CN115305240B - Method for culturing organoids and organoid culture device - Google Patents
Method for culturing organoids and organoid culture device Download PDFInfo
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- CN115305240B CN115305240B CN202211211359.4A CN202211211359A CN115305240B CN 115305240 B CN115305240 B CN 115305240B CN 202211211359 A CN202211211359 A CN 202211211359A CN 115305240 B CN115305240 B CN 115305240B
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Abstract
The present invention provides a method of culturing an organoid, the method comprising: terminating division of the trophoblast cells to obtain the treated trophoblast cells; performing adherent culture on the treated trophoblast cells in a culture container to obtain adherent trophoblast cells; covering the liquid gel containing the organoid seed cells and the hydrogel on the adhered trophoblast cells, and solidifying the liquid gel to obtain an organoid growing gel layer; adding a liquid-phase supplementary culture medium on the organoid growth gel layer to obtain a liquid-phase culture medium layer, and culturing. The invention also provides a device for cultivating the organoid. Through the technical scheme, the invention has advantages in modeling efficiency, model stability, tumor organoid cancer cell abundance maintenance and clinical relevance to the medicine, and further provides a system which is more similar to a human body for medicine screening and evaluation.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a method for culturing organoids and a device for culturing organoids.
Background
Organoid technology is a technology that cultures 3D cells in vitro. The technology can realize high-success-rate culture of cells derived from embryonic stem cells, IPS induced pluripotent stem cells, adult cells or patients with diseases in vitro, and can retain some tissue structural characteristics, genetic characteristics and pharmacodynamic biological characteristics in tumor bodies.
The tumor organoids are characterized in that: highly simulating the internal characteristics of the tumor; the modeling success rate is high, the cost is low, and the speed is high; can realize high-flux drug, target spot and biomarker screening; the living tissue cells can be frozen, a living specimen library is established, the living cells are recovered at any time, and the living tissue cells can be derived from a genetic database, a drug effect database, a clinical database, a proteome database and an expression profile database, so that the living tissue cell has expansibility. Therefore, the tumor organoid technology has high practical value in the fields of tumor personalized medicine, new medicine research and development and health big data.
The existing organoid culture method adopts an embedding method, wherein cells are mixed with hydrogel, and then dropwise added into a cell culture dish, and after the hydrogel is solidified, a culture medium containing components such as growth factors is added around the hydrogel.
However, the modeling efficiency and stability of organoids need to be further improved.
Disclosure of Invention
The invention aims to further improve the modeling efficiency and stability of organoids.
To achieve the above object, the present invention provides a method of culturing an organoid, the method comprising: terminating division of the trophoblast cells to obtain the treated trophoblast cells; performing adherent culture on the treated trophoblast cells in a culture container to obtain adherent trophoblast cells; covering the liquid gel containing the organoid seed cells and the hydrogel on the adhered trophoblast cells, and solidifying the liquid gel to obtain an organoid growing gel layer; adding a liquid-phase supplementary culture medium on the organoid growth gel layer to obtain a liquid-phase culture medium layer, and culturing.
The invention also provides an organ-like culture apparatus comprising: a culture vessel suitable for the adherent growth of trophoblast cells; adherent trophoblast cells grown on the inner bottom surface of the culture vessel; the trophoblast cells are after termination of division treatment; an organoid growing gel layer overlying the adherent trophoblast cells; the organoid growing gel layer is formed by solidifying liquid gel, wherein the liquid gel contains organoid seed cells and hydrogel; a liquid-phase culture substrate layer overlying the organoid growth gel layer.
Through the technical scheme, the invention has advantages in modeling efficiency, model stability, tumor organoid cancer cell abundance maintenance and clinical relevance to medicines, and further provides a system which is more similar to a human body for screening and evaluating tumor immune medicines.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 is a graph comparing organoid modeling success rates using an embedding method and a multi-layer culture method.
FIG. 2 is a graph comparing organoid cell viability proliferation in embedding and multilaminate cultures.
FIG. 3 is a graph comparing organoid area proliferation of multi-layer culture and multi-layer culture.
FIG. 4 is a photograph of a bright field organoid cultured by an embedding method.
FIG. 5 is a photograph of a bright field organoid cultured by a multi-layer culture method.
FIG. 6 is a plot of HE staining of organoids cultured using an embedding method.
FIG. 7 is a chart of HE staining of organoids cultured using a multi-layer culture method.
FIG. 8 is a comparison of NGS sequencing versus embedding and multilayering mutation maintenance.
Fig. 9 is a graph showing comparison of organoid EGFR L858R mutant abundance maintenance using an embedding method and a multi-layer culture method.
Fig. 10 is a graph comparing the activation of organoids HER2 and EGFR in tissue samples, embedding and multilaminate cultures.
FIG. 11 is a graph showing the comparison of maximum inhibition of breast cancer organoids by herceptin under conditions of embedding and multilayering.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The present invention provides a method of culturing an organoid, the method comprising: terminating division of the trophoblast cells to obtain the treated trophoblast cells; performing adherent culture on the treated trophoblast cells in a culture container suitable for the adherent growth of the trophoblast cells to obtain adherent trophoblast cells; covering the liquid gel containing the organoid seed cells and the hydrogel on the adhered trophoblast cells, and solidifying the liquid gel to obtain an organoid growing gel layer; adding a liquid-phase supplementary culture medium on the organoid growth gel layer to obtain a liquid-phase culture medium layer, and culturing.
Wherein, optionally, the trophoblast cells comprise at least one of STO (mouse embryo fibroblasts), MEF (mouse embryo fibroblasts), BRL (rat hepatocytes), CAF (tumor-associated fibroblasts), human endometrial stromal cells, mouse endometrial stromal cells.
Wherein optionally, the cleavage termination treatment comprises a mitomycin-C treatment or a gamma-ray treatment. In the mitomycin-C treatment, the final concentration of mitomycin-C is 5-20 mug/mL.
Wherein, optionally, the culture vessel comprises a polylysine-treated culture dish or a polylysine-treated culture well plate; the culture medium for the adherent cultureAt least one of MEM medium, DMEM medium, RPMI-640 medium and F12 medium added with 1-10 vol% of serum; the treated trophoblast cells have an seeded cell density of 0.5-2×10 in adherent culture 4 cells/cm -2 。
Wherein, optionally, the liquid gel contains organoid medium, organoid seed cells, and hydrogel; the organoid seed cells are at least one of primary tumor cells, PDX-separated cancer cells, organoid passage cells, embryonic stem cells, induced reprogramming cells and normal adult cells, and the seed cell density of the organoid seed cells in the liquid gel is 0.5-5×10 6 cells/mL; the hydrogel is contained in the liquid gel in an amount of 10-90% (v/v). The hydrogels may be liquid at 1-10 ℃ and may also be solid at 20-40 ℃, such as those available from Corning under the trade designation 356231.
Wherein, optionally, the organoid medium is a growth factor-attenuated medium (MLC-CM medium) containing Ad-DMEM/F12 medium and further containing 0.5-2-fold concentrations of B27 additive, 1-1.5 mM N-acetylcysteine, 5-20 mM nicotinamide, 5-20ng/mL Noggin,50-200ng/mL R-spondin-1,3-10ng/mL Wnt-3a,1-5ng/mL human recombinant EGF,1-10 ng/mL human recombinant FGF-2,1-10ng/mL human recombinant FGF-7,5-20ng/mL human recombinant FGF-10, 300-700 nM A83-01,0.5-2mM SB202190 and 3-7 mM Y-27632.
Wherein, optionally, the temperature of the liquid gel is 1-10 ℃; the temperature of the organoid gel layer is 20-40 ℃.
Wherein, the temperature of the supplementary culture medium is optionally 20-40 ℃, and is at least one of MLC-CM culture medium and MMC-CM culture medium.
Wherein, optionally, the thickness ratio of the organoid growing gel layer to the liquid phase medium layer is 1: (0.5-5).
The invention also provides an organ-like culture apparatus comprising: a culture vessel suitable for the adherent growth of trophoblast cells; adherent trophoblast cells grown on the inner bottom surface of the culture vessel; the trophoblast cells are after termination of division treatment; an organoid growing gel layer overlying the adherent trophoblast cells; the organoid growing gel layer is formed by solidifying liquid gel, wherein the liquid gel contains organoid seed cells and hydrogel; a liquid-phase culture substrate layer overlying the organoid growth gel layer.
Alternatively, the culture vessel suitable for cell attachment growth comprises a polylysine-treated culture dish or a polylysine-treated culture well plate.
The present invention is further illustrated by the following examples, but the present invention is not limited by the examples.
Example 1
This example illustrates the multi-layered organoid culture process of the present invention using lung cancer tissue as an example.
200mm of lung cancer operation tissue 3 Placed in tissue preservation solution, and transported to a tissue culture chamber at 4 ℃. Washing with pre-chilled sterile PBS+5% FBS for 2 times, adding 1ml of proteolytic liquid (DMEM+Collagenase IV+DispaseII+hyaluronidase), cutting with scissors, and performing enzymolysis on a shaker at 37deg.C for 30-60 min. The digested tissue was observed under a microscope, 10 mL of Ad-DMEM/F12 medium was added, and the mixture was filtered through a 70 μm cell strainer. Centrifugation at 200g for 5 min, the filtered primary tumor cells were resuspended as organoid seed cells.
Fibroblasts from ATCC under the trade designation CRL-2804 (M-7) were used as trophoblasts, which were plated in T75 cell flasks and treated with 10 ug/ml mitomycin for 3 hours according to conventional methods until the growth density reached 75%. Cells treated with mitomycin were washed 2 times to remove any mitomycin involved, digested with pancreatin and counted. Then in an ultra-low adsorption culture dish, performing plating and adherence culture, wherein the density of plated inoculated cells is 1 multiplied by 10 4 cells/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The adherent culture time was 12 hours, and the culture medium for adherent culture was MEM medium supplemented with 10% FBS,1% L-Glutamine,1% NEAA, and 1% diabody, to give adherent trophoblast cells.
MLC-CM medium was prepared with the formulation Ad-DMEM/F12,1 XB 27 supplement, 1.25 mM N-Acetylcysteine, 10 mM nicotinamide, 10ng/mL Noggin, 100ng/mL R-spondin-1, 6ng/mL Wnt-3a, 2ng/mL human recombinant EGF, 5ng/mL human recombinant FGF-2, 5ng/mL human recombinant FGF-7,10 ng/mL human recombinant FGF-10, 500 nM A83-01, 1mM SB202190 and 5 mM Y-27632.
The hydrogel thawed and pre-chilled at 4℃was removed from the refrigerator (available from Corning under the trade designation 356231), diluted to 80% strength by weight with MLC-CM medium pre-chilled at 4℃and placed on ice. Organoid seed cells were adjusted to a cell concentration of 1.6X10 by using MLC-CM medium pre-chilled at 4 ℃ 6 Cells/ml. And mixing the cell suspension with the diluted hydrogel in equal volume to obtain liquid gel containing organoid seed cells and the hydrogel.
Covering the trophoblast cells after adherence with liquid gel containing organoid seed cells and hydrogel, and placing in a cell incubator at 37 ℃ for incubation for 30 minutes, so that the liquid gel is solidified, and thus the organoid growing gel layer is obtained.
The MLC-CM culture medium is used as a liquid-phase culture medium layer to be preheated at 37 ℃ and then dripped on the organoid growth gel layer to obtain the liquid-phase culture medium layer, and the culture is carried out for 7-14 days at 37 ℃.
Comparative example 1
This comparative example illustrates the process of culturing organoids by matrix embedding.
Organoid seed cells were identical to example 1, without using trophoblast cells, and the cell culture plates were ultra-low adsorption plates.
MMC-CM medium was formulated as Ad-DMEM/F12,1 XB 27 supplement, 1.25 mM N-Acetylcysteine, 10 mM nicotinamide, 100ng/mL Noggin, 1ug/mL R-spondin-1, 60ng/mL Wnt-3a, 50ng/mL human recombinant EGF,20 ng/mL human recombinant FGF-2, 50ng/mL human recombinant FGF-7,100 ng/mL human recombinant FGF-10, 500 nM A83-01, 1mM SB202190 and 5 mM Y-27632.
The hydrogel melted at 4℃and precooled was taken out of the refrigerator, diluted to a concentration of 80% by weight with MMC-CM medium precooled at 4 ℃,placed on ice. Organoid seed cells were adjusted to a cell concentration of 1.6x10 with MMC-CM medium pre-chilled at 4 ℃ 6 Cells/ml. And mixing the cell suspension with the diluted hydrogel in equal volume to obtain liquid gel containing organoid seed cells and the hydrogel.
Directly inoculating the liquid gel containing organoid seed cells and hydrogel onto an ultralow adsorption culture dish, and placing the liquid gel in a cell incubator at 37 ℃ for incubation for 30 minutes, so that the liquid gel is solidified, and the organoid growth gel embedding body is obtained.
Preheating MMC-CM culture medium at 37deg.C, dripping onto organoid growth gel embedding body to obtain liquid phase culture medium layer, and culturing at 37deg.C for 7-14 days.
Test example 1
This test example tests the difference in the culture efficiency of the multi-layered method of example 1 and the embedding method of comparative example 1 for various cancer species.
The tumor operation sample is stored in a preservation solution at 4 ℃, and after the tumor operation sample is transported to a laboratory, the corresponding tissue enzymolysis solution is used for enzymolysis, and the primary organoid modeling culture is carried out according to the traditional embedding method and the multilayer method with the same primary cell quantity. Modeling was defined to be successful with the ability to pass more than 3 passages as an evaluation criterion. Of 20 cases of non-small cell lung cancer, the embedding method was 18 cases of success, and the multilayer method was 19 cases of success; of 22 cases of gastric cancer, the embedding method has 16 cases of successful modeling, and the multilayer method has 18 cases of successful modeling; of the colorectal cancers, the embedding method and the multilayer method have 27 cases of successful modeling in 30 cases; among 45 cases of breast cancer, the embedding method has 33 cases of successful modeling, and the multilayer method has 36 cases of successful modeling; of 25 pancreatic cancer samples, the embedding method has 18 cases of successful modeling, and the multilayer method has 21 cases of successful modeling; of 24 ovarian cancer tissues, the embedding method has 18 cases of successful modeling, and the multilayer method has 20 cases of successful modeling; of 36 liver cancer samples, the embedding method has 10 cases of successful modeling, and the multilayer method has 18 cases of successful modeling.
Thus, although the multilayer method reduces the use amount of the exogenous recombinant protein factor, the trophoblast cells provide more diverse microenvironments, and the success rate of organoid modeling is higher than that of the traditional embedding method. (Table 1 and FIG. 1).
Further comparing the differences in proliferation efficiency of modeled organoids under the embedding and multilaminate conditions.
In 96-well plates, 8000 single cells were plated according to the embedding method and the multi-layer method, respectively, at 9 time points of 1, 2, 3, 4, 5, 6, 7, 8, and 9 days, respectively, the organoid image area was calculated by measuring ATP released from the cells and photographing 2 methods.
ATP measurement adopts a 3D-CTG kit of Promega company, a cracking reagent is directly added in an embedding method for cracking organoids, a gel layer is transferred without taking part of a trophoblast in a multilayer method, the gel layer is transferred into another 96-well plate, a 3D-CTG cell cracking solution is added, and after cracking for 10 minutes, a chemiluminescent value is read on an enzyme-labeled instrument.
The method for calculating the area of the organoid is to collect pictures of 10 fields of view of the repeated holes at different time points, calculate the average value of the image area of the organoid by ImageJ software and calculate the average sum of the organoid areas in each detection hole.
In this comparative example, organoid proliferation efficiency was significantly higher in the multilayer culture than in the embedding method, as examined from ATP levels (FIG. 2). However, the difference between the two sets of data acquired by the organoid image area method was not significant, and the organoid area cultured by the embedding method was slightly higher than that cultured by the multilayering method (fig. 3).
In this organ-like culture case, cells cultured by the embedding method are observed to grow more in a vesicle shape, and the organ-like cultured by the multilayer method is a mixture of vesicles and a thick-layer spheroid state, compared with the culture photographs of the embedding method and the multilayer method derived from the same tissue. This difference in organoid morphology accounts for the measurement differences with the ATP method and the area calculation method. The actual number of cells in the multilaminate method is greater than the organoids envisaged by the embedding method.
Table 1: success rate test of culture in non-small cell lung cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer and liver cancer
| Tumor type | Total number of samples | Number of successful cases of embedding method | Number of successful cases of multilayer method | Modeling success rate of embedding method | Multi-layer method modeling success rate |
| Non-small cell lung cancer | 20 | 18 | 19 | 90.0 | 95.0 |
| Stomach cancer | 22 | 16 | 18 | 72.7 | 81.8 |
| Colorectal cancer | 30 | 27 | 27 | 90.0 | 90.0 |
| Breast cancer | 45 | 33 | 36 | 73.3 | 80.0 |
| Pancreatic cancer | 25 | 18 | 21 | 72.0 | 84.0 |
| Ovarian cancer | 24 | 19 | 20 | 79.2 | 83.3 |
| Liver cancer | 36 | 10 | 18 | 27.8 | 50.0 |
Test example 2
The ability of the multilayering method of example 1 and the embedding method of comparative example 1 to specifically amplify tumors in non-small cell lung cancer was compared in this test embodiment.
When cultured by embedding, normal epithelial cells have higher growth advantage than tumor cells. The possible reason is that the normal epithelial cell genome is more stable, and in the case of a large number of growth factor stimuli, it is possible to activate signaling pathways that were not activated originally, and enter mitosis rapidly. However, tumor cells are not stable in their chromosomes due to a large number of mutations, and rely on abnormal activation of signal pathways, and higher growth advantages cannot be obtained in the presence of high concentrations of protein factors.
However, the embedding and multilaminate cultures were morphologically different from organoids of the same tissue sample (FIGS. 4 and 5).
Organoid pellet under different culture conditions was first collected and HE stained, and it was found that organoid vesicles were thinner in the embedding method and thicker in the multi-layer culture method. The former appears more in normal epithelial cell organoids, the latter increases frontal morphology similar to that of tumor organoids (FIGS. 6 and 7)
Genetic stability test: organoid culture genetic stability of 2 non-small cell lung cancer tissues (Pt 110 and Pt 112) was compared by NGS sequencing. NGS sequencing (detection of mutations and copy number changes of 673 tumor-associated genes) was performed on original tissue samples, 1 st generation cultures, 3 rd generation cultures, 5 th generation cultures, and 10 th generation cultures, respectively, under the conditions of the embedding method and the multilayering method. In Pt110 tissue, KRASG 12D-driven mutations and amplification of MYCN were found, as well as some other non-driven mutations (passenger mutations), with the multilayering method retaining both driven and non-driven mutations at passage 10, whereas in the embedding method KRAS mutations were detected at passage 3, but lost at passage 5. Many non-driving mutations also change after passage 5. In the case of Pt110, P53 and EGFR, PIK3CA, CDKN2A were found to amplify 4 important driving mutations (amplifications), which were well maintained by the multilayering method in 10-generation culture, and the non-driving mutations were changed, whereas none of these 4 driving mutations could be detected by the embedding method in 10-generation culture. The genetic stability of the multilayer culture organoids was also superior to that of the embedding method in terms of non-driven mutations (FIG. 8).
If normal epithelial cells would occupy a major position during culture, a research system driven mutation (e.g., EGFR L858R) can be used to examine changes in tumor abundance.
The abundance of L858R was compared in different culture generations in one tissue carrying the EGFRL858R mutation. The experiment uses ARMS-PCR method to detect EGFR L858R mutation, and GAPDH as internal reference. In the embedding method, the difference of EGFR generation 1 is not large, the Ct difference is increased by 1.16, but the expression of EGFR 858R is not detected by generation 5 and generation 10. In the multilayering method, the abundance of EGFRL858R in the 1 st, 5 th and 10 th generation cultures did not differ much (fig. 9).
Thus, it can be seen that the multilaminate approach better maintains organoid genome stability in the 3D case using low concentrations of exogenous recombinant protein factors in combination with the microenvironment provided by the trophoblast cells.
Test example 3
The effect of the multi-layer method of example 1 and the embedding method of comparative example 1 on molecular level in organoid culture was compared in this test example. It was mainly examined whether there was a difference in the organoid molecular signaling pathway and the response to the drug under the culture conditions of the embedding method and the multilaminate method.
Growth factors well above physiological concentrations may cause activation of signal pathways that would not normally occur. High concentrations of EGF may not only result in activation of the EGFR pathway, but may also cause multimerization of other ERBB family molecules, thereby initiating downstream signaling pathways. Sustained activation of high concentrations of protein factors may also lead to endocytosis of the receptor molecule, creating a unique signaling pathway within the cell.
This test example examined EGFR (Tyr 1068) and HER2 (Tyr 1248) phosphorylation. Comparative studies were performed using breast cancer samples that were clinically sensitive to herceptin. Breast cancer tissue samples, breast cancer embedding organoids cultures, breast cancer multilaminate organoids cultures were lysed by protein lysates containing protease inhibitors and phospholipase inhibitors, separated in SDS-PAGE gels, and specific antibodies detected the phosphorylated forms and total proteins of EGFR and HER 2. It was found that under the multi-layered culture conditions of example 1, the EGFR activation and HER2 activation levels were similar to those in the tissue samples, whereas the EGFR phosphorylation levels were very high in the embedding method of comparative example 1, with aberrant HER2 activation (fig. 10).
Abnormal activation of HER2 may lead to endocytosis of HER2 and changes in drug sensitivity. In the herceptin-sensitive breast cancer organoids, the embedding method of comparative example 1 and the multilayer method of example 1 were examined for sensitivity to herceptin under 3-day and 5-day drug treatment conditions, respectively. It was found that the drug sensitivity (drug sensitivity) was higher than that of the embedding method (0.18 ug/ml vs. 0.23ug/ml;0.20ug/ml vs. 0.30 ug/ml) under the multi-layer method conditions of example 1; the multilayer method of example 1 exhibited significantly higher drug inhibition than the embedding method of comparative example 1 in terms of maximum inhibition (efficacy). This suggests that the organoids cultured by the method of example 1 are more sensitive to drugs in the HER2 pathway because of the differences in drug sensitivity due to high concentrations of exogenous factor activation (fig. 11).
The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations of the invention are not described in detail in order to avoid unnecessary repetition.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (3)
1. A method of culturing an organoid, the method comprising:
terminating division of the trophoblast cells to obtain the treated trophoblast cells;
performing adherent culture on the treated trophoblast cells in a culture container to obtain adherent trophoblast cells;
covering the liquid gel containing the organoid seed cells and the hydrogel on the adhered trophoblast cells, and solidifying the liquid gel to obtain an organoid growing gel layer;
adding a liquid-phase supplementary culture medium on the organoid growth gel layer to obtain a liquid-phase culture medium layer, and culturing;
the liquid gel contains an organoid medium, organoid seed cells and a hydrogel; the volume content of the hydrogel in the liquid gel is 10% -90%; the hydrogel is liquid at 1-10 ℃ and solid at 20-40 ℃;
the temperature of the liquid gel is 1-10 ℃; the temperature of the organoid gel layer is 20-40 ℃; the temperature of the supplementary culture medium is 20-40 ℃;
the thickness ratio of the organoid growth gel layer to the liquid phase culture medium layer is 1: (0.5-5);
the organoid medium and the supplemental medium are growth factor-attenuated media;
the growth factor-attenuated medium contains Ad-DMEM/F12 medium and also contains 0.5-2 times the concentration of B27 additive, 1-1.5N-acetylcysteine of mM, 5-20N-acetylcysteine of mM, 1-20ng/mL Noggin,50-200ng/mL R-spondin-1,3-10ng/mL Wnt-3a,1-5ng/mL human recombinant EGF,1-10 ng/mL human recombinant FGF-2,1-10ng/mL human recombinant FGF-7,5-20ng/mL human recombinant FGF-10, A83-01,0.5-2mM SB202190 of 300-700 nM and Y-27632 of 3-7 mM;
the culture medium for the adherence culture is at least one of MEM culture medium, DMEM culture medium, RPMI-640 culture medium and F12 culture medium added with 1-10% of serum; the treated trophoblast cells have an seeded cell density of 0.5-2×10 in adherent culture 4 cells/cm 2 ；
The organoid seed cells are primary tumor cells, and the seed cell density of the organoid seed cells in the liquid gel is 0.5-5×10 6 cells/mL；
The trophoblast cells include at least one of M-7 fibroblasts or mouse embryonic fibroblasts or tumor-associated fibroblasts.
2. The method of claim 1, wherein the terminating split treatment is a mitomycin-C treatment; in the mitomycin-C treatment, the final concentration of mitomycin-C is 5-20 mug/mL.
3. The method of claim 1, wherein the hydrogel comprises at least one of matrigel, collagen gel, synthetic hydrogel.
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