic is PrimocinTMThe addition of this component helps to avoid contamination of primary cells, and in particular, infection with hepatitis B virus. In the method for culturing liver cancer organoid, R-Spondin is added, and the component is helpful for formation of liver cancer organoid. In the method for culturing the liver cancer organoid, N-acetylcysteine is required to be added, and the component is beneficial to maintaining the regular and mellow appearance of the liver cancer organoid.
As a further proposal of the invention, the liver cancer tissue taken out by the operation is cut into 1mm2Rinsing the tissue fragments with the size of the tissue fragments by using PBS (phosphate buffer solution) containing double antibodies, centrifuging for 2min at 100g, collecting the tissue fragment sediment, adding tissue digestive juice with the volume of 2-3 times of that of the tissue fragment sediment, fully and uniformly mixing, digesting for 30-60 min at 37 ℃, observing the digestion state every 10min, shaking forcefully until the tissue is observed to be in a dispersion state, stopping digestion, and collecting cells;
centrifuging the separated primary tumor cell suspension at 1000rpm/min for 3 minutes, removing supernatant, collecting precipitate, adding a proper amount of special culture medium for heavy suspension, purifying cells by using a differential adherence method, inoculating the purified cells, inoculating the cells into an ultra-low adsorption 96-pore plate U-shaped culture plate according to the density of 2500 cells/pore, supplementing the special culture medium of 150 mu L in the table 1 into each pore, placing the culture plate at 37 ℃ and 5% CO2CulturingCulturing in a box;
cells can form 3D cell balls within 24 hours after inoculation due to gravity aggregation 30 minutes after inoculation, then the cell balls are assembled and become more and more compact, the cells reach stability after 3-4 days, the volume begins to increase, the complete culture medium is replaced once a day, the cultured balls are complete and regular in shape, round and full, the boundaries are clear, and the stability of the cell ball shape and the cell ball activity can be maintained after continuous culture for 10 days.
As a further embodiment of the invention, the tissue digest is 0.1% collagenase type I + 0.1% collagenase type IV + Hank's solution.
Compared with the prior art, the invention has the beneficial effects that: the special liver tumor organoid culture medium provided by the invention aims at the growth characteristics of tumor cells from liver cancer patients, realizes the in vitro proliferation of the primary tumor cells, can quickly construct a stent-free liver tumor organoid, and can form 3D cell balls within 24 hours. The culture medium and the culture method for the liver tumor organoid without the stent can promote the long-term stable growth of the liver tumor organoid, and the formed organoid keeps the heterogeneity of tumor tissues of patients, so that the culture medium and the culture method are suitable for drug sensitivity detection of in vitro liver cancer chemotherapeutic drugs and tumor-related research. The invention provides an effective in-vitro research model for in-vitro drug sensitivity screening of chemotherapeutic drugs, can accurately predict the reaction of patients to drugs, is beneficial to individualized treatment of clinical patients, reduces drug resistance and improves curative effect.
Drawings
FIG. 1 is a graph showing the effect of establishing organoid system according to the present invention (where a is 1 day after inoculation, b is 5 days after inoculation, and c is 10 days after inoculation).
FIG. 2 is a graph showing the difference in appearance and morphology between organoids formed in the absence of different components in complete medium according to the present invention (where a is a special complete medium, b is R-Spondin-deficient medium, and c is N-acetylcysteine-deficient medium).
FIG. 3 is a chart of immunofluorescence identification results of primary hepatocellular carcinoma cells cultured in general in example 2D of the present invention (where a is CEA antigen identification result, b is CK7 antigen identification result, c is p53 antigen identification result, and D is CD44 antigen identification result).
FIG. 4 is a graph showing immunofluorescence assay results of cultured stentless organoid cytospheres according to example 3D of the present invention (where a is the CEA antigen assay result, b is the CK7 antigen assay result, c is the p53 antigen assay result, and D is the CD44 antigen assay result).
FIG. 5 is a graph showing the cell activity inhibition rate of 2D conventional culture (A) and organoid 3D culture (B) after 7 days of administration of the example of the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely explained below with reference to the drawings in the embodiments of the present invention.
Preparing a primary liver cancer cell organoid:
cutting the liver cancer tissue into pieces of 1mm2The tissue fragments with the size are rinsed clean by PBS solution containing double antibody, and centrifuged for 2min at 100g to collect the tissue fragment precipitate. Adding 2-3 times volume of tissue digestive juice (0.1% collagenase I + 0.1% collagenase IV + Hank's solution), mixing, digesting at 37 deg.C for 30-60 min, observing digestion state every 10min, shaking with force until tissue dispersion state is observed, stopping digestion, and collecting cells.
Centrifuging the separated primary tumor cell suspension at 1000rpm/min for 3min, removing supernatant, collecting precipitate, adding appropriate amount of special culture medium for resuspension, purifying cells by differential adherence method, inoculating the purified cells, inoculating to U-shaped culture plate (SUMITOMO BAKE L ITE, or other similar commercially available products) of ultra-low adsorption 96-well plate at 2500 cells/well density, supplementing the special culture medium of Table 1 to 150 μ L per well, and adding 5% CO at 37 deg.C2Culturing in an incubator.
Cells can form organoid spheres within 24 hours after inoculation 30 minutes due to gravity aggregation, then the cell spheres begin to be assembled and become more and more compact, and the cells reach stability after 3 to 4 days, and the volume begins to increase. Complete medium was changed once a day. The experimental result is shown in figure 1, the technology of the invention can effectively culture the liver cancer primary cells stent-free organoid spheres, and organoids can be formed within 24h after inoculation, the cultured spheres have complete and regular shapes, are mellow and full, have clear boundaries, and can maintain the stability of the shapes and the activities of the cell spheres after continuous culture for 10 days.
In a special complete medium, PrimocinTMThe method is necessary to add, and because clinically obtained liver cancer tissues are mainly from patients with hepatitis B, hepatitis B virus can cause the failure of extraction and separation of primary tumor cells of the patients.
Comparing the effects of R-Spondin and N-acetylcysteine in the establishment of organoid models;
the experiments were divided into 3 groups: a special complete medium group, an R-Spondin-lacking medium group (the composition of the other components is completely consistent with that of the special complete medium group) and an N-acetylcysteine-lacking medium group (the composition of the other components is completely consistent with that of the special complete medium group). And (3) separating the obtained primary liver cancer tumor cell suspension, randomly and equally dividing into 3 parts, and suspending and counting by using three different culture media respectively after centrifugation. Inoculating the culture medium into an ultra-low adsorption 96-well plate U-shaped culture plate according to the density of 2500 cells/well, and inoculating not less than 9 multiple wells in each experimental group.
The result is shown in figure 2, the organoid cells cultured by the special complete culture medium according to the invention have round, complete and complete spherical state and clear boundary line; when the component R-Spondin is lacked, the edge of the formed cell ball has disintegration phenomenon, and the integrity of the ball is reduced; in the absence of N-acetylcysteine, the regularity of the spheroids of the formed cells is reduced, and even one-hole and multi-sphere phenomena occur.
Identifying surface antigens of primary liver cancer cells and tumor organs;
the expression distribution of four antigens including CEA (carcinoembryonic antigen), CK7 (cytokeratin 7), p53 (suppressor gene p53 antigen) and CD44 (polypeptide antigen) is identified for the cultured stable primary liver cancer cells (2D) and the formed organoids (scaffold-free 3D cytospheres). The CD44 antigen can be used as a surface marker of hepatocellular carcinoma stem cells.
Murine anti-human monoclonal CEA antibodies were purchased from EastCoast Bio; murine anti-human monoclonal antibodies CK7, p53, CD44 were purchased from Abcam; the secondary donkey anti-murine 488 antibody was purchased from Southern Biotech; DAPI was purchased from bi yun sky.
The immunofluorescence assay employs an indirect immunofluorescence method, and the results are shown in FIGS. 3 and 4. The four surface antigens of CEA, CK7, p53 and CD44 are expressed in primary cells and organoid cytospheres, the expression of CEA and CD44 on the surface of the primary cells is stronger, and the expression of CK7 and p53 is weaker; on organoids, the fluorescence signal intensity of the 4 antigens is obviously enhanced, and the expression of CEA and CD44 is relatively stronger. The immunofluorescence result shows that the primary cells obtained by separating from the liver cancer tissue are liver cancer primary cells, and the dryness of the primary cells can be enhanced after the organoid cell balls are cultured by the primary cells, so that the advantages of the organoid cell balls in structure and primary characteristics are reflected.
Drug sensitivity screening;
the sensitivity of 2D conventional culture of liver cancer primary cells in vitro and 3D cell balls of liver cancer primary cell organoids established by the culture system of the patent to drugs is studied and compared by adopting a bioluminescent tumor in vitro drug sensitivity detection technology (ATP-TCA). The inoculation densities of a common 96-well plate (2D conventional culture) and an organoid culture 96-well plate (non-stent organoid 3D culture) are 2500 cells/well, the inoculation of the organoid culture 96-well plate is centrifuged for 3min, 1000rpm, first-line drugs gemcitabine, oxaliplatin, paclitaxel, sorafenib and the combination of the two drugs for treating liver cancer are selected by chemotherapeutic drugs, the administration concentrations are set according to the Plasma Peak Concentration (PPC), the gemcitabine is 25 mu g/m L, the oxaliplatin is 1.0 mu g/m L, the paclitaxel is 13.8 mu g/m L, the sorafenib is 10 mu g/m L, the administration concentration gradients are set to be 0%, 25%, 50%, 100% and 200% of the PPC are respectively administered for the days 3, 5 and 7 days, the cell activity inhibition rate of the drugs is calculated, and the sensitivity of the ATP-day is evaluated according to the drug sensitivity test results of the ATP-day 2.
TABLE 2 evaluation results of drug sensitivity
The minimum time from the inoculation of the organoid to the acquisition of the drug sensitivity result is only 10 days, the organoid can reach a stable state after 3 days, the drug sensitivity screening experiment is started, and the drug sensitivity result can be obtained after 7 days of drug administration. According to the evaluation standard of ATP-TCA drug sensitivity, after 7 days of administration, the organoid has drug resistance to gemcitabine, oxaliplatin and the combined administration reaction of gemcitabine and oxaliplatin; the drug resistance is realized on the reaction of paclitaxel, the mild sensitivity is realized on the reaction of sorafenib, and the strong sensitivity is realized on the combined administration reaction of the paclitaxel and the sorafenib. The primary liver cancer cells in the 2D common culture state are strongly sensitive to gemcitabine and combined drug delivery reaction, and are resistant to oxaliplatin reaction; the drug resistance to paclitaxel reaction and the strong sensitivity to sorafenib and the drug combination reaction of the sorafenib and the sorafenib are achieved. Compared with the drug sensitive screening results of 2D cell culture and organoid 3D cell balls, the method prompts that organoids are better correlated with clinic, and the organoid 3D cell ball drug sensitive screening can obtain more accurate results.
In addition, from the detection result, the liver cancer cells in 2D common culture and organoid 3D cell sphere states both react to oxaliplatin and paclitaxel to form drug resistance, and the individualized drug resistance characteristics of the patient to the two drugs are reflected; the administration scheme containing sorafenib shows a remarkable inhibition effect on the activity of liver cancer cells, which is consistent with the wide application of sorafenib in clinical liver cancer treatment.
The foregoing is a preferred embodiment of the present invention, and it will be apparent to those skilled in the art that variations, modifications, substitutions and alterations can be made in the embodiment without departing from the principles and spirit of the invention.
Claims (9)
1. A culture medium for constructing a liver tumor stent-free organoid is prepared from serum substitutes N2 and B27, Noggin, diabody, epidermal growth factor, fibroblast growth factor, selective ROCK1 inhibitor, A L K5 inhibitor, p38 MAPK inhibitor, N-acetylcysteine, R-Spondin protein, nicotinamide, Wnt3A cytokine, hepatocyte growth factor, PrimocinTMFetal bovine serum and basal medium Advanced DMEM/F12.
2. The culture medium for constructing a stentless organoid of liver tumor according to claim 1, wherein the final concentrations of the serum substitutes N2 and B27 are 1% and 2%, respectively, the final concentration of Noggin is 100ng/m L, the final concentration of the diabody is 1-2%, the final concentration of the epidermal growth factor is 50-100ng/m L, the final concentration of the fibroblast growth factor is 10-20ng/m L, the final concentration of the selective ROCK1 inhibitor is 10-20 μmol/L1, the final concentration of the A L K5 inhibitor is 0.5-1.0 μmol/L, the final concentration of the p38 MAPK inhibitor is 5-15 μmol/L, the final concentration of the N-acetylcysteine is 0.5-1.5 mmol/L, the final concentration of the R-Spondin protein is 100ng/m 200ng/m, the final concentration of the Wnt 1 is 100ng/m 59638, the final concentration of the Wnt 11-50 Wnt 1 inhibitor is 10-20 μmol/L1, the final concentration of the A L K5 inhibitor is 0.0.0-1, the final concentration of the MAPK inhibitor is 5-1, the final concentration of the Wnt-50-3, the Wnt-6-3 is 200-6TMThe final concentration of the compound is 100-200 mu g/m L, the final concentration of fetal calf serum is 5 percent, and the balance is Advanced DMEM/F12.
3. The culture medium for constructing a stentless organoid for liver tumors as claimed in claim 1, wherein the dual antibody is supplemented with a GlutaMax component that facilitates growth of organoid 3D cell balls.
4. A method for culturing the organs without the stent of liver tumor includes such steps as inoculating the primary liver tumor cells to an ultra-low adsorption U-shaped 96-well plate, adding said culture medium, centrifugal treating at 1000rpm for 3min to promote the aggregation of primary liver tumor cells, and culturing.
5. The method of claim 4, wherein the number of primary liver tumor cells inoculated is in the range of 1000-12000 cells/well.
6. The method as claimed in claim 4, wherein the number of primary cells inoculated after purification is in the range of 2000-6000 cells/well.
7. The method of claim 4, wherein the aggregated cell pellet morphology is formed within 24 hours after the cells are seeded and the well plate is centrifuged, and the culture medium is changed every 2 days for 4 days before the seeding and every day for 4 days after the centrifugation to maintain the stability of the primarily formed cell pellets.
8. The method of claim 4, wherein the liver cancer tissue is cut into pieces of 1mm2Rinsing the tissue fragments with the size of the tissue fragments by using PBS (phosphate buffer solution) containing double antibodies, centrifuging for 2min at 100g, collecting the tissue fragment sediment, adding tissue digestive juice with the volume of 2-3 times of that of the tissue fragment sediment, fully and uniformly mixing, digesting for 30-60 min at 37 ℃, observing the digestion state every 10min, shaking forcefully until the tissue is observed to be in a dispersion state, stopping digestion, and collecting cells;
centrifuging the separated primary tumor cell suspension at 1000rpm/min for 3min, removing supernatant, collecting precipitate, adding proper amount of special culture medium for resuspension, purifying cells by differential adherence, inoculating the purified cells, inoculating to U-shaped culture plate of ultra-low adsorption 96-well plate at density of 2500 cells/well, supplementing special culture medium to each well by 150 mu L, adding 5% CO at 37 deg.C2Culturing in an incubator, and forming organoid balls within 24 hours; and then the cell balls are assembled and become more and more compact, the stability is achieved after 3-4 days, the volume is increased, the complete culture medium is replaced once a day, the shapes of the cultured balls are complete and regular, round and full, the boundaries are clear, and the stability of the shapes and the activities of the cell balls can be still maintained after continuous culture for 10 days.
9. The stentless liver tumor organoid culture method of claim 4, wherein the tissue digest is 0.1% collagenase type I + 0.1% collagenase type IV + Hank's solution.
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