CN111903603B - Transplant for constructing bile duct cancer xenograft model and preparation method and application thereof - Google Patents
Transplant for constructing bile duct cancer xenograft model and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a transplant for constructing a bile duct cancer xenograft model and a preparation method and application thereof, wherein the preparation method comprises the following steps: a. mixing matrigel and a bile duct cancer culture medium to obtain a bile duct cancer culture medium-matrigel mixture; b. mixing the bile duct cancer tissue after enzymolysis with the bile duct cancer culture medium-matrigel mixture, and performing culture and amplification to obtain the transplant containing the bile duct cancer organoid. According to the method, the transplant containing the bile duct cancer organoid is obtained by using the tissue culture of the bile duct cancer after enzymolysis, and the success rate and the required time for constructing the bile duct cancer xenograft model by using the transplant are high.
Description
Technical Field
The disclosure relates to the technical field of biomedicine, in particular to a transplant for constructing a bile duct cancer xenograft model and a preparation method and application thereof.
Background
Cholangiocarcinoma refers to a malignancy of the bile duct that originates in the extrahepatic bile duct, including the hepatic portal area to the lower end of the common bile duct. The causes of the disease may be related to diseases such as bile duct calculi, primary sclerosing cholangitis and the like. The clinical application can adopt the methods of operation treatment, radiation treatment, chemical treatment and the like, but the effect is not obvious and the prognosis is poor. In order to improve the therapeutic effect of cholangiocarcinoma, intensive studies on the pathogenic mechanism, drug resistance, etc. of cholangiocarcinoma are required, and an animal xenograft model is an important tool for the studies.
In the related art, a tumor tissue block of a primary focus or a metastatic focus of a patient with bile duct cancer is used as a transplant, and the transplant is transplanted into subcutaneous or orthotopic tissues of an immunodeficiency mouse, so that a bile duct cancer xenograft model is constructed.
However, when the tumor tissue mass of the primary focus or the metastatic focus of a patient with bile duct cancer is used as a transplant, the obtained tissue quantity is limited, the success rate of constructing a bile duct cancer xenograft model is low, and the required time is long.
Disclosure of Invention
The invention aims to provide a transplant for constructing a bile duct cancer xenograft model, a preparation method and application thereof.
In order to achieve the above object, the present disclosure provides, in a first aspect, a method for preparing a graft for constructing a biliary duct cancer xenograft model, the method comprising:
a. mixing matrigel and a bile duct cancer culture medium to obtain a bile duct cancer culture medium-matrigel mixture;
b. mixing the bile duct cancer tissue after enzymolysis with the bile duct cancer culture medium-matrigel mixture, and performing culture and amplification to obtain the transplant containing the bile duct cancer organoid.
Optionally, the cholangiocarcinoma medium contains RPMI 1640 medium and growth factors including penicillin, streptomycin, Y-276322 HCl, Y-27632, nicotinic acid and bovine serum albumin.
Optionally, in the cholangiocarcinoma culture medium, the concentration of penicillin is 100-150U/mL, the concentration of streptomycin is 100-150 μ g/mL, the concentration of Y-276322 HCl is 5-10 μ M, the concentration of Y-27632 is 5-10 μ M, the concentration of nicotinic acid is 5-15 mM, and the concentration of bovine serum albumin is 0.3-0.5 g/100 mL.
Optionally, when the matrigel and the cholangiocarcinoma medium are mixed in the step a, the cholangiocarcinoma medium is used in an amount of 3-5 parts by volume relative to 1 part by volume of the matrigel.
Optionally, the mixing the bile duct cancer tissue after enzymolysis with the bile duct cancer culture medium-matrigel mixture in the step b comprises:
mixing the bile duct cancer tissue subjected to enzymolysis with the bile duct cancer culture medium-matrigel mixture to enable the bile duct cancer tissue subjected to enzymolysis to be suspended in the bile duct cancer culture medium-matrigel mixture; wherein the content of the first and second substances,
the dosage of the bile duct cancer culture medium-matrigel mixture is 0.5-1 mL relative to 30000-40000 bile duct cancer tissue cells after enzymolysis.
Optionally, the preparation method of the bile duct cancer tissue after enzymolysis includes:
(1) mixing the bile duct cancer tissue fragments with an enzymolysis liquid and carrying out enzymolysis to obtain an enzymolysis material, wherein the bile duct cancer tissue comprises bile duct cancer primary focus tissue and/or bile duct cancer metastatic focus tissue; the enzymatic hydrolysate contains RPMI 1640 culture medium, 2-5% of fetal bovine serum albumin and 1-3 mg/mL of collagen hydrolase; the dosage of the enzymolysis liquid is 5-10 parts by volume relative to 1 part by weight of the bile duct cancer tissue fragments;
(2) centrifuging the enzymolysis material, collecting the precipitate, and obtaining the bile duct cancer tissue after enzymolysis, wherein the centrifugation conditions comprise: the centrifugal force is 300-500 g, and the centrifugal time is 3-5 min.
Optionally, the transplant containing the bile duct cancer organoid comprises a bile duct cancer organoid and a wrapping layer wrapping the surface of the bile duct cancer organoid; the diameter of the bile duct cancer organoid is 1.5-3 mm, and the thickness of the wrapping layer is 1-3 mm.
In a second aspect, the present disclosure provides a graft for constructing a bile duct cancer xenograft model, the graft being prepared by the method of any one of the first aspect.
In a third aspect, the present disclosure provides use of the graft of the second aspect in constructing a biliary tract cancer xenograft model.
In a fourth aspect, the present disclosure provides a method of producing a biliary duct cancer xenograft mouse model, the method comprising:
implanting the graft of the second aspect into subcutaneous and/or orthotopic tissues of an immunodeficient mouse to obtain the bile duct cancer xenograft mouse model.
According to the technical scheme, the method provided by the disclosure comprises the steps of obtaining the transplant containing the bile duct cancer organoid by using the tissue culture of the bile duct cancer after enzymolysis, and obtaining a high success rate under the condition of a small amount of tissues and short time for constructing the xenograft model when the transplant is used for constructing the bile duct cancer xenograft model.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
A first aspect of the present disclosure provides a method of preparing a graft for constructing a biliary duct cancer xenograft model, the method comprising: a. mixing matrigel and a bile duct cancer culture medium to obtain a bile duct cancer culture medium-matrigel mixture; b. mixing the bile duct cancer tissue after enzymolysis with the bile duct cancer culture medium-matrigel mixture, and performing culture and amplification to obtain the transplant containing the bile duct cancer organoid.
In the related technology, a tumor tissue block of a primary focus or a metastatic focus of a bile duct cancer patient is directly transplanted to subcutaneous or orthotopic tissues of an immune deficiency type mouse as a transplant to construct a bile duct cancer xenograft model. However, the inventors of the present disclosure found that directly using tumor tissue blocks of primary or metastatic focus of patients with bile duct cancer as transplant has high requirements on the quality, activity and quantity of transplanted tissues, resulting in low success rate and long required time for constructing bile duct cancer xenograft models.
In the technical scheme, the transplant containing the bile duct cancer organoid is obtained by using the tissue culture of the bile duct cancer after enzymolysis, and then the transplant is used for constructing the bile duct cancer xenograft model. Moreover, the biological characteristics of the bile duct cancer organoid obtained by culturing the bile duct cancer tissue after enzymolysis are similar to those of the bile duct cancer tissue, so that the adaptation degree of a xenograft model constructed by the transplant to the bile duct cancer tissue of a patient is higher, and the research value is higher.
Meanwhile, in the method, a large number of transplants containing the bile duct cancer organoids can be obtained in a short time only by a small amount of bile duct cancer tissues, so that the preparation of a large number of transplants can be simultaneously carried out by using the method, the number of the finally constructed xenograft models can be effectively increased, and more resources can be provided for clinical treatment or scientific research.
In addition, in the method, the matrigel and the bile duct cancer culture medium are mixed uniformly to obtain the bile duct cancer culture medium-matrigel mixture, and then the bile duct cancer culture medium-matrigel mixture and the bile duct cancer tissue after enzymolysis are mixed uniformly and cultured, so that the contact area between the bile duct cancer tissue after enzymolysis and the matrigel and bile duct cancer culture medium can be effectively increased, and the success rate of culture of bile duct cancer organoids is improved.
According to the present disclosure, a medium that facilitates proliferation and growth of bile duct cancer tissue after enzymatic hydrolysis can be used for bile duct cancer culture of the present disclosure. Preferably, the culture medium for bile duct cancer selected by the disclosure at least comprises RPMI 1640 culture medium and growth factors, wherein the growth factors at least comprise penicillin, streptomycin, Y-276322 HCl, Y-27632, nicotinic acid and bovine serum albumin. Under the above preferred conditions, the preferred cholangiocarcinoma culture medium is developed and prepared by the present inventors aiming at the cell structure of the cholangiocarcinoma tissue mass, and the cholangiocarcinoma culture medium can sufficiently promote the proliferation and growth of cholangiocarcinoma cells and simultaneously inhibit the proliferation and growth of other cells in the tissue mass, so the cholangiocarcinoma culture medium can effectively improve the culture success rate of cholangiocarcinoma organs.
According to the present disclosure, the concentration of each growth factor in the cholangiocarcinoma medium may vary within a certain range, for example, the concentration of penicillin in the cholangiocarcinoma medium may be 100 to 150U/mL, the concentration of streptomycin may be 100 to 150 μ g/mL, the concentration of Y-276322 HCl may be 5 to 10 μ M, the concentration of Y-27632 may be 5 to 10 μ M, the concentration of nicotinic acid may be 5 to 15mM, and the concentration of bovine serum albumin may be 0.3 to 0.5g/100 mL.
Wherein Y-276322 HCl (Y-27632 dihydrochloride) is ROCK inhibitor with CAS number of 146986-50-7 and molecular formula of C14H23Cl2N3O。
Preferably, the matrigel selected by the present disclosure can be, for example, Curtrex 3-D Culture Matrix BME.
According to the present disclosure, in order to further improve the success rate of culturing the cholangiocarcinoma organoids, preferably, when the matrigel and the cholangiocarcinoma culture medium are mixed in step a, the dosage of the cholangiocarcinoma culture medium may be 3-5 parts by volume relative to 1 part by volume of the matrigel.
According to the disclosure, the mixing of the bile duct cancer tissue after enzymolysis and the bile duct cancer culture medium-matrigel mixture in the step b at least may include: mixing the bile duct cancer tissue after enzymolysis with the bile duct cancer culture medium-matrigel mixture, so that the bile duct cancer tissue after enzymolysis is suspended in the bile duct cancer culture medium-matrigel mixture. The bile duct cancer tissue after enzymolysis is suspended in the bile duct cancer culture medium-matrigel mixture, so that the bile duct cancer tissue after enzymolysis is in full contact with the bile duct cancer culture medium and the matrigel, and the bile duct cancer tissue after enzymolysis can obtain sufficient nutrient substances and is easier to adhere to the matrigel, thereby being beneficial to improving the culture success rate of the bile duct cancer organoid.
Preferably, when the bile duct cancer tissue subjected to enzymolysis in the step b is mixed with the bile duct cancer culture medium-matrigel mixture, the dosage of the bile duct cancer culture medium-matrigel mixture is 0.5-1 mL for 30000-40000 bile duct cancer tissue cells subjected to enzymolysis.
According to the present disclosure, the bile duct cancer tissue after enzymolysis used in the present disclosure may be obtained by subjecting the bile duct cancer tissue to enzymolysis. Illustratively, the preparation method of the bile duct cancer tissue after enzymolysis can comprise the following steps: (1) mixing the bile duct cancer tissue fragments with an enzymolysis liquid and carrying out enzymolysis to obtain an enzymolysis material, wherein the bile duct cancer tissue comprises bile duct cancer primary focus tissue and/or bile duct cancer metastatic focus tissue; the enzymatic hydrolysate contains RPMI 1640 culture medium, 2-5% of fetal bovine serum albumin and 1-3 mg/mL of collagen hydrolase; the dosage of the enzymolysis liquid is 5-10 parts by volume relative to 1 part by weight of the bile duct cancer tissue fragments; (2) centrifuging the enzymolysis material, collecting the precipitate, and obtaining the bile duct cancer tissue after enzymolysis, wherein the centrifugation conditions comprise: the centrifugal force is 300-500 g, and the centrifugal time is 3-5 min.
According to the present disclosure, the transplant containing the cholangiocarcinoma organoid can comprise a cholangiocarcinoma organoid and a coating layer coated on the surface of the cholangiocarcinoma organoid; the diameter of the bile duct cancer organoid can be 1.5-3 mm, and the thickness of the wrapping layer can be 1-3 mm. The bile duct cancer organoid and the coating layer are taken out of the culture system and used as a transplant, so that the activity of the bile duct cancer organoid in the transplant can be guaranteed, and the success rate of constructing a bile duct cancer xenograft model is effectively improved.
A second aspect of the present disclosure provides a graft for constructing a bile duct cancer xenograft model, the graft being prepared by the method of any one of the first aspects.
A third aspect of the present disclosure provides use of the graft described in the second aspect in constructing a bile duct cancer xenograft model.
A fourth aspect of the present disclosure provides a method of producing a biliary duct cancer xenograft mouse model, the method comprising: implanting the graft of the second aspect into subcutaneous and/or orthotopic tissues of an immunodeficient mouse to obtain the bile duct cancer xenograft mouse model. The method for implanting the graft into the subcutaneous and/or in situ tissue of the immunodeficient mouse may be conventional in the art and will not be described herein.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
The starting materials, reagents, instruments and equipment referred to in the examples of the present disclosure may be obtained by purchase, unless otherwise specified.
Where specific experimental temperatures are not noted in the examples of the present disclosure, the experimental temperatures are all room temperature (20-25 ℃). The sources of reagents used in the examples of the disclosure are as follows:
RPMI 1640 medium was purchased from Hyclone, USA; cultrex 3-D Culture Matrix BME was purchased from Trevigen, USA; fetal bovine serum albumin (FBS) was purchased from inner mongolia jinyuan kang bioengineering ltd; collagen hydrolase (collagen) was purchased from Sigma-Aldrich, usa; Penicillin-Streptomycin (Penicillin-Streptomycin) was purchased from shanghai bio-engineering gmbh; y-276322 HCl (p160ROCK inhibitor Y-27632dihydrochloride) and collagenase type II (collagene type II) from Invitrogen, USA; nicotinamide (Nicotinamide) was purchased from Sigma; bovine Serum Albumin (BSA) was purchased from Sigma.
The cholangiocarcinoma culture medium used in the embodiment of the present disclosure is a dedicated culture medium for cholangiocarcinoma which is configured autonomously, and includes:
RPMI 1640 medium, 150U/mL penicillin, 150ug/mL streptomycin, 10. mu.M Y-276322 HCl, 5. mu.M Y-27632(Abmole, Y-27632), 5mM nicotinamide, and 0.4g/100mL bovine serum albumin.
Example 1
This example illustrates the preparation of the graft of the present disclosure.
(1) Bile duct cancer tissue preservation
The primary lesion surgical tissue of fresh cholangiocarcinoma was preserved in the cholangiocarcinoma medium described above and transported to the operating room in 48 hours with a 4 ℃ cold chain.
(2) Preparation of bile duct cancer tissue after enzymolysis
The medium was removed and the cholangiocarcinoma tissue was placed in a 6cm petri dish. Preparing an RPMI 1640 culture medium containing 2% FBS and 2mg/mL collagen hydrolase as an enzymolysis liquid, filtering and sterilizing the enzymolysis liquid, carrying out warm bath at 37 ℃, adding the enzymolysis liquid into a culture dish containing bile duct cancer tissues, wherein 8mL of enzymolysis liquid is correspondingly added into 1g of bile duct cancer tissues. Shearing the bile duct cancer tissue blocks into minced meat by using disinfected and sterilized scissors, transferring the sheared tissue blocks and the enzymolysis liquid into a 24-hole plate by using a 10mL pipette, putting the 24-hole plate into a 37-DEG C constant-temperature incubator for enzymolysis for 2 hours, and blowing and beating the enzymolysis tissue by using a pipettor every 30 minutes in the enzymolysis process to promote the enzymolysis effect. And then, centrifuging the enzymolysis liquid for 3min under the centrifugation condition of 300g, removing the supernatant, adding 10ml of bile duct cancer culture medium into the precipitate, and carrying out heavy suspension to obtain the bile duct cancer tissue subjected to enzymolysis.
(3) Graft preparation
Matrigel (Cultrex 3-D Culture Matrix BME) was mixed into a homogenate using a 4 ℃ pre-chilled pipette tip. And then uniformly mixing pulpous Matrix gum (Cultrex 3-D Culture Matrix BME) with the bile duct cancer Culture medium precooled at 4 ℃ by using a pipette tip precooled at 4 ℃ to obtain a bile duct cancer Culture medium-Matrix gum mixture, wherein the volume ratio of the Matrix gum to the bile duct cancer Culture medium is 1: 4.
And uniformly mixing the bile duct cancer tissue subjected to enzymolysis with the bile duct cancer culture medium-matrigel mixture to obtain a culture to be cultured, wherein each milliliter of the culture to be cultured contains 30000 bile duct cancer tissue cells subjected to enzymolysis. The 96-well plate was placed on ice, and 200. mu.L of the above culture to be cultured was added to each well, and then the whole 96-well plate was placed in a 37 ℃ incubator for 72 hours. After the culture, microscopic examination was performed, and the cholangiocarcinoma organoids having a diameter of not less than 1.5mm in each well were removed together with the coating layer having a thickness of not less than 1mm around them, to obtain a graft a of this example.
Example 2
The graft B of the present disclosure was prepared by the method of example 1, except that the tissue mass of the primary focus of cholangiocarcinoma in example 1 was replaced with the tissue mass of the metastatic focus of cholangiocarcinoma from the same patient in this example.
Comparative example
Cutting primary bile duct cancer focus tissue from the same patient to 2.5mm3To obtain a graft C. Cutting bile duct cancer metastasis tissue from the same patient to 2.5mm3To obtain a graft D.
Xenograft mouse models a, b, c, and D of cholangiocarcinoma were constructed using grafts A, B, C and D, respectively.
The construction method of the bile duct cancer xenograft mouse model comprises the following steps: selecting an immune-deficient NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju) mouse which is fed based on an SPF (specific pathogen free) environment, carrying out abdominal anesthesia, disinfecting and skin preparation on the lateral skin of forelimbs on two sides of the mouse, then cutting the skin by using a disinfection scalpel, lightly implanting the transplant by using a capillary glass tube, wiping clean and oozing blood, pasting a wound by using a medical adhesive tape, and removing the transplant after the wound is completely healed. The same treatment was done on the opposite side. And observing the mouse every day, and obtaining a bile duct cancer xenograft model after the transplant forms tumor.
1. Test of the incubation period of transplanted tumor and the time required for transplanted tumor to grow to passable
The test method comprises the following steps:
(1) the mice were observed daily and the size of the mouse graft tumor was measured using a vernier caliper with the graft tumor having a major diameter of t and a minor diameter of r according to V ═ t × r2Calculating the approximate volume of the transplanted tumor when the approximate volume is equal to or greater than 1.2mm3The time is the end of the incubation period, and the days of the incubation period of the transplanted tumor are calculated. 10 cases were tested per group and the average was calculated.
(2) The transplanted tumor grows to 500mm from the date of completion of transplantation to the P1 generation3The time of (a) is the time required for the transplanted tumor to grow to be passable. 10 cases were tested per group and the average was calculated.
The test results are shown in table 1.
TABLE 1
| Group of | Mean incubation period (sky) | Growth to passable average time (day) |
| Graft A | 3.12±0.94 | 71.13±1.83 |
| Graft B | 2.62±0.86 | 68.59±1.04 |
| Graft C | 8.46±1.01 | 93.21±3.77 |
| Graft D | 4.32±0.97 | 87.54±2.56 |
As can be seen from table 1, the incubation period and the average time to growth passable of the transplanted tumors obtained by transplantation with the graft a are both significantly shorter than those of the graft C; the incubation period and the average time to growth to passability of the transplants obtained with transplant B were significantly shorter than with transplant D. The transplants prepared by the method of the present disclosure can shorten the time required for constructing a biliary duct cancer xenograft mouse model.
2. Drug sensitivity detection of transplantable tumors
Culturing human bile duct cancer cell strain FRH-0201 by conventional method at 1 × 107And (4) constructing a bile duct cancer mouse model e by the inoculation amount of each cell.
From the day of completion of transplantation, mice were observed daily, and the size of mouse graft tumor was measured using a vernier caliper, the graft tumor having a long diameter of t and a short diameter of r according to V ═ t × r2The approximate volume of the transplanted tumor was calculated. When the approximate volume of the transplanted tumor is equal to or greater than 100mm3According to the injection amount of 0.2 mL/mouse, 10mg/kg of adriamycin, 10mg/kg of cis-platinum, 4mg/kg of mitomycin, 20mg/kg of taxol and 120mg/kg of cyclophosphamide are respectively injected into the tail part of each group of mice, a control group is correspondingly arranged, and the mice of the control group are only injected with the corresponding solvent with the same amount once a day and subjected to the drug effect evaluation on the 18 th day. The evaluation index of the antitumor drug effect was T/C, which is the relative tumor proliferation rate, wherein T/C (%) is the relative tumor volume of experimental mice/the relative tumor volume of control mice × 100%.
The test results are shown in table 2.
TABLE 2
As can be seen from Table 2, the relative tumor proliferation rate of mouse a is similar to that of mouse c, and the relative tumor proliferation rate of mouse b is similar to that of mouse d under the action of five chemotherapeutic drugs, i.e., adriamycin, cisplatin, mitomycin, taxol and cyclophosphamide. In addition, the relative tumor proliferation rates of mice a, b, c, d were all lower than that of mouse e.
This shows that compared with cell lines, the biological characteristics of the bile duct cancer tissue are not changed by the preparation method of the present disclosure, and the biological characteristics of the transplant prepared by the method of the present disclosure are similar to the biological characteristics of the bile duct cancer native tissue, so that the compatibility between the xenograft model constructed by the transplant and the bile duct cancer tissue of the patient is higher, and the research value is higher.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (6)
1. A method for preparing a graft for constructing a biliary duct cancer xenograft model, the method comprising:
a. mixing matrigel and a bile duct cancer culture medium to obtain a bile duct cancer culture medium-matrigel mixture;
b. mixing the bile duct cancer tissue subjected to enzymolysis with the bile duct cancer culture medium-matrigel mixture, and performing culture and amplification to obtain a transplant containing bile duct cancer organoids;
the transplant containing the bile duct cancer organoid comprises a bile duct cancer organoid and a coating layer coated on the surface of the bile duct cancer organoid;
mixing the bile duct cancer tissue subjected to enzymolysis with the bile duct cancer culture medium-matrigel mixture in the step b, wherein the step b comprises the following steps:
mixing the bile duct cancer tissue subjected to enzymolysis with the bile duct cancer culture medium-matrigel mixture to enable the bile duct cancer tissue subjected to enzymolysis to be suspended in the bile duct cancer culture medium-matrigel mixture; wherein the content of the first and second substances,
the dosage of the bile duct cancer culture medium-matrigel mixture is 0.5-1 mL relative to 30000-40000 bile duct cancer tissue cells after enzymolysis;
the preparation method of the bile duct cancer tissue after enzymolysis comprises the following steps:
(1) mixing the bile duct cancer tissue fragments with an enzymolysis liquid and carrying out enzymolysis to obtain an enzymolysis material, wherein the bile duct cancer tissue comprises bile duct cancer primary focus tissue and/or bile duct cancer metastatic focus tissue; the enzymatic hydrolysate contains an RPMI 1640 culture medium, 2-5% of fetal bovine serum albumin and 1-3 mg/mL of collagen hydrolase; the dosage of the enzymolysis liquid is 5-10 parts by volume relative to 1 part by weight of the bile duct cancer tissue fragments;
(2) centrifuging the enzymolysis material, collecting the precipitate, and obtaining the bile duct cancer tissue after enzymolysis, wherein the centrifugation conditions comprise: the centrifugal force is 300-500 g, and the centrifugal time is 3-5 min.
2. The method of claim 1, wherein the bile duct cancer culture medium comprises RPMI 1640 medium and growth factors, the growth factors comprising penicillin, streptomycin, Y-276322 HCl, Y-27632, nicotinic acid, and bovine serum albumin.
3. The method according to claim 2, wherein the bile duct cancer culture medium contains 100-150U/mL of penicillin, 100-150 μ g/mL of streptomycin, 5-10 μ M of Y-276322 HCl, 5-10 μ M of Y-27632, 5-15 mM of nicotinic acid and 0.3-0.5 g/100mL of bovine serum albumin.
4. The method according to claim 1, wherein the amount of the cholangiocarcinoma medium used in the step a is 3-5 parts by volume based on 1 part by volume of the matrigel when mixing the matrigel and the cholangiocarcinoma medium.
5. The method of claim 1, wherein the bile duct cancer organoid has a diameter of 1.5-3 mm and the coating has a thickness of 1-3 mm.
6. A method of producing a biliary duct cancer xenograft mouse model, the method comprising:
implanting the graft prepared by the method of any one of claims 1 to 5 into subcutaneous and/or orthotopic tissues of an immunodeficient mouse to obtain the bile duct cancer xenograft mouse model.
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| WO2019121872A1 (en) * | 2017-12-20 | 2019-06-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the diagnosis and treatment of liver cancer |
| CN110656086B (en) * | 2019-10-11 | 2021-06-29 | 纳肽得(青岛)生物医药有限公司 | In vitro culture method of cancer organoid |
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