CN114214283B - Method for culturing liver tumor organoids - Google Patents
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- CN114214283B CN114214283B CN202111678849.0A CN202111678849A CN114214283B CN 114214283 B CN114214283 B CN 114214283B CN 202111678849 A CN202111678849 A CN 202111678849A CN 114214283 B CN114214283 B CN 114214283B
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Abstract
The invention provides a method for culturing liver tumor organoids, belonging to the organoid culture field. The invention provides a liver tumor organoid culture container, which is a cell culture container with a liver extracellular matrix imprinting block array on the inner bottom surface; the area of the bottom surface not covered by the extracellular matrix of the liver is blocked by a blocking agent; the liver extracellular matrix imprinting block array consists of circular lattice points with the diameter of 50-300 mu m, and the distance between each two circular lattice points is 25-500 mu m. The invention also provides a culture method of the liver tumor organoids, which shortens the culture time, simplifies the culture mode, effectively controls the particle size and arrangement of the liver tumor organoids, and improves the repeatability and stability of drug screening. The cultured liver tumor organoids are used for drug screening, so that the method has higher efficiency and safety, and can be used for personalized treatment and guiding clinical medication.
Description
Technical Field
The invention belongs to the field of organoid culture, and particularly relates to a method for culturing liver tumor organoids.
Background
China is a large country of liver diseases, and is the country with the highest incidence rate of viral hepatitis and liver cancer and the largest number of patients in the world. According to the incidence of malignant tumors in China and the mortality report, the primary liver cancer is the fourth in incidence of malignant tumors in China, the mortality is the second, and the incidence of primary liver cancer in China accounts for 1/2 of the world. Wherein, the hepatocellular carcinoma (hepatocellular carcinoma, HCC) accounts for 85-90% of the primary liver cancer. Chemotherapy and immunotherapy appear to be the most effective treatments at present. Therefore, screening effective new drugs against liver cancer and making personalized medical treatment schemes for liver cancer patients are urgent clinical demands.
The establishment of an effective and stable tumor model with human tumor morphology and molecular characteristics has important significance for screening anti-tumor drugs. In recent years, more and more tumor models have emerged, such as commercial tumor cell lines, human tumor cell lines (PDC) and human tumor xenograft models (PDX), etc. Among them, commercial tumor cell lines and PDC are becoming fundamental research models for tumor research and drug screening due to their ease of manipulation and culture. However, commercial tumor cell lines and PDC cannot fully reveal the drug response in vivo due to the susceptibility to loss of tumor cell heterogeneity and their in vivo characteristics during in vitro culture. PDX retains the molecular and morphological characteristics of the primary tumor and reveals the heterogeneity of the primary tumor. However, PDX transplantation is inefficient and has a long experimental period, while originating from nude mice lacking an immune system, and specific tumor mutations may occur in the nude mice. Limiting the application of the drug in anti-tumor drug screening and tumor accurate treatment. Thus, there is an urgent need for a tumor model that more closely approximates the characteristics of the body. With the advent of oncology organoids, this need was met.
The tumor organoid is used as an in vitro cell 3D culture model, can highly simulate the structure and function of in vivo tumor tissues, can show the interaction between cells and matrixes, and has similar pathophysiological characteristics with in vivo differentiated tumor tissues in an ideal state. The human tumor organoids (PDO) retain individual tumor heterogeneity, and the screening of targeted drugs by PDO guides clinical drugs, thus showing great value in personalized treatment. The stability and fidelity of tumor organoids as drug screening models can be divided into three aspects: first, the tumor organoids retain the heterogeneity of the primary tumor during long-term culture, wherein the results of different generations of tumor organoids on anti-tumor drug screening are substantially consistent. Second, the results of drug screening of the tumor organoids are consistent with clinical medication of patients, which indicates that the tumor organoids have great potential value for accurate treatment. Thirdly, the tumor organoid can be regarded as a 'cell line' in the long-term culture process, and the same tumor organoid cell line is subjected to drug screening, so that the same tumor organoid has consistent drug screening results.
The tumor organoids can be passaged for a long time without losing or changing genetic information during subculture. The heterogeneity between intratumoral and external is preserved. At present, colorectal cancer, non-small cell lung cancer, breast cancer and pancreatic cancer tumor organoid models have been widely applied to drug evaluation, biomarker identification, biological research and personalized treatment. In the aspect of liver organoids, huch research team utilizes Lgr5+ stem cells to construct liver organoids to realize in vitro culture for up to 6 months, and meanwhile, corresponding tumor organoid models are established through clinical samples such as hepatocellular carcinoma, cholangiocarcinoma, hepatocellular cholangiocarcinoma and the like, and the histological and genetic characteristics of organ site specificity of the models can be maintained through methods such as histology, immunohistochemistry, genomics and the like. It can be seen that the continuous development of liver PDO technology provides a powerful technical support for realizing personalized treatment of liver cancer patients.
At present, the main culture mode of liver PDO is that Huch research team utilizes Matrigel to carry out 3D package culture in 96 or 384 pore plates, and the culture mode has the defects of complicated operation, higher cost, small scale, different organoid particle sizes, non-identical plane, difficult analysis and observation and the like, and cannot be produced and used in batch in large scale, thus bringing a plurality of inconveniences for screening anti-tumor drugs and evaluating drug effect. Meanwhile, the time for the first passage of liver PDO is about 4 weeks, and the time taken to prepare a sufficient amount of PDO for drug screening by amplification can be as much as 12 weeks. However, the time window for clinical neoadjuvant therapy and postoperative chemotherapy is typically 2-3 weeks.
Therefore, the method for quickly and simply operating and having low cost is found, the liver tumor organoids which are uniform in culture size, grow on the same plane and are convenient to analyze and observe are cultivated, and the method has important significance for realizing quick large-scale cultivation of the liver tumor organoids.
Disclosure of Invention
The invention aims to provide a method for culturing liver tumor organoids.
The invention provides a liver tumor organoid culture container, which is a cell culture container with an extracellular matrix imprinting block array on the inner bottom surface;
the area of the bottom surface not covered by the extracellular matrix of the liver is blocked by a blocking agent;
the liver extracellular matrix imprinting block array consists of circular lattice points with the diameter of 50-300 mu m, and the distance between each two circular lattice points is 25-500 mu m.
Further, the liver extracellular matrix blot block array consists of circular lattice points with the diameter of 100 μm, and the interval between each circular lattice point is 50 μm.
Further, the area of the liver extracellular matrix imprinting block array is 1-10 cm 2 ；
Preferably, the area of the liver extracellular matrix blotting block array is 4cm 2 。
Further, the cell culture container is a culture dish, a culture box, a culture plate or a culture bottle;
and/or the blocking agent is Pluronic F-127;
preferably, the mass fraction of Pluronic F-127 is 1-5%.
Further, the liver extracellular matrix is porcine liver extracellular matrix;
preferably, the preparation method of the liver extracellular matrix of the pig comprises the following steps:
(1) Preparing a pig whole liver decellularized scaffold by adopting a Triton-SDS-Triton portal vein infusion method;
(2) Freeze-drying and pulverizing the obtained decellularized liver stent, and performing enzymolysis by pepsin.
Preferably, the Triton-SDS-Triton portal vein infusion method is to take a new pig hepatic vein cannula, freeze and thaw the liver after removing blood, infuse with 1% Triton X-100 for 3 hours, then with 1% Sodium Dodecyl Sulfate (SDS) for 6 hours, infuse with 1% Triton X-100 for 3 hours, and finally with PBS for 2 hours to balance the decellularized liver, thus obtaining the decellularized liver scaffold. The flow rate throughout the course was 200mL/min during the perfusion.
Preferably, during enzymolysis, the concentration of the liver extracellular matrix obtained after crushing is 10mg/mL; the mass ratio of pepsin to liver extracellular matrix powder is 1:10. In addition, the pH was 2 during the enzymolysis, the temperature was room temperature, and the enzymolysis time was 72 hours.
Further, the concentration of the extracellular matrix of the liver is 0.05-0.5 mg/mL;
preferably, the concentration of the extracellular matrix of the liver is 0.1-0.5 mg/mL.
The liver tumor organoid culture container is prepared by adopting a PDMS micro-pattern printing technology: firstly, a silicon wafer with a specific pattern is etched by using laser as a template, a micro-array PDMS stamp with a convex circular micro-pattern with a specific size is obtained by reverse molding, liver extracellular matrix glue is coated on the surface of the PDMS stamp, the liver extracellular matrix glue is stamped on the bottom surface of a culture container to obtain an extracellular matrix micro-pattern array, and then the rest of the culture container is sealed by using a sealing agent to obtain the liver tumor organoid culture container.
The invention also provides a liver tumor organoid culture method, which comprises the following steps:
inoculating the liver cancer cells into the culture container, culturing for 4-6 h, washing off unattached cells, and culturing for 3-5 days to obtain the liver tumor organoid.
Further, the inoculation density of the liver cancer cells is 10 4～ 10 6 A personal/culture container;
and/or, the liver cancer cells are derived from a human;
preferably, the inoculation density of the liver cancer cells is 10 5 A culture vessel.
The invention also provides a liver tumor organoid, which is prepared by the method.
The invention also provides application of the liver tumor organoid in constructing a liver cancer model or screening medicines.
The invention provides a liver tumor organoid culture method, which adopts a liver ECM pattern microarray with specific lattice shape, size and density for culture, shortens the in-vitro amplification culture time of the liver tumor organoid, simplifies the culture mode of the liver tumor organoid, and effectively overcomes the defects of complicated culture operation, higher cost, small scale, different organoid particle sizes, difficult analysis and observation and the like of the existing tumor organoids. Effectively controls the grain size and arrangement of liver tumor organoids and improves the repeatability and stability of drug screening. The liver decellularized ECM provides a tumor microenvironment for liver tumor organoid culture, is favorable for maintaining tumor heterogeneity and simulates in-vivo liver cancer tissues. The liver tumor organoid is used for drug screening, has higher efficiency and safety, and simultaneously PDO can be used for personalized treatment to guide clinical medication.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 shows the course of culture of liver tumor organoids according to the present invention.
Fig. 2 shows the results after decellularization of whole pig liver: a is the general appearance of the liver after decellularization; b is H & E staining after decellularization; c is an electron microscope image after decellularization; d is red dyeing of the decellularized sirius; e is the dyeing of the type I collagen fiber after decellularization; f is the dyeing of the IV type collagen fiber after decellularization; g is the detection of the collagen content before and after decellularization; h is the detection of glycosaminoglycan (GAG) content before and after decellularization; * Represents p <0.05 compared to the control group; scale = 100 μm; in the figure, CONTROL is a CONTROL group without decellularization, and DLS is a liver without decellularization.
FIG. 3 shows the ECM powder obtained after decellularization of the liver (left) and the ECM gel obtained after enzymolysis (right).
FIG. 4 is a patterned microarray of spot shapes and sizes: a is a PDMS circular array dot pattern microarray; b is ECM circular lattice point pattern micro array; c is a PDMS square lattice point pattern microarray; d is ECM square lattice point pattern microarray; A. the diameters of the round lattice points or the side lengths of the square lattice points in B, C, D are sequentially 50 mu m, 75 mu m, 100 mu m, 150 mu m, 200 mu m and 300 mu m from left to right; scale in the figure: 200 μm.
FIG. 5 shows the results of liver tumor organoid culture: a is a circular lattice microarray with different diameters for culturing liver cancer cells for 6 hours; b is a circular lattice microarray with different diameters for culturing liver cancer cells for 3 days; c is the micro array culture of liver cancer cells with square lattice points with different side lengths for 6 hours; d is the liver cancer cells cultured by the different side length square lattice microarray for 3 days (scale: 100 μm).
FIG. 6 shows FDA staining and functional detection results of liver tumor organoids cultured in circular array spots of different diameters: a is FDA dyeing; b is a function test (scale: 50 μm).
Fig. 7 is FDA staining and functional detection of liver tumor organoids: a is paclitaxel; b is epirubicin; c is disulfiram.
Detailed Description
Unless otherwise indicated, the materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
The culture route of the liver tumor organoid is shown in figure 1, and comprises the following steps:
(1) Preparation of liver decellularized ECM: porcine whole liver decellularization scaffold was prepared using Triton-SDS-Triton portal vein infusion method. The obtained decellularized liver scaffold (extracellular matrix of liver) is further freeze-dried, crushed by a ball mill, and subjected to enzymolysis by pepsin to obtain the extracellular matrix (ECM) gel of the liver.
(2) Preparing a pattern micro-array master plate by adopting a PDMS micro-pattern printing technology: a silicon wafer with a specific pattern is etched by using laser as a template, the etching precision reaches 1 mu m, the etching area reaches 12 inches at maximum, and a Polydimethylsiloxane (PDMS) seal is obtained by reverse molding. The extracellular matrix gel (called extracellular matrix gel or matrigel) is coated on the surface of a seal, then a PDMS micro-pattern printing technology is utilized to lay an extracellular matrix gel pattern micro-array on the bottom of a cell culture dish which is subjected to non-attachment treatment, pluronic F-127 seals the space at the bottom of the rest culture dish, so that the added cells can only grow on the extracellular matrix gel pattern in an attached mode. And obtaining the liver decellularized ECM pattern microarray chip.
(3) Culturing liver tumor organoids: liver decellularized ECM pattern microarray chip is used for culturing liver tumor organoids, so that the in vitro amplification culture time is shortened, and the particle size and arrangement of the liver tumor organoids can be effectively controlled.
Example 1 method for culturing liver tumor organoids of the present invention
The method for culturing liver tumor organoids comprises the following steps:
(1) Preparation of pig liver decellularized extracellular matrix glue: after taking fresh pig liver, 10L of perfusate (NaCl 8.3g/L, KCl 0.5g/L, HEPES 2.4g/L, EGTA 0.95 g/L) was perfused through a venous cannula to remove blood from the liver. The liver was then frozen and thawed, perfused with 1% triton X-100 for 3 hours, followed by 1% Sodium Dodecyl Sulfate (SDS) for 6 hours, further perfused with 1% triton X-100 for 3 hours, and finally perfused with PBS for 2 hours to equilibrate the decellularized liver scaffolds (liver extracellular matrix). The flow rate throughout the course was 200mL/min during the perfusion. Decellularized Liver Scaffolds (DLSs) were then cut cubes of 1X 1cm were freeze-dried. Further pulverizing with ball mill to obtain liver extracellular matrix powder. The liver extracellular matrix powder is then digested with pepsin to obtain decellularized liver extracellular matrix gel (extracellular matrix gum). During enzymolysis, the extracellular matrix powder of the liver is dissolved in water with the concentration of 10mg/mL, and then pepsin is added for enzymolysis; the mass ratio of pepsin to liver extracellular matrix powder is 1:10; the pH value is 2 during enzymolysis, the temperature is room temperature, and the enzymolysis time is 72 hours.
The pig liver is white and transparent after decellularization, and after immunofluorescence, histochemical and ELISA, extracellular matrix protein is well reserved (figure 2), and further, the decellularized liver extracellular matrix gel is obtained through crushing and enzymolysis (figure 3).
(2) A silicon wafer with a specific pattern is etched by using laser as a template, and a PDMS stamp is obtained by reverse molding, so that a pattern microarray with convex circular array points (each circular array point has a diameter of 100 μm, the distance between each circular array point is 50 μm, and the microarray size is 4 cm) 2 ) And a PDMS stamp.
(3) The matrigel concentration is regulated by PBS, 1mL of the extracellular matrigel with the concentration of 100 mug/mL for decellularization of the pig liver is coated on the surface of the PDMS seal, and after incubation for 20 minutes at room temperature, the superfluous matrigel is washed off and dried in an incubator at 37 ℃.
(4) The PDMS stamp was pressed against the inner bottom surface of the cell non-attached culture dish, and a force of 0.2 newton was applied for 10 minutes, and the stamp was removed after the pattern was formed on the bottom surface of the culture dish.
(5) Pluronic F-127 (Prulinix F-127, a polypropylene glycol and ethylene oxide addition polymer (polyether)) was added in a mass fraction of 1% and incubated at room temperature for 2 hours, and the space on the inner bottom surface of the remaining dish was closed to obtain an "extracellular matrix pattern microarray" having an adhesive effect on cells. Before the prepared microarray culture dish is used, PBS is used for cleaning for 3 times, so that the culture dish substrate cannot be completely dried, and then cells are inoculated.
(6) Human hepatoma cells (HepG 2, inoculum size 1.5X10) were inoculated into a petri dish 5 Dish) for 6 hours at 37 ℃, washing off unattached cells, and culturing for 3 days to form macroscopic liver tumor organoids. The liver tumor organoids are all arranged on the same plane, and have uniform size and controllable quantity.
Human hepatoma cells were defined to attach to "extracellular matrix patterned microarrays" for ingrowth. The culture medium used in the step is DMEM high-sugar culture medium, and contains 10% of fetal calf serum and 1% of diabody.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 screening of Pattern microarrays in the liver tumor organoid culture method of the invention
An extracellular matrix patterned microarray was prepared by the method described in example 1, except that the shape (the spots may be circular spots or square spots) and size (the diameter of the circular spots or the side length of the square spots is selected from 50. Mu.m, 75. Mu.m, 100. Mu.m, 150. Mu.m, 200. Mu.m, or 300. Mu.m) of spots in the patterned microarray were changed, the interval between each spot was 50. Mu.m, and the microarray size was 4cm 2 . Each pattern microarray is shown in fig. 4.
Liver tumor organoids were cultured using the above extracellular matrix patterned microarray according to the method described in example 1, the patterned microarray was washed 1 time with PBS before use to ensure that the culture substrate was not completely dried, and human hepatoma cells were inoculated (HepG 2, inoculum size 1.5X10 5 /dish). The patterned microarray was grown on spots defined in the extracellular matrix gel patterned microarray by restricting the growth and adhesion space of cells on the bottom of the cell culture dish, and cultured for 3 days to form liver tumor organoids (fig. 5).
The optimal shape and size of the spots in the microarray is selected according to the morphology of the liver tumor organoids and the dead-alive function. The research finds that: (1) liver tumor organoids cultured using circular lattice points had better morphology than square lattice points, favoring tumor study (fig. 5); (2) with the increase of the diameter of the circular lattice point, the number of dead cells in the cultured liver tumor organoids is gradually increased, and according to the fluorescent quantitative PCR analysis, the functions of the liver tumor organoids are firstly increased and then weakened with the increase of the diameter of the circular lattice point. Finally, a circular array spot with a diameter of 100 μm was selected as the optimal array spot for culturing liver tumor organoid pattern microarrays (FIG. 6).
Test example 2 screening evaluation of liver tumor organoid micropattern chip drug
The liver tumor organoids with the diameter of 100 μm cultured in example 1 were selected, and the antitumor drugs paclitaxel, epirubicin and disulfiram which are commonly used in the market were selected, and the liver tumor organoids were used for drug screening evaluation, with the drug concentration of 5-100 μg/ml. FDA staining analysis showed that: the liver tumor organoid cultured by the invention can effectively screen the influence of medicine types, medicine doses and medicine action time on liver tumor. The study shows that the drug type, the drug dosage and the drug action time have obvious drug sensitivity differences, wherein the anti-liver tumor drug effect of the apparent doxorubicin is optimal (figure 7). The research shows that after the liver tumor organoid is controlled and arranged, the result of drug screening can be better analyzed and observed, and the liver tumor organoid pattern microarray chip is applied to the potential value of personalized treatment and anti-tumor drug screening.
The experiment shows that the liver decellularized ECM gel can be used as a biological ink for pattern microarray printing, and the printing efficiency is high. By comparing the shape, the size and the condition of the lattice points in the microarray to the shape, the death and the function of the cultured liver tumor organoid, the optimal lattice point in the microarray is selected to be a circular lattice point with the diameter of 100 mu m. Namely, the optimal pattern microarray for culturing liver tumor organoids is: circles with a diameter of 100 μm are used as lattice points, and the interval between each circular lattice point is 50 μm.
Then, three common antitumor drugs (taxol, epirubicin and disulfiram) in the market are selected for drug screening evaluation, and experimental results show that the liver tumor organoid cultured by the invention can effectively screen the influence of drug types, drug doses and drug action time on liver tumors. The liver tumor organoid has dependence on drug type, drug dosage and drug action time, wherein epirubicin has strongest drug sensitivity. Meanwhile, the research discovers that the liver tumor organoids are cultured by means of ECM pattern microarrays, and the observation and analysis of drug response conditions are facilitated. The micro-pattern chip of liver tumor organoid is shown to have great potential value in personalized medicine screening, guiding clinical medicine application according to laboratory data results.
In summary, the invention provides a liver tumor organoid culture method, which adopts a liver ECM pattern microarray with specific lattice shape, size and density for culture, shortens the in-vitro amplification culture time of the liver tumor organoid, simplifies the liver tumor organoid culture mode, and effectively overcomes the defects of complicated operation, higher cost, small scale, different organoid particle sizes, difficult analysis and observation and the like of the existing tumor organoids. Effectively controls the grain size and arrangement of liver tumor organoids and improves the repeatability and stability of drug screening. The liver decellularized ECM provides a tumor microenvironment for liver tumor organoid culture, is favorable for maintaining tumor heterogeneity and simulates in-vivo liver cancer tissues. The liver tumor organoid is used for drug screening, has higher efficiency and safety, and simultaneously PDO can be used for personalized treatment to guide clinical medication.
Claims (13)
1. A liver tumor organoid culture container, characterized in that: the cell culture container is provided with a liver extracellular matrix imprinting block array on the inner bottom surface;
the area of the bottom surface not covered by the extracellular matrix of the liver is blocked by a blocking agent;
the liver extracellular matrix imprinting block array consists of circular lattice points with the diameter of 100 mu m, and the interval between each circular lattice point is 50 mu m;
the concentration of the extracellular matrix of the liver is 0.05-0.5 mg/mL.
2. The liver tumor organoid culture container of claim 1 wherein: the area of the liver extracellular matrix imprinting block array is 1-10 cm 2 。
3. The liver tumor organoid culture container of claim 2, wherein: the area of the liver extracellular matrix imprinting block array is 4cm 2 。
4. A liver tumor organoid culture vessel according to any one of claims 1 to 3, wherein: the cell culture container is a culture dish, a culture box, a culture plate or a culture bottle;
and/or the blocking agent is Pluronic F-127.
5. The liver tumor organoid culture container of claim 4 wherein: the mass fraction of Pluronic F-127 is 1-5%.
6. A liver tumor organoid culture vessel according to any one of claims 1 to 3, wherein: the liver extracellular matrix is porcine liver extracellular matrix.
7. The liver tumor organoid culture container of claim 6 wherein: the preparation method of the pig liver extracellular matrix comprises the following steps:
(1) Preparing a pig whole liver decellularized scaffold by adopting a Triton-SDS-Triton portal vein infusion method;
(2) Freeze-drying and pulverizing the obtained decellularized liver stent, and performing enzymolysis by pepsin.
8. The liver tumor organoid culture container of claim 1 wherein: the concentration of the extracellular matrix of the liver is 0.1-0.5 mg/mL.
9. A liver tumor organoid culture method is characterized in that: the method comprises the following steps:
inoculating liver cancer cells into the culture container according to any one of claims 1-8, culturing for 4-6 h, washing off unattached cells, and culturing for 3-5 days to obtain liver tumor organoids.
10. The culture method according to claim 9, wherein: the inoculation density of the liver cancer cells is 10 4 ～10 6 A personal/culture container;
and/or, the liver cancer cells are derived from human.
11. The culture method according to claim 10, wherein: the inoculation density of the liver cancer cells is 10 5 A culture vessel.
12. A liver tumor organoid, characterized in that: a liver tumor organoid prepared by the method of any one of claims 9 to 11.
13. Use of the liver tumor organoid of claim 12 in constructing a liver cancer model or in drug screening.
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