Method for culturing three-dimensional model of liver cancer organoid

Abstract

The invention discloses a culture method of a liver cancer organoid three-dimensional model, which comprises the following steps: S1, washing a liver cancer sample from a patient, cutting into pieces, digesting with digestive juice to obtain liver cancer cells, resuspending liver cancer cell precipitates by using a liver cancer organoid isolation medium, inoculating the liver cancer cells into a low-adsorption culture plate, and culturing to obtain a liver cancer organoid three-dimensional model; waiting for aggregation of cell clusters; s2, after the cell clusters are gathered, the culture solution and the cell clusters are transferred into a centrifugal tube, supernate is sucked and discarded after centrifugation, Matrigel matrigel is added and evenly mixed, and the mixture is inoculated into a non-low-adsorption culture plate preheated in advance; s3, placing the non-low-adsorption culture plate in an incubator at 37 DEG C to incubate for a preset time, then turning over the culture plate, and then inverting the culture plate to continue incubation; s4, after incubation is finished, a liver cancer organoid isolation medium is added for culture for a period of time, and then a liver cancer organoid amplification medium is used for continuous culture until passage. Not only can rapid agglomeration growth of liver cancer cells be realized, but also long-term stable passage of subsequent liver cancer organs can be ensured.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a method for culturing a three-dimensional liver cancer organoid model. Background Art

[0002] Liver cancer models are crucial for understanding the occurrence and development of liver cancer, new drug development, and personalized diagnosis and treatment. Although traditional two-dimensional cell culture models are easy to operate, they cannot simulate the key characteristics and complex microenvironment of liver cancer tissue. Therefore, researchers are gradually turning from traditional two-dimensional cell culture methods to more complex three-dimensional liver cancer models. This shift aims to more accurately capture the physiological characteristics of liver cancer in order to create a liver cancer cell growth model that is closer to the real in vivo environment.

[0003] As an innovative three-dimensional cell culture technology, the liver cancer organoid model successfully simulates the tissue structure and complex interactions between cells of liver cancer. This model provides a more realistic platform for in-depth research on key biological characteristics of liver cancer, such as heterogeneity, invasiveness, and drug resistance. Although the liver cancer organoid model has shown significant advantages in simulating the tumor microenvironment, it still faces some challenges in existing culture technologies. For example, although the traditional matrix gel embedding method can support the long-term growth of liver cancer organoids, its growth rate is slow in the early stage of culture, which to some extent limits the model's potential for application in rapid screening and early drug testing.

[0004] Therefore, it is necessary to provide a new culture method for a three-dimensional liver cancer organoid model that can not only achieve rapid cluster growth of liver cancer cells, but also ensure the subsequent long-term and stable passaging of liver cancer organoids. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for culturing a three-dimensional model of liver cancer organoids, which can not only achieve rapid cluster growth of liver cancer cells, but also ensure the subsequent long-term and stable propagation of liver cancer organoids.

[0006] In order to solve the above technical problems, the present invention provides a method for culturing a three-dimensional liver cancer organoid model, comprising the following steps: S1: washing, chopping and digesting a patient-derived liver cancer sample to obtain liver cancer cells, resuspending the liver cancer cell pellet with a liver cancer organoid separation medium, and then inoculating the liver cancer cells into a low-adsorption culture plate for culture until the cell clusters are aggregated; S2: after the cell clusters are aggregated, the culture medium and the cell clusters are transferred to a centrifuge tube, the supernatant is discarded after centrifugation, Matrigel matrix gel is added and mixed, and then inoculated into a preheated non-low-adsorption culture plate; S3: the non-low-adsorption culture plate is first placed upright in a 37°C incubator for a predetermined time, and then the culture plate is turned over and inverted to continue incubation; S4: after the incubation is completed, the liver cancer organoid separation medium is added for a period of time, and then the liver cancer organoid expansion medium is replaced and continued to be cultured until passage.

[0007] Furthermore, the step S1 comprises: washing the patient-derived liver cancer sample with 1×PBS for 3-5 times, and then cutting it into pieces with a volume of 0.5 mm using a surgical instrument. 3 -1mm 3 The minced tissue was then added to a digestion solution containing 75 U / ml IV collagenase, 0.5 U / mL DNase I (deoxyribonuclease I) and 125 g / mL Dispase, and incubated at 37°C for 30 min-60 min to obtain liver cancer cells.

[0008] Furthermore, step S1 includes: setting the centrifuge temperature to 4°C, the centrifugal speed to 200g, centrifuging for 4-6 minutes, discarding the supernatant, and then resuspending the liver cancer cell pellet with liver cancer organoid isolation medium, inoculating the liver cancer cells into a 24-well low-adsorption culture plate and culturing them for 2-3 days until they aggregate into cell clusters.

[0009] Furthermore, step S2 includes: using a pipette to gently aspirate the cell mass and transfer it to a 15 ml centrifuge tube, setting the centrifuge temperature to 4°C, the centrifugal speed to 200g, centrifuging for 4-6 minutes, and discarding the supernatant; adding Matrigel matrix gel and gently mixing, and inoculating it into a preheated 24-well non-low-adsorption culture plate.

[0010] Furthermore, the step S3 comprises: placing the non-low-adsorption culture plate upright in a 37° C. incubator for 2-4 minutes, and then turning the non-low-adsorption culture plate upside down and continuing to incubate for 9-11 minutes.

[0011] Furthermore, the step S4 comprises: adding liver cancer organoid isolation medium after the incubation is completed and culturing for 4-5 days, and then changing to liver cancer organoid expansion medium and continuing culturing until passaging.

[0012] Furthermore, the culture medium for liver cancer organoid isolation was: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL Penicillin / Streptomycin, 1-20mM Nicotinamide, 1-20mM N-Acetylcysteine, 20-200μg / ml Primocin, 40-100 ng / ml EGF, 20-60ng / ml HGF, 20-200ng / ml FGF-10, 1-2×B-27 (Vatamin-Free), 1-2×N-2, 10-100nM gastrin I, 10-100uM Forskolin, 1-20μM Y-27632, 1-10μM A83-01, 1-10nM Dexamethasone, and the basal culture medium was Advanced DMEM / F12.

[0013] Furthermore, the liver cancer organoid expansion medium is: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL Penicillin / Streptomycin, 1-20mM Nicotinamide, 1-20mM N-Acetylcysteine, 20-200μg / ml Primocin, 100-800ng / ml R-spondin-1, 40-100 ng / ml EGF, 20-60ng / ml HGF, 20-200ng / ml FGF-10, 1-2×B-27 (Vatamin-Free), 1-2×N-2, 10-100nM gastrin I, 10-100uM Forskolin, 1-20μM Y-27632, 1-10μM A83-01, and the basal medium is Advanced DMEM / F12.

[0014] Compared with the prior art, the present invention has the following advantages and effects: the method for culturing a three-dimensional model of liver cancer organoids provided by the present invention achieves rapid aggregation and morphogenesis of tumor organoids by optimizing the culture conditions, and shortens the time of organoid formation compared to traditional methods, thereby shortening the culture time. The present invention can obtain a sufficient number of tumor organoids in a relatively short period of time, significantly improves experimental efficiency and throughput, accelerates scientific research and drug screening processes, and also reduces the costs of reagents and consumables, thereby improving experimental efficiency and reducing economic costs. The present invention solves the problem that organoids are difficult to maintain for a long time in low-adsorption culture plates, achieves long-term and stable culture of tumor organoids, retains the good biological characteristics of the source tissue, and is conducive to in-depth research on the biological behavior of tumors. Therefore, the method for culturing a three-dimensional model of liver cancer organoids provided by the present invention can not only achieve rapid aggregation growth of liver cancer cells, but also ensure the subsequent long-term and stable passage of liver cancer organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a flow chart of the culture of a three-dimensional liver cancer organoid model in an embodiment of the present invention; Figure 2 (a), Figure 2 (b), Figure 2 (c) are bright field images of liver cancer organoids cultured for 10 days in the embodiment of the present invention, comparative example 1, and comparative example 2, respectively; Figure 3 The average diameter size diagram of the three-dimensional liver cancer organoid model in the embodiment of the present invention and the comparative example; Figure 4 A diagram showing the number of cells in liver cancer organoids after digestion in the examples and comparative examples of the present invention; Figure 5 (a) Figure 5 (b) are H&E staining images of liver cancer organoids in the examples of the present invention and the comparative examples, respectively; Figure 6 (a) Figure 6 (b) are immunohistological staining images of liver cancer organoids in the embodiment of the present invention and the comparative example, respectively. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0017] See also Figure 1 The present invention provides a method for culturing a three-dimensional liver cancer organoid model, comprising the following steps: S1: The liver cancer cells were obtained by washing, cutting and digesting the patient-derived liver cancer samples with digestion solution. The liver cancer cell pellet was resuspended in liver cancer organoid isolation medium and then inoculated into low-adsorption culture plates for culture until the cells aggregated. The patient-derived liver cancer samples were first washed with 1× PBS for 3-5 times and then cut into 0.5 mm2 pieces with surgical instruments. 3 -1mm 3 The minced tissue was then added to a digestive solution containing 75U / ml IV collagenase, 0.5U / mL DNase I (deoxyribonuclease I) and 125g / mL Dispase (dispersase) and incubated at 37°C for 30-60min to obtain liver cancer cells. The centrifuge was set at 4°C and 200g for 4-6min. The supernatant was discarded and the liver cancer cell pellet was resuspended in liver cancer organoid separation medium. The liver cancer cells were inoculated into a 24-well low-adsorption culture plate and cultured for 2-3 days until they aggregated into cell clusters. Low-adsorption culture plates refer to plates that are coated with special amphiphilic polymers on the surface of the plate, which can effectively inhibit cell attachment and meet the needs of suspended cell culture.

[0018] The culture medium for liver cancer organoid isolation is: 2-20mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 0.5-2.0×Glutamax, 50-200U / mL Penicillin / Streptomycin, 1-20mM Nicotinamide, 1-20mM N-Acetylcysteine, 20-200μg / ml Primocin, 40-100 ng / ml EGF (Epidermal Growth Factor), 20-60ng / ml HGF (Hepatocyte Growth Factor), 20-200ng / ml FGF-10, 1-2× B-27 (Vatamin-Free, without vitamin additives), 1-2×N-2, 10-100nM gastrin I, 10-100uM Forskolin (adenylate cyclase activator), 1-20μM Y-27632, 1-10μM A83-01, 1-10nM Dexamethasone, and the basic culture medium is Advanced DMEM / F12.

[0019] Among them, the buffer HEPES can stabilize the pH value and ensure the effectiveness and stability of the culture medium. Glutamax additive is a substitute for L-glutamine, with better stability and can improve cell health. Penicillin / streptomycin dual antibiotics can effectively inhibit the growth of most Gram-positive and Gram-negative bacteria, thereby effectively controlling bacterial contamination of cells; Nicotinamide (Nicotinamide) functions to improve cell viability. The antioxidant is N-acetylcysteine ​​(N-Acetylcysteine), which helps to reduce oxidative stress and cell damage. Primocin antibiotics can effectively prevent bacterial, mycoplasma and fungal contamination, while cytotoxicity is controlled to a lower level. Epidermal growth factor EGF can promote cell proliferation. Hepatocyte growth factor HGF promotes cell proliferation, migration and morphogenesis. B-27 (vitamin-free additive) is a nutrient and a substitute for serum. Additive N-2 is a chemically defined serum-free additive, mainly used for the growth and expression of late mitotic neurons in neuroblastoma and peripheral nervous system (PNS) and central nervous system (CNS) sources. Gastrin I can promote cell proliferation. Forskolin (adenylate cyclase activator) can activate adenylate cyclase, which is used to increase the level of intracellular cAMP and promote cell proliferation. Y-27632 is a rho-associated protein kinase (ROCK inhibitor) that can target and bind to the catalytic sites of ROCK-1 and ROCK-2, thereby inhibiting their kinase activity. It has the function of protecting cells from anoikis and is of great significance for maintaining the cell activity of primary cells. A83-01 is an inhibitor of the Activin / NODAL / TGF-β pathway, inhibits the activity of ALK5 / 4 / 7 kinases, and maintains the self-renewal of liver cancer cells. Dexamethasone can induce cell maturation and increase cell differentiation. Advanced DMEM / F12 is a widely used basal medium that can culture mammalian cells with reduced fetal bovine serum (FBS); compared with classic DMEM / F-12, the amount of serum added can be reduced by 50–90% without changes in cell growth rate or morphology.

[0020] S2: After the cell clusters are aggregated, the culture medium and cell clusters are transferred to a centrifuge tube. After centrifugation, the supernatant is discarded, Matrigel matrix gel is added and mixed, and then inoculated into a preheated non-low-adsorption culture plate; specifically, the cell clusters are gently transferred to a 15ml centrifuge tube with a pipette, the centrifuge temperature is set to 4°C, the centrifugal speed is 200g, centrifuge for 4-6 minutes, and the supernatant is discarded; Matrigel matrix gel is added and mixed gently, and inoculated into a preheated 24-well non-low-adsorption culture plate. Non-low-adsorption culture plates are the most common type of cell culture plates. Their surfaces have not been specially treated or the treatment degree is low, and they have a high adsorption capacity for cells and other biological molecules.

[0021] S3: The non-low-adsorption culture plate is first placed upright in a 37° C. incubator for a predetermined time, and then the culture plate is flipped and then inverted to continue incubation. Specifically, the non-low-adsorption culture plate is first placed upright in a 37° C. incubator for 2-4 minutes, and then the non-low-adsorption culture plate is flipped and incubated for 9-11 minutes.

[0022] S4: After the incubation, add the liver cancer organoid isolation medium and culture for a period of time, then change to the liver cancer organoid expansion medium and continue to culture until passaging. Specifically, after the incubation, add the liver cancer organoid isolation medium and culture for 4-5 days, then change to the liver cancer organoid expansion medium and continue to culture until passaging.

[0023] The culture medium for liver cancer organoid expansion is: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mLPenicillin / Streptomycin, 1-20mM Nicotinamide, 1-20mM N-Acetylcysteine, 20-200μg / ml Primocin, 100-800ng / ml R-spondin-1, 40-100 ng / ml EGF, 20-60ng / ml HGF, 20-200ng / ml FGF-10, 1-2× B-27 (Vatamin-Free), 1-2×N-2, 10-100nM gastrin I, 10-100uMForskolin, 1-20μM Y-27632, 1-10μM A83-01, and the basal culture medium is Advanced DMEM / F12.

[0024] Among them, R-spondin-1 recombinant protein can activate the Wnt-β-catenin signaling pathway and promote cell proliferation.

[0025] Comparative Example 1 Patient-derived liver cancer samples (surgical tissue, puncture or gastrointestinal endoscopic biopsy samples) were washed 3-5 times with 1× PBS and then cut into minced meat (0.5 mm 3 -1mm 3 ). Next, add the chopped tissue to the digestion solution (containing 75 U / mL IV collagenase, 0.5 U / mL DNase I and 125g / mL Dispase) and incubate at 37°C for 30min-60min to obtain liver cancer cells. Set the centrifuge temperature to 4°C, the centrifugal speed to 200g, centrifuge for 5min, and discard the supernatant. Next, resuspend the cell pellet with liver cancer organoid isolation medium and inoculate it into a 24-well low-adsorption culture plate for 1 week, then change to liver cancer organoid expansion medium and continue culturing until passage.

[0026] The hepatoma organoid isolation culture medium and the hepatoma organoid expansion culture medium are the same as those in the embodiment of the present invention.

[0027] Comparative Example 2 Patient-derived liver cancer samples (surgical tissue, puncture or gastrointestinal endoscopic biopsy samples) were washed 3-5 times with 1× PBS and then cut into minced meat (0.5 mm 3 -1mm 3 ). Next, add the chopped tissue to the digestion solution (containing 75 U / mL IV collagenase, 0.5 U / mL DNase I and 125g / mL Dispase), and incubate at 37°C for 30min-60min to obtain liver cancer cells. Set the centrifuge temperature to 4°C, the centrifugal speed to 200g, centrifuge for 5min, and discard the supernatant. Next, resuspend the cell pellet with Matrigel matrix gel, inoculate it into a preheated 24-well non-low-adsorption culture plate, place it upright at 37°C for 3min, then flip the culture plate upside down and continue incubation for 10min. After the incubation, add liver cancer organoid isolation medium and culture for 7 days, then change to liver cancer organoid expansion medium and continue culture until passage.

[0028] The hepatoma organoid isolation culture medium and the hepatoma organoid expansion culture medium are the same as those in the embodiment of the present invention.

[0029] Figure 2-4 This shows that the liver cancer organoids cultured in this example are superior to those in the comparative example in terms of cell morphology, diameter size, cell number, etc. Figure 5-6 This indicates that the liver cancer organoids cultured in this example retain the good biological properties of the source tissue.

[0030] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for culturing a three-dimensional liver cancer organoid model, characterized in that: The steps include: S1: The liver cancer cells are obtained by washing, chopping and digesting the liver cancer samples from patients with digestive fluid. The liver cancer cell pellet is resuspended in liver cancer organoid isolation medium, and then the liver cancer cells are inoculated into low-adsorption culture plates for culture until they aggregate into cell clusters. S2: After the cell clusters are aggregated, the culture medium and cell clusters are transferred to a centrifuge tube. After centrifugation, the supernatant is discarded, Matrigel matrix gel is added and mixed, and then inoculated into a preheated non-low-adsorption culture plate; S3: placing the non-low-adsorption culture plate upright in a 37° C. incubator for a predetermined time, then turning the culture plate over and then inverting it to continue incubation; S4: After the incubation, add liver cancer organoid isolation medium and culture for a period of time, then change to liver cancer organoid expansion medium and continue culture until passaging.

2. The method for culturing a three-dimensional liver cancer organoid model according to claim 1, characterized in that: The step S1 comprises: washing the patient-derived liver cancer sample with 1×PBS for 3-5 times, and then cutting it into pieces with a volume of 0.5 mm using a surgical instrument. 3 -1mm 3 The minced tissue was then added to a digestion solution containing 75 U / ml IV collagenase, 0.5 U / mL DNase I (deoxyribonuclease I) and 125 g / mL Dispase, and incubated at 37°C for 30 min-60 min to obtain liver cancer cells.

3. The method for culturing a three-dimensional liver cancer organoid model according to claim 1, characterized in that: The step S1 comprises: setting the centrifuge temperature to 4°C and the centrifugal speed to 200g for 4-6 minutes, discarding the supernatant, and then resuspending the liver cancer cell pellet with liver cancer organoid isolation medium, inoculating the liver cancer cells into a 24-well low-adsorption culture plate, and culturing them for 2-3 days until they aggregate into cell clusters.

4. The method for culturing a three-dimensional liver cancer organoid model according to claim 1, characterized in that: The step S2 comprises: using a pipette to gently aspirate the cell mass and transfer it to a 15 ml centrifuge tube, setting the centrifuge temperature to 4°C, the centrifugal speed to 200g, centrifuging for 4-6 minutes, and discarding the supernatant; adding Matrigel matrix gel and gently mixing, and inoculating it into a preheated 24-well non-low-adsorption culture plate.

5. The method for culturing a three-dimensional liver cancer organoid model according to claim 1, characterized in that: The step S3 comprises: placing the non-low-adsorption culture plate upright in a 37° C. incubator for 2-4 minutes, and then turning the non-low-adsorption culture plate upside down for further incubation for 9-11 minutes.

6. The method for culturing a three-dimensional liver cancer organoid model according to claim 1, characterized in that: The step S4 comprises: adding liver cancer organoid isolation medium after the incubation is completed and culturing for 4-5 days, and then changing to liver cancer organoid expansion medium and continuing culturing until passage.

7. The method for culturing a three-dimensional liver cancer organoid model according to claim 1, characterized in that: The culture medium for liver cancer organoid isolation is: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL Penicillin / Streptomycin, 1-20mM Nicotinamide, 1-20mM N-Acetylcysteine, 20-200μg / ml Primocin, 40-100 ng / ml EGF, 20-60ng / ml HGF, 20-200ng / ml FGF-10, 1-2× B-27 (Vatamin-Free), 1-2×N-2, 10-100nM gastrin I, 10-100uM Forskolin, 1-20μM Y-27632, 1-10μM A83-01, 1-10nM Dexamethasone, and the basal culture medium is Advanced DMEM / F12.

8. The method for culturing a three-dimensional liver cancer organoid model according to claim 1, characterized in that: The culture medium for liver cancer organoid expansion is: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL Penicillin / Streptomycin, 1-20mM Nicotinamide, 1-20mM N-Acetylcysteine, 20-200μg / ml Primocin, 100-800ng / ml R-spondin-1, 40-100 ng / ml EGF, 20-60ng / ml HGF, 20-200ng / ml FGF-10, 1-2× B-27 (Vatamin-Free), 1-2×N-2, 10-100nM gastrin I, 10-100uM Forskolin, 1-20μM Y-27632, 1-10μM A83-01, and the basal culture medium is Advanced DMEM / F12.

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