In-vitro culture method of hepatoblastoma organoid

Abstract

The invention relates to the technical field of organoid construction, and discloses an in-vitro culture method of hepatoblastoma organoid, which comprises the following steps: preparing solidified gel drops dispersed with hepatoblastoma cells; the method comprises the following steps: adding growth factors including a fibroblast growth factor, a eriochin and N-acetyl-L-cysteine into a basic culture medium to prepare a constructed culture solution; the constructed culture solution is adopted to immerse and solidify the gel drops, and the hepatoblastoma organoid is obtained through culture. According to the method, firstly, the hepatoblastoma cells are prepared into the solidified gel drops, then the specific construction culture solution is adopted for in-vitro culture, successful construction and subsequent stable passage of the hepatoblastoma organoid are achieved, operation is easy and safe, consumed time is short, cost is low, and the method is suitable for large-scale popularization and application. And a model capable of better simulating real organs of a human body is provided for research on pathogenesis and diagnosis and treatment means of hepatoblastoma.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid construction, and in particular to an in vitro culture method of hepatoblastoma organoids. Background Art

[0002] Hepatoblastoma is a malignant tumor that mainly occurs in children. Its pathological characteristics and difficulty in treatment make it very important to study the pathogenesis of hepatoblastoma and develop effective treatments. Currently, the research methods for hepatoblastoma are two-dimensional cell culture and animal models, which are difficult to fully simulate the complex pathological characteristics of hepatoblastoma.

[0003] As an emerging three-dimensional cell culture method, organoid technology can simulate the tissue structure, cell type and physiological functions of real organs, such as respiration, excretion, secretion and perception, by culturing three-dimensional structures and functions similar to real organs in vitro, thus helping to better understand the characteristics and functions of organs and explore their changes in health and disease. In addition, organoids can be cultured using cells of human origin, so they can more accurately reflect the response of human organs to drugs and the effects of diseases, which not only makes the research results more reliable, but also reduces the use of animals and avoids the ethical and moral issues brought about by animal experiments. Therefore, the establishment of a hepatoblastoma organoid model will provide a new approach for the study of hepatoblastoma and help the study of the pathogenesis, diagnosis and treatment of hepatoblastoma.

[0004] The existing method for constructing hepatoblastoma is to start from embryonic liver cells, construct embryonic liver organoids, and then use lentiviruses expressing specific genes to infect the embryonic liver organoids to transform into hepatoblastoma (such as patent CN116004722A). This method has the problems of complex operation, long construction cycle, high cost, and high laboratory safety level requirements. Summary of the invention

[0005] In order to solve the technical problems of the existing hepatoblastoma organoid construction method, which is complicated in operation, long in construction cycle, high in cost, and high in laboratory safety level requirements, the present invention provides an in vitro culture method of hepatoblastoma organoid. The method of the present invention can achieve the successful construction of hepatoblastoma organoid, and the operation is relatively simple and safe, the time required to obtain the organoid is short, and the cost is low.

[0006] The specific technical scheme of the present invention is: An in vitro culture method for hepatoblastoma organoids, comprising the steps of: I: Preparation of solidified gel droplets with dispersed hepatoblastoma cells; II: adding growth factors including fibroblast growth factor, forskolin and N-acetyl-L-cysteine ​​to the basal medium to prepare the constructed culture medium; III: Hepatoblastoma organoids were cultured by immersing the solidified gel droplets in the construction culture medium.

[0007] In the present invention, by first preparing hepatoblastoma cells into solidified gel droplets, and then using a construction culture medium containing specific components for in vitro culture, the construction of hepatoblastoma organoids is successfully achieved, and the hepatoblastoma organoids obtained thereby have a clear and complete structural morphology, can maintain stability during the growth process, and can be stably passaged in the construction culture medium. The method of the present invention is used to construct hepatoblastoma organoids, the operation is relatively simple, and the time required to obtain the organoids is relatively short.

[0008] In the construction culture medium used in the present invention, experiments have shown that fibroblast growth factor, forskolin and N-acetyl-L-cysteine ​​have an important influence on the construction of hepatoblastoma organoids. When any one of them is missing, the organoids will grow and develop slowly, have poor activity, or even fail to be constructed.

[0009] Preferably, in step II, in the constructed culture medium, the content of fibroblast growth factor is 105-320 ng / mL, the content of forskolin is 45-55 ng / mL, and the content of N-acetyl-L-cysteine ​​is 1-2 mmol / L.

[0010] Preferably, in step II, the fibroblast growth factor includes recombinant human fibroblast growth factor-10 (FGF-10) and basic fibroblast growth factor (bFGF), and the contents in the constructed culture medium are 100-300 ng / mL and 5-20 ng / mL, respectively.

[0011] Preferably, in step II, the growth factor further comprises: Growth factor name The content in the culture medium 4-Hydroxyethylpiperazineethanesulfonic acid 5~10mmol / L L-Glutamine 5~15mmol / L Serum-free supplement 1.5~4.5%vol Epidermal Growth Factor 50–100 ng / mL TGF-β type I receptor inhibitors 0.3~1.0μmol / L Bone morphogenetic protein antagonists 250~500 ng / mL Wnt signaling pathway enhancers 100–250 ng / mL Gastrin 10–30 ng / mL Rho-associated protein kinase inhibitors 5~15μM Furthermore, the serum-free additives include serum-free NSC-27 additives and serum-free N-2 additives, and their contents in the constructed culture medium are 1-3% vol and 0.5-1.5% vol, respectively.

[0012] In constructing the culture medium, each growth factor has a different effect on hepatoblastoma cells. The present invention adopts an overall system composed of the above-mentioned growth factors, and the growth factors cooperate with each other, thereby achieving the successful construction and stable propagation of hepatoblastoma organoids.

[0013] Although some culture media for constructing other tumor organoids have been reported in the prior art, the culture medium formula is not universal among different tumor organoids. In addition, the metabolic activities within cells are extremely complex. The components in the constructed culture medium affect the formation of organoids by affecting the metabolic activities of cells. The factors involved are multifaceted, intertwined and complex, which brings great difficulty to the research.

[0014] Preferably, in step II, the basal culture medium is DMEM / F12 culture medium.

[0015] Preferably, in step I, the volume of the solidified gel droplet is 50-100 μL, and each solidified gel droplet contains 1.5×10 5 ~2.5×10 5 indivual.

[0016] Preferably, in step I, the step of preparing hepatoblastoma cells comprises: washing and chopping human hepatoblastoma tissue, digesting it with collagenase I and dispase II in sequence, and collecting hepatoblastoma cells from the product.

[0017] Furthermore, after digestion with collagenase I and dispase II in sequence, the digestion products are collected, the red blood cells therein are lysed, the clumped tissues are filtered out, and the hepatoblastoma cells are collected from the filtrate.

[0018] Preferably, step I comprises: dispersing the hepatoblastoma cells in matrix gel, adding the matrix gel dropwise into a culture container, and forming solidified gel droplets containing dispersed hepatoblastoma cells after solidification.

[0019] Preferably, in step III, each solidified gel droplet uses 800-1000 μL of the culture medium, and the culture conditions are as follows: the ambient temperature is 35-37°C, the ambient CO 2 The concentration was 4-6%, and the culture medium was replaced every 2-3 days.

[0020] Preferably, in step III, after the hepatoblastoma organoids are constructed, they are subcultured, and the steps include: i: dissociating the hepatoblastoma organoids into cells; ii: preparing solidified gel droplets in which the cells obtained in step i are dispersed; iii: using the constructed culture medium to immerse the solidified gel droplets obtained in step ii for subculture.

[0021] After the hepatoblastoma organoids are constructed in the present invention, the construction culture fluid formula used can also be used for the subsequent stable propagation of the hepatoblastoma organoids.

[0022] Preferably, in step i, the hepatoblastoma organoids are dissociated into cells using Tryple digestion solution.

[0023] Preferably, in step ii, the cell content in the solidified gel droplets is set according to a passage ratio of 1:2 to 3.

[0024] Preferably, in step iii, each solidified gel droplet uses 800-1000 μL of the constructed culture medium, and the conditions for the subculture are as follows: the ambient temperature is 35-37°C, the ambient CO 2 The concentration is 4-6%.

[0025] Compared with the prior art, the present invention has the following advantages: In the present invention, by first preparing hepatoblastoma cells into solidified gel droplets and then using a specific construction culture medium for in vitro culture, the successful construction of hepatoblastoma organoids and the subsequent stable passaging are achieved. The operation is simple and safe, the time consumption is short, and the cost is low. This provides a model that can better simulate real human organs for the study of the pathogenesis, diagnosis and treatment methods of hepatoblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of constructing hepatoblastoma organoids in Example 1. Figure 1 A is the image taken under a microscope on day 0 of culture; Figure 1 B is the image taken under a microscope on the second day of culture; Figure 1 C is the image taken under a microscope on the 6th day of culture; Figure 1 D is an image taken under a microscope on the 10th day of culture.

[0027] Figure 2 The results of subculture of hepatoblastoma organoids in Example 1. Figure 2 A is the image taken under a microscope on day 0 of subculture; Figure 2 B is the image taken under a microscope on the second day of subculture; Figure 2 C is an image taken under a microscope on the 10th day of subculture.

[0028] Figure 3 This is the hepatoblastoma organoid obtained in Example 2.

[0029] Figure 4 This is the hepatoblastoma organoid obtained in Example 3.

[0030] Figure 5 Comparison of the construction of hepatoblastoma organoids with and without the addition of FGF-10. Figure 5A is an image taken under a microscope on the 6th day of culture according to the method of Example 1; Figure 5 B is an image taken under a microscope on the 6th day of cultivation according to the method of Comparative Example 1.

[0031] Figure 6 Comparison of the construction of hepatoblastoma organoids with and without forskolin. Figure 6 A is an image taken under a microscope on the 6th day of culture according to the method of Example 1; Figure 6 B is an image taken under a microscope on the 6th day of cultivation according to the method of Comparative Example 2.

[0032] Figure 7 Comparison of hepatoblastoma organoid construction with and without N-acetyl-L-cysteine. Figure 7 A is an image taken under a microscope on the 6th day of culture according to the method of Example 1; Figure 7 B is an image taken under a microscope on the 6th day of cultivation according to the method of Comparative Example 3.

[0033] Figure 8 Comparison of hepatoblastoma organoid construction using different basal culture media. Figure 8 A is an image taken under a microscope on the 6th day of culture according to the method of Example 1; Figure 8 B is an image taken under a microscope on the 6th day of cultivation according to the method of Comparative Example 4. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the embodiments.

[0035] An in vitro culture method for hepatoblastoma organoids, comprising the steps of: I: Preparation of solidified gel droplets with dispersed hepatoblastoma cells; II: adding growth factors including fibroblast growth factor, forskolin and N-acetyl-L-cysteine ​​to the basal medium to prepare the constructed culture medium; III: Hepatoblastoma organoids were cultured by immersing the solidified gel droplets in the construction culture medium.

[0036] In some specific embodiments, step I comprises: dispersing hepatoblastoma cells in matrix gel, adding the matrix gel dropwise into a culture container, and forming solidified gel droplets containing dispersed hepatoblastoma cells after solidification.

[0037] In some specific embodiments, in step I, the preparation step of hepatoblastoma cells includes: washing and chopping human hepatoblastoma tissue, digesting it with collagenase I and dispase II in sequence, collecting the digestion products, lysing the red blood cells therein, filtering out the clumped tissue, and collecting hepatoblastoma cells from the filtrate.

[0038] In some specific embodiments, in step I, the volume of the solidified gel droplet is 50-100 μL, and each solidified gel droplet contains 1.5×10 5 ~2.5×10 5 indivual.

[0039] In some specific embodiments, in step II, the basal culture medium is DMEM / F12 culture medium.

[0040] In some specific embodiments, in step II, in the constructed culture medium, the content of fibroblast growth factor is 105-320 ng / mL, the content of forskolin is 45-55 ng / mL, and the content of N-acetyl-L-cysteine ​​is 1-2 mmol / L.

[0041] In some specific embodiments, in step II, the fibroblast growth factor includes recombinant human fibroblast growth factor-10 and basic fibroblast growth factor, and the contents of the recombinant human fibroblast growth factor-10 and basic fibroblast growth factor in the constructed culture medium are 100-300 ng / mL and 5-20 ng / mL, respectively.

[0042] In some embodiments, in step II, the growth factor further comprises: Growth factor name The content in the culture medium 4-Hydroxyethylpiperazineethanesulfonic acid 5~10mmol / L L-Glutamine 5~15mmol / L Serum-free supplement 1.5~4.5%vol Epidermal Growth Factor 50–100 ng / mL TGF-β type I receptor inhibitors 0.3~1.0μmol / L Bone morphogenetic protein antagonists 250~500 ng / mL Wnt signaling pathway enhancers 100–250 ng / mL Gastrin 10–30 ng / mL Rho-associated protein kinase inhibitors 5~15μM In the above table, the serum-free additives include serum-free NSC-27 additives and serum-free N-2 additives, and their contents in the constructed culture medium are 1-3% vol and 0.5-1.5% vol, respectively.

[0043] In some specific embodiments, in step III, each solidified gel droplet uses 800-1000 μL of the constructed culture solution, and the culture conditions are as follows: the ambient temperature is 35-37°C, the ambient CO 2 The concentration was 4-6%, and the culture medium was replaced every 2-3 days.

[0044] In some specific embodiments, in step III, after constructing the hepatoblastoma organoid, subculture is performed, and the steps include: i: Hepatoblastoma organoids were dissociated into cells using Tryple digestion solution; ii: preparing a solidified gel droplet in which the cells obtained in step i are dispersed, wherein the cell content in the solidified gel droplet is set according to a passage ratio of 1:2 to 3; iii: Use the constructed culture solution to immerse the solidified gel droplets obtained in step ii, and use 800-1000 μL of the constructed culture solution for each solidified gel droplet for subculture. The subculture conditions are as follows: the ambient temperature is 35-37°C, the ambient CO 2 The concentration is 4-6%.

[0045] The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0046] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0047] The sources of materials (brands and product numbers) used in the embodiments and comparative examples of the present invention are provided below, but the protection scope of the present invention is not limited thereto: DMEM / F12 medium: Gibco, 11320033; L-glutamine: Gibco, 35050061; N-acetyl-L-cysteine: Yisheng, 50303ES05; TGF-β type I receptor inhibitor A83-01: Yisheng, 53002ES03; 4-Hydroxyethylpiperazineethanesulfonic acid: Yisheng, 60110ES60; Serum-free N-2 supplement: Yisheng, 60706ES08; Serum-free NSC-27 supplement: Yisheng, 60703ES10; Gastrin: Yisheng, 53007ES03; Bone morphogenetic protein antagonist Noggin: Yisheng, 92528ES08; Wnt signaling pathway enhancer R-Spondin-1: Yisheng, 92278ES20; Epidermal growth factor (EGF): Sino Biological, 10605-HNAE; Forskolin: Yisheng, 51001ES60; Basic fibroblast growth factor (bFGF): Sino Biological, 11609-HNCH; Recombinant human fibroblast growth factor-10 (FGF-10): Sino Biological, 10395-HNAE; Rho-associated protein kinase inhibitor (ROCK inhibitor) Y27632: Yisheng, 53006ES10.

[0048] Example 1 Follow the steps below to culture hepatoblastoma organoids in vitro (establishment and passaging): Step 1: Preparation of human hepatoblastoma cells After removing the connective tissue and necrotic parts in the human hepatoblastoma tumor tissue, wash it three times with pre-cooled PBS buffer supplemented with antibiotics, then transfer it to an ice box and evenly cut it into tissue fragments that can be sucked up with a 1mL pipette tip and do not stick.

[0049] Collagenase I was added to the DMEM / F12 medium to obtain a digestion solution I having a collagenase I content of 2 mg / mL, which was set aside. Dispase II was added to the DMEM / F12 medium to obtain a digestion solution II having a dispase II content of 2 μg / mL, which was set aside.

[0050] Transfer the tissue fragments to a 15 mL centrifuge tube, add digestion solution I, mix well with a pipette, and then transfer to a shaker, treat at 37°C for 45 min, then add two times the volume of pre-cooled PBS buffer to dilute, terminate the reaction, centrifuge and discard the supernatant. Add digestion solution II to the centrifuge tube with the precipitate, mix well with a pipette, transfer to a shaker, treat at 37°C for 20 min, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0051] Add 5 mL of red blood cell lysis solution to the centrifuge tube with the precipitate, mix with a pipette, and let stand at room temperature for 5 minutes. After the reaction, add 3 times the volume of pre-cooled PBS buffer to dilute and terminate the reaction. Mix with a pipette, filter with a filter with a pore size of 100 μm, centrifuge the collected filtrate at 1500 rpm for 5 minutes, and discard the supernatant. Add DMEM / F12 culture medium to the centrifuge tube with the precipitate to resuspend the cells. After completing the cell counting, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the precipitate to obtain human hepatoblastoma cells.

[0052] Step 2: Construction of hepatoblastoma organoids Add L-glutamine, N-acetyl-L-cysteine, TGF-β type I receptor inhibitor A83-01, 4-hydroxyethylpiperazineethanesulfonic acid, serum-free N-2 additive, serum-free NSC-27 additive, gastrin, bone morphogenetic protein antagonist Noggin, Wnt signaling pathway enhancer R-Spondin-1, epidermal growth factor, forskolin, basic fibroblast growth factor and recombinant human fibroblast growth factor-10 to liquid DMEM / F12 culture medium, fully dissolve and mix to obtain construction culture solution for use. The content of each component in the construction culture solution is as follows: On an ice box, add Matrigel to the human hepatoblastoma cells prepared in step 1 at a volume of 100 μL / 1.5×10 5 Cells were resuspended and then added to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets containing dispersed hepatoblastoma cells.

[0053] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2 The cells were cultured under the conditions of 10% CO 2 and 10% CO 2 , and the culture medium was replaced every 3 days. After 12 days of culture, the culture medium in the 24-well cell culture plate was removed to obtain hepatoblastoma organoids.

[0054] During the above culture process, the growth of hepatoblastoma organoids photographed at different culture times (days 0, 2, 6, and 10) is shown in Figure 1 The results showed that this example can obtain hepatoblastoma organoids relatively quickly, and the obtained organoids can maintain a stable, clear and complete morphological structure during the growth process.

[0055] Step 3: Passaging Hepatoblastoma Organoids After completing step 2, add PBS buffer to the 24-well cell culture plate containing hepatoblastoma organoids for three washes, and then add 1×Tryple digestion solution at a volume of 1.5 mL / well, and mix well by pipetting. Transfer the 24-well cell culture plate to an incubator and incubate at 37°C, 5% CO 2The cells were allowed to stand for 15 minutes under the conditions to allow the organoids to dissociate into cells, and then DMEM / F12 medium was added at a volume of 1.5 mL / well to dilute the cells to terminate the reaction. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded and transferred to an ice box. Matrigel matrix gel was added at a subculture ratio of 1:2 to resuspend the cell pellet, and then added dropwise to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets with dispersed organoid dissociated cells.

[0056] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2 Cultivated under conditions.

[0057] During the above culture process, the growth of hepatoblastoma organoids photographed at different culture times (days 0, 2, and 10) is shown in Figure 2 The results showed that the hepatoblastoma organoids constructed in this example can be stably subcultured in the constructed culture medium, and the hepatoblastoma organoids after subculture can maintain a relatively fast growth rate and a stable, clear and complete morphological structure.

[0058] Example 2 Follow the steps below to culture (construct) hepatoblastoma organoids in vitro: Step 1: Preparation of human hepatoblastoma cells After removing the connective tissue and necrotic parts in the human hepatoblastoma tumor tissue, wash it three times with pre-cooled PBS buffer supplemented with antibiotics, then transfer it to an ice box and evenly cut it into tissue fragments that can be sucked up with a 1mL pipette tip and do not stick.

[0059] Collagenase I was added to the DMEM / F12 medium to obtain a digestion solution I having a collagenase I content of 2 mg / mL, which was set aside. Dispase II was added to the DMEM / F12 medium to obtain a digestion solution II having a dispase II content of 2 μg / mL, which was set aside.

[0060] Transfer the tissue fragments to a 15 mL centrifuge tube, add digestion solution I, mix well with a pipette, and then transfer to a shaker, treat at 37°C for 45 min, then add two times the volume of pre-cooled PBS buffer to dilute, terminate the reaction, centrifuge and discard the supernatant. Add digestion solution II to the centrifuge tube with the precipitate, mix well with a pipette, transfer to a shaker, treat at 37°C for 20 min, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0061] Add 5 mL of red blood cell lysis solution to the centrifuge tube with the precipitate, mix with a pipette, and let stand at room temperature for 5 minutes. After the reaction, add 3 times the volume of pre-cooled PBS buffer to dilute and terminate the reaction. Mix with a pipette, filter with a filter with a pore size of 100 μm, centrifuge the collected filtrate at 1500 rpm for 5 minutes, and discard the supernatant. Add DMEM / F12 culture medium to the centrifuge tube with the precipitate to resuspend the cells. After completing the cell counting, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the precipitate to obtain human hepatoblastoma cells.

[0062] Step 2: Construction of hepatoblastoma organoids Add L-glutamine, N-acetyl-L-cysteine, TGF-β type I receptor inhibitor A83-01, 4-hydroxyethylpiperazineethanesulfonic acid, serum-free N-2 additive, serum-free NSC-27 additive, gastrin, bone morphogenetic protein antagonist Noggin, Wnt signaling pathway enhancer R-Spondin-1, epidermal growth factor, forskolin, basic fibroblast growth factor and recombinant human fibroblast growth factor-10 to liquid DMEM / F12 culture medium, fully dissolve and mix to obtain construction culture solution for use. The content of each component in the construction culture solution is as follows: On an ice box, add Matrigel to the human hepatoblastoma cells prepared in step 1 at a volume of 100 μL / 1.5×10 5 Cells were resuspended and then added to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets containing dispersed hepatoblastoma cells.

[0063] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2 The culture medium was replaced every 3 days. After 12 days of culture, the culture medium in the 24-well cell culture plate was removed to obtain hepatoblastoma organoids (see Figure 3 ).

[0064] Example 3 Follow the steps below to culture (construct) hepatoblastoma organoids in vitro: Step 1: Preparation of human hepatoblastoma cells After removing the connective tissue and necrotic parts in the human hepatoblastoma tumor tissue, wash it three times with pre-cooled PBS buffer supplemented with antibiotics, then transfer it to an ice box and evenly cut it into tissue fragments that can be sucked up with a 1mL pipette tip and do not stick.

[0065] Collagenase I was added to the DMEM / F12 medium to obtain a digestion solution I having a collagenase I content of 2 mg / mL, which was set aside. Dispase II was added to the DMEM / F12 medium to obtain a digestion solution II having a dispase II content of 2 μg / mL, which was set aside.

[0066] Transfer the tissue fragments to a 15 mL centrifuge tube, add digestion solution I, mix well with a pipette, and then transfer to a shaker, treat at 37°C for 45 min, then add two times the volume of pre-cooled PBS buffer to dilute, terminate the reaction, centrifuge and discard the supernatant. Add digestion solution II to the centrifuge tube with the precipitate, mix well with a pipette, transfer to a shaker, treat at 37°C for 20 min, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0067] Add 5 mL of red blood cell lysis solution to the centrifuge tube with the precipitate, mix with a pipette, and let stand at room temperature for 5 minutes. After the reaction, add 3 times the volume of pre-cooled PBS buffer to dilute and terminate the reaction. Mix with a pipette, filter with a filter with a pore size of 100 μm, centrifuge the collected filtrate at 1500 rpm for 5 minutes, and discard the supernatant. Add DMEM / F12 culture medium to the centrifuge tube with the precipitate to resuspend the cells. After completing the cell counting, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the precipitate to obtain human hepatoblastoma cells.

[0068] Step 2: Construction of hepatoblastoma organoids Add L-glutamine, N-acetyl-L-cysteine, TGF-β type I receptor inhibitor A83-01, 4-hydroxyethylpiperazineethanesulfonic acid, serum-free N-2 additive, serum-free NSC-27 additive, gastrin, bone morphogenetic protein antagonist Noggin, Wnt signaling pathway enhancer R-Spondin-1, epidermal growth factor, forskolin, basic fibroblast growth factor and recombinant human fibroblast growth factor-10 to liquid DMEM / F12 culture medium, fully dissolve and mix to obtain construction culture solution for use. The content of each component in the construction culture solution is as follows: On an ice box, add Matrigel to the human hepatoblastoma cells prepared in step 1 at a volume of 100 μL / 1.5×10 5 Cells were resuspended and then added to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets containing dispersed hepatoblastoma cells.

[0069] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2 The culture medium was replaced every 3 days. After 12 days of culture, the culture medium in the 24-well cell culture plate was removed to obtain hepatoblastoma organoids (see Figure 4 ).

[0070] Comparative Example 1 In this comparative example, recombinant human fibroblast growth factor-10 was not added to the culture medium, and the remaining operations were the same as those in Example 1. Specifically, the steps for in vitro culture (construction) of hepatoblastoma organoids in this comparative example are as follows: Step 1: Preparation of human hepatoblastoma cells After removing the connective tissue and necrotic parts in the human hepatoblastoma tumor tissue, wash it three times with pre-cooled PBS buffer supplemented with antibiotics, then transfer it to an ice box and evenly cut it into tissue fragments that can be sucked up with a 1mL pipette tip and do not stick.

[0071] Collagenase I was added to the DMEM / F12 medium to obtain a digestion solution I having a collagenase I content of 2 mg / mL, which was set aside. Dispase II was added to the DMEM / F12 medium to obtain a digestion solution II having a dispase II content of 2 μg / mL, which was set aside.

[0072] Transfer the tissue fragments to a 15 mL centrifuge tube, add digestion solution I, mix well with a pipette, and then transfer to a shaker, treat at 37°C for 45 min, then add two times the volume of pre-cooled PBS buffer to dilute, terminate the reaction, centrifuge and discard the supernatant. Add digestion solution II to the centrifuge tube with the precipitate, mix well with a pipette, transfer to a shaker, treat at 37°C for 20 min, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0073] Add 5 mL of red blood cell lysis solution to the centrifuge tube with the precipitate, mix with a pipette, and let stand at room temperature for 5 minutes. After the reaction, add 3 times the volume of pre-cooled PBS buffer to dilute and terminate the reaction. Mix with a pipette, filter with a filter with a pore size of 100 μm, centrifuge the collected filtrate at 1500 rpm for 5 minutes, and discard the supernatant. Add DMEM / F12 culture medium to the centrifuge tube with the precipitate to resuspend the cells. After completing the cell counting, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the precipitate to obtain human hepatoblastoma cells.

[0074] Step 2: Construction of hepatoblastoma organoids Add L-glutamine, N-acetyl-L-cysteine, TGF-β type I receptor inhibitor A83-01, 4-hydroxyethylpiperazineethanesulfonic acid, serum-free N-2 additive, serum-free NSC-27 additive, gastrin, bone morphogenetic protein antagonist Noggin, Wnt signaling pathway enhancer R-Spondin-1, epidermal growth factor, forskolin and basic fibroblast growth factor to liquid DMEM / F12 culture medium, fully dissolve and mix to obtain construction culture solution for use. The content of each component in the construction culture solution is as follows: On an ice box, add Matrigel to the human hepatoblastoma cells prepared in step 1 at a volume of 100 μL / 1.5×10 5 Cells were resuspended and then added to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets containing dispersed hepatoblastoma cells.

[0075] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2 The cells were cultured under 1% paraformaldehyde and 1% paraformaldehyde, and the culture medium was replaced every 3 days.

[0076] In the above culture process, the growth of hepatoblastoma organoids photographed on the 6th day of culture is shown in Figure 5 B, the results show that: compared with the situation on the 6th day of culture during the construction of hepatoblastoma organoids in Example 1 ( Figure 5 A) compared to the comparative example cultured to the 6th day ( Figure 5B) It was observed that fewer organoids were clearly formed and smaller in size. This indicates that if recombinant human fibroblast growth factor-10 is not added to the culture medium during the construction of hepatoblastoma organoids, the growth and development of hepatoblastoma organoids will be slow and the construction success rate will be low.

[0077] Comparative Example 2 In this comparative example, forskolin was not added to the culture medium, and the remaining operations were the same as those in Example 1. Specifically, the steps for in vitro culture (construction) of hepatoblastoma organoids in this comparative example are as follows: Step 1: Preparation of human hepatoblastoma cells After removing the connective tissue and necrotic parts in the human hepatoblastoma tumor tissue, wash it three times with pre-cooled PBS buffer supplemented with antibiotics, then transfer it to an ice box and evenly cut it into tissue fragments that can be sucked up with a 1mL pipette tip and do not stick.

[0078] Collagenase I was added to the DMEM / F12 medium to obtain a digestion solution I having a collagenase I content of 2 mg / mL, which was set aside. Dispase II was added to the DMEM / F12 medium to obtain a digestion solution II having a dispase II content of 2 μg / mL, which was set aside.

[0079] Transfer the tissue fragments to a 15 mL centrifuge tube, add digestion solution I, mix well with a pipette, and then transfer to a shaker, treat at 37°C for 45 min, then add two times the volume of pre-cooled PBS buffer to dilute, terminate the reaction, centrifuge and discard the supernatant. Add digestion solution II to the centrifuge tube with the precipitate, mix well with a pipette, transfer to a shaker, treat at 37°C for 20 min, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0080] Add 5 mL of red blood cell lysis solution to the centrifuge tube with the precipitate, mix with a pipette, and let stand at room temperature for 5 minutes. After the reaction, add 3 times the volume of pre-cooled PBS buffer to dilute and terminate the reaction. Mix with a pipette, filter with a filter with a pore size of 100 μm, centrifuge the collected filtrate at 1500 rpm for 5 minutes, and discard the supernatant. Add DMEM / F12 culture medium to the centrifuge tube with the precipitate to resuspend the cells. After completing the cell counting, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the precipitate to obtain human hepatoblastoma cells.

[0081] Step 2: Construction of hepatoblastoma organoids Add L-glutamine, N-acetyl-L-cysteine, TGF-β type I receptor inhibitor A83-01, 4-hydroxyethylpiperazineethanesulfonic acid, serum-free N-2 additive, serum-free NSC-27 additive, gastrin, bone morphogenetic protein antagonist Noggin, Wnt signaling pathway enhancer R-Spondin-1, epidermal growth factor, basic fibroblast growth factor and recombinant human fibroblast growth factor-10 to liquid DMEM / F12 culture medium, fully dissolve and mix to obtain construction culture solution for use. The content of each component in the construction culture solution is as follows: On an ice box, add Matrigel to the human hepatoblastoma cells prepared in step 1 at a volume of 100 μL / 1.5×10 5 Cells were resuspended and then added to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets containing dispersed hepatoblastoma cells.

[0082] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2 The cells were cultured under the conditions of 10% CO 2 and 10% CO 2 , and the culture medium was replaced every 3 days. After 12 days of culture, the culture medium in the 24-well cell culture plate was removed to obtain hepatoblastoma organoids.

[0083] In the above culture process, the growth of hepatoblastoma organoids photographed on the 6th day of culture is shown in Figure 6 B, the results show that: compared with the situation on the 6th day of culture during the construction of hepatoblastoma organoids in Example 1 ( Figure 6 A) compared to the comparative example cultured to the 6th day ( Figure 6 B) It was observed that fewer organoids were formed, and most of the formed organoids were small in size. This indicates that if forskolin is not added to the culture medium during the construction of hepatoblastoma organoids, the growth and development of hepatoblastoma organoids will be slow, and the construction success rate will be low.

[0084] Comparative Example 3 In this comparative example, N-acetyl-L-cysteine ​​was not added to the culture medium, and the remaining operations were the same as those in Example 1. Specifically, the steps for in vitro culture (construction) of hepatoblastoma organoids in this comparative example are as follows: Step 1: Preparation of human hepatoblastoma cells After removing the connective tissue and necrotic parts in the human hepatoblastoma tumor tissue, wash it three times with pre-cooled PBS buffer supplemented with antibiotics, then transfer it to an ice box and evenly cut it into tissue fragments that can be sucked up with a 1mL pipette tip and do not stick.

[0085] Collagenase I was added to the DMEM / F12 medium to obtain a digestion solution I having a collagenase I content of 2 mg / mL, which was set aside. Dispase II was added to the DMEM / F12 medium to obtain a digestion solution II having a dispase II content of 2 μg / mL, which was set aside.

[0086] Transfer the tissue fragments to a 15 mL centrifuge tube, add digestion solution I, mix well with a pipette, and then transfer to a shaker, treat at 37°C for 45 min, then add two times the volume of pre-cooled PBS buffer to dilute, terminate the reaction, centrifuge and discard the supernatant. Add digestion solution II to the centrifuge tube with the precipitate, mix well with a pipette, transfer to a shaker, treat at 37°C for 20 min, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0087] Add 5 mL of red blood cell lysis solution to the centrifuge tube with the precipitate, mix with a pipette, and let stand at room temperature for 5 minutes. After the reaction, add 3 times the volume of pre-cooled PBS buffer to dilute and terminate the reaction. Mix with a pipette, filter with a filter with a pore size of 100 μm, centrifuge the collected filtrate at 1500 rpm for 5 minutes, and discard the supernatant. Add DMEM / F12 culture medium to the centrifuge tube with the precipitate to resuspend the cells. After completing the cell counting, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the precipitate to obtain human hepatoblastoma cells.

[0088] Step 2: Construction of hepatoblastoma organoids Add L-glutamine, TGF-β type I receptor inhibitor A83-01, 4-hydroxyethylpiperazineethanesulfonic acid, serum-free N-2 additive, serum-free NSC-27 additive, gastrin, bone morphogenetic protein antagonist Noggin, Wnt signaling pathway enhancer R-Spondin-1, epidermal growth factor, forskolin, basic fibroblast growth factor and recombinant human fibroblast growth factor-10 to liquid DMEM / F12 culture medium, fully dissolve and mix to obtain construction culture solution for use. The content of each component in the construction culture solution is as follows: On an ice box, add Matrigel to the human hepatoblastoma cells prepared in step 1 at a volume of 100 μL / 1.5×10 5Cells were resuspended and then added to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets containing dispersed hepatoblastoma cells.

[0089] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2 The cells were cultured under the conditions of 10% CO 2 and 10% CO 2 , and the culture medium was replaced every 3 days. After 12 days of culture, the culture medium in the 24-well cell culture plate was removed to obtain hepatoblastoma organoids.

[0090] In the above culture process, the growth of hepatoblastoma organoids photographed on the 6th day of culture is shown in Figure 7 B, the results show that: compared with the situation on the 6th day of culture during the construction of hepatoblastoma organoids in Example 1 ( Figure 7 A) compared to the comparative example cultured to the 6th day ( Figure 7 B) It was observed that most of the formed organoids were small in size, indicating that if N-acetyl-L-cysteine ​​was not added to the culture medium during the construction of hepatoblastoma organoids, the growth and development of hepatoblastoma organoids would be slow.

[0091] Comparative Example 4 In this comparative example, when preparing the culture medium, the basic culture medium used was RPMI-1640 culture medium, and the rest of the operations were the same as those in Example 1. Specifically, the steps for in vitro culture (construction) of hepatoblastoma organoids in this comparative example are as follows: Step 1: Preparation of human hepatoblastoma cells After removing the connective tissue and necrotic parts in the human hepatoblastoma tumor tissue, wash it three times with pre-cooled PBS buffer supplemented with antibiotics, then transfer it to an ice box and evenly cut it into tissue fragments that can be sucked up with a 1mL pipette tip and do not stick.

[0092] Collagenase I was added to the DMEM / F12 medium to obtain a digestion solution I having a collagenase I content of 2 mg / mL, which was set aside. Dispase II was added to the DMEM / F12 medium to obtain a digestion solution II having a dispase II content of 2 μg / mL, which was set aside.

[0093] Transfer the tissue fragments to a 15 mL centrifuge tube, add digestion solution I, mix well with a pipette, and then transfer to a shaker, treat at 37°C for 45 min, then add two times the volume of pre-cooled PBS buffer to dilute, terminate the reaction, centrifuge and discard the supernatant. Add digestion solution II to the centrifuge tube with the precipitate, mix well with a pipette, transfer to a shaker, treat at 37°C for 20 min, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0094] Add 5 mL of red blood cell lysis solution to the centrifuge tube with the precipitate, mix with a pipette, and let stand at room temperature for 5 minutes. After the reaction, add 3 times the volume of pre-cooled PBS buffer to dilute and terminate the reaction. Mix with a pipette, filter with a filter with a pore size of 100 μm, centrifuge the collected filtrate at 1500 rpm for 5 minutes, and discard the supernatant. Add DMEM / F12 culture medium to the centrifuge tube with the precipitate to resuspend the cells. After completing the cell counting, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the precipitate to obtain human hepatoblastoma cells.

[0095] Step 2: Construction of hepatoblastoma organoids L-glutamine, N-acetyl-L-cysteine, TGF-β type I receptor inhibitor A83-01, 4-hydroxyethylpiperazineethanesulfonic acid, serum-free N-2 additive, serum-free NSC-27 additive, gastrin, bone morphogenetic protein antagonist Noggin, Wnt signaling pathway enhancer R-Spondin-1, epidermal growth factor, forskolin, basic fibroblast growth factor and recombinant human fibroblast growth factor-10 were added to the liquid RPMI-1640 culture medium, fully dissolved and mixed to obtain a construction culture solution for use. The content of each component in the construction culture solution is as follows: On an ice box, add Matrigel to the human hepatoblastoma cells prepared in step 1 at a volume of 100 μL / 1.5×10 5 Cells were resuspended and then added to a 24-well cell culture plate to form a gel drop in the center of each well. 100 μL was added to each well. The 24-well cell culture plate was transferred to an incubator at 37°C and 5% CO. 2 The mixture was allowed to stand for 15 min under the conditions to allow the matrix gel to solidify and form solidified gel droplets containing dispersed hepatoblastoma cells.

[0096] In a 24-well cell culture plate, slowly add the culture solution along the side wall at a volume of 800 μL / well to immerse the solidified gel droplet. Transfer the 24-well cell culture plate to an incubator at 37°C and 5% CO. 2The cells were cultured under the conditions of 10% CO 2 and 10% CO 2 , and the culture medium was replaced every 3 days. After 12 days of culture, the culture medium in the 24-well cell culture plate was removed to obtain hepatoblastoma organoids.

[0097] In the above culture process, the growth of hepatoblastoma organoids photographed on the 6th day of culture is shown in Figure 8 B, the results show that: compared with the situation on the 6th day of culture during the construction of hepatoblastoma organoids in Example 1 ( Figure 8 A) compared to the comparative example cultured to the 6th day ( Figure 8 B) No obvious organoids were observed. This indicates that the choice of basal culture medium will affect the construction effect during the construction of hepatoblastoma organoids. Hepatoblastoma organoids can be successfully obtained when DMEM / F12 is used as the basal culture medium, while when RPMI-1640 is used as the culture medium, it is not easy to form organoids with clear and complete morphological structures, and the construction success rate is low.

[0098] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for culturing hepatoblastoma organoids in vitro, characterized in that the steps include: I: Preparation of solidified gel droplets with dispersed hepatoblastoma cells; II: Growth factors including fibroblast growth factor, forskolin and N-acetyl-L-cysteine ​​are added to the basal medium to prepare the constructed culture medium; III: Hepatoblastoma organoids were cultured by immersing the solidified gel droplets in the construction culture medium.

2. The in vitro culture method according to claim 1, characterized in that: In step II, in the constructed culture medium, the content of fibroblast growth factor is 105-320 ng / mL, the content of forskolin is 45-55 ng / mL, and the content of N-acetyl-L-cysteine ​​is 1-2 mmol / L.

3. The in vitro culture method according to claim 1 or 2, characterized in that: In step II, the fibroblast growth factors include recombinant human fibroblast growth factor-10 and basic fibroblast growth factor, and the contents of the recombinant human fibroblast growth factor-10 and basic fibroblast growth factor in the culture medium are 100-300 ng / mL and 5-20 ng / mL, respectively.

4. The in vitro culture method according to claim 1 or 2, characterized in that: In step II, the growth factor is also include: 。 5. The in vitro culture method according to claim 4, characterized in that: The serum-free additives include serum-free NSC-27 additives and serum-free N-2 additives, and the contents of the serum-free additives in the constructed culture medium are 1-3% vol and 0.5-1.5% vol respectively.

6. The in vitro culture method according to claim 1, characterized in that: In step I, the volume of the solidified gel droplet is 50-100 μL, and each solidified gel droplet contains 1.5×10 5 ~2.5×10 5 indivual.

7. The in vitro culture method according to claim 1, characterized in that: In step III, each solidified gel droplet uses 800-1000 μL of the construction culture medium, and the culture conditions are as follows: the ambient temperature is 35-37° C., the ambient CO 2 concentration is 4-6%, and the construction culture medium is replaced every 2-3 days.

8. The in vitro culture method according to claim 1, characterized in that: In step III, after constructing the hepatoblastoma organoid, subculture is performed, and the steps include: i: Dissociation of hepatoblastoma organoids into cells; ii: preparing solidified gel droplets in which the cells obtained in step i are dispersed; iii: using the constructed culture medium to immerse the solidified gel droplets obtained in step ii for subculture.

9. The in vitro culture method according to claim 8, characterized in that: In step i, the hepatoblastoma organoids are dissociated into cells using Tryple digestion solution.

10. The in vitro culture method according to claim 8, characterized in that: In step ii, the cell content in the solidified gel droplets is set according to a passage ratio of 1:2 to 3.

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