A tumor organoid culture hydrogel with clear components and its preparation method and application

Abstract

The present invention relates to the field of organoid culture technology, and in particular to a tumor organoid culture hydrogel with clear components and its preparation method and application, wherein the tumor organoid culture hydrogel with clear components includes a gel precursor and laminin 521; the gel precursor is prepared by dissolving sodium alginate and gelatin in PBS; the concentration of laminin 521 is 0.10-0.15mg / ml. The hydrogel components provided by the present invention are clear, and the performance of its cultured tumor organoids is comparable to Matrigel, which overcomes the technical problems that Matrigel has complex components, batch differences, and expensive prices, resulting in the conversion of tumor patient-derived organoid models and limited applications. In addition, the hydrogel components provided by the present invention have a high degree of industrialization, good stability, and are easy to obtain, and standardized and economical production can be achieved, providing support for the construction of tumor organoid models.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and more specifically, to a tumor organoid culture hydrogel with clear components, and a preparation method and application thereof. Background Art

[0002] Current treatments for malignant tumors have limited efficacy and severe side effects, and there are large differences in anti-tumor efficacy between individuals. Therefore, appropriate tumor models are an important issue that needs to be addressed in current oncology research.

[0003] Organoids are three-dimensional organisms cultured in vitro that can simulate the structure and function of original tissues or organs to a high degree. Compared with traditional two-dimensional cell culture and patient-derived tumor xenograft animal models (PDX), they can better preserve the gene mutation spectrum of parental tumors and have broad application prospects in new drug development, stem cell research, and tumor research.

[0004] The three elements of organoid culture are: a medium (ECM) that supports three-dimensional growth, a culture medium suitable for the development of the corresponding organoids, and small molecule inhibitors of key signaling pathways. However, the current ECM for organoid culture relies heavily on animal-derived matrix gel Matrigel, a substance extracted from the basement membrane protein of EHS mouse osteosarcoma. Its components are extremely complex, including more than 1,800 unique proteins from proteomics analysis, which are quite different from human tissues, making it extremely challenging to identify factors affecting organoid culture. In addition, batch-to-batch differences in Matrigel and species differences with humans can affect the drug sensitivity results of tumor organoids. In addition, Matrigel is expensive, and it is very costly when conducting large-scale tumor organoid construction and preclinical drug screening. The above factors have limited its translational application in tumor organoid culture.

[0005] Sodium alginate and gelatin are currently the most common industrial hydrogel components, with adjustable mechanical properties and easy cross-linking, and exhibit viscoelastic behavior that simulates tissue. However, the number and size of organoids successfully cultured in alginate-gelatin are significantly lower than those in Matrigel. Studies have shown that some subtypes of laminin can promote tumor cell adhesion and migration; however, some laminins inhibit tumor growth by inhibiting tumor cell proliferation and invasion. Therefore, the type and concentration of laminin are important factors affecting the growth rate of organoids. Screening the appropriate concentration of laminin subtypes as one of the components of the new hydrogel is an effective solution to improve the growth of tumor organoids in industrial hydrogels. Summary of the invention

[0006] The purpose of the present invention is to provide a tumor organoid culture hydrogel with clear ingredients, which is used to solve the technical problems that the existing animal-derived matrix gel Matrigel has complex ingredients, batch differences, and high prices, which lead to limited transformation and application of tumor patient-derived organoid models.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A tumor organoid culture hydrogel with clear ingredients, including a gel precursor and laminin 521; the gel precursor is prepared by dissolving sodium alginate and gelatin in PBS; the concentration of laminin 521 is 0.10-0.15 mg / ml.

[0009] Preferably, the concentration of sodium alginate in the gel precursor is 0.5-1.5% (w / v), and the concentration of gelatin in the gel precursor is 0.1-0.3% (w / v).

[0010] Preferably, the concentration of sodium alginate in the gel precursor is 1% (w / v), the concentration of gelatin in the gel precursor is 0.25% (w / v), and the concentration of laminin 521 is 0.10 mg / ml.

[0011] Another object of the present invention is to provide a method for preparing a tumor organoid culture hydrogel with a clear composition, which comprises the following steps: weighing 0.050-0.150g of sodium alginate and 0.010-0.030g of gelatin, adding them to 10ml of PBS and stirring, heating them in a 60°C water bath for 30 min, and sterilizing them after natural cooling to obtain a gel precursor; adding 1-1.5mg of laminin 521 to the gel precursor to obtain the tumor organoid culture hydrogel with a clear composition.

[0012] Preferably, the viscosity of sodium alginate is 200-500 mPa·s, and the viscosity of gelatin is 25-45 mPa·s.

[0013] Another object of the present invention is to provide an application of a tumor organoid culture hydrogel with a clear composition, specifically to use the aforementioned tumor organoid culture hydrogel with a clear composition for the culture of tumor organoids.

[0014] Preferably, the tumor organoids include gastric cancer organoids, colorectal cancer organoids, lung cancer organoids, and osteosarcoma organoids.

[0015] Preferably, the tumor organoid culture method comprises the following steps: resuspending the tumor cells in a mixture consisting of a pre-cooled tumor organoid culture medium and a tumor organoid culture hydrogel with a clear composition in a volume ratio of 1:1.5, inoculating them on a 12-well low-adhesion cell culture plate with 1 ml of 75 mM CaCl2 solution attached to the bottom, and placing them in a cell culture incubator at 37°C and 5% carbon dioxide for solidification for 30 minutes; removing the CaCl2 solution in the well plate, adding 1 ml of the corresponding tumor organoid culture medium to each well for culture, and obtaining tumor organoids; replacing the corresponding tumor organoid culture medium every 2-3 days, and observing the growth morphology of the organoids under a light microscope.

[0016] The present invention has the following beneficial effects:

[0017] (1) Clear composition: The composition of Matrigel is extremely complex, including more than 1,800 unique proteins from proteomics analysis, while the hydrogel components provided by the present invention are three clear components: sodium alginate, gelatin and laminin.

[0018] (2) Standardization and economy: Matrigel is an animal-derived matrix gel extracted from the basement membrane protein of EHS mouse osteosarcoma, which shows frequent batch-to-batch differences and is expensive. The new hydrogel component provided by the present invention has a high degree of industrialization, good stability, and is easy to obtain. It can achieve standardized and economical production and provide support for the construction of tumor organoid models. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a morphological image of a hydrogel for culturing tumor organoids with defined components;

[0020] Figure 2 This is a bright-field photograph of cells in tumor organoids. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0022] Example 1: Synthesis of a novel hydrogel

[0023] (1) Determination of sodium alginate and gelatin concentrations: 0.050g-0.150g sodium alginate (viscosity 200-500 mPa•s) and 0.010g-0.030g gelatin (viscosity 25-45mPa•s) were weighed using an electronic analytical balance, added to 10ml PBS (pH = 7.4), stirred, and heated in a 60℃ water bath for 30 min. After cooling naturally to room temperature, high-pressure steam sterilization was performed to obtain a sodium alginate-gelatin gel precursor (hereinafter referred to as gel precursor) containing sodium alginate (mass fraction range 0.5%-1.5%) and gelatin (mass fraction range 0.1%-0.3%). The gelling ability of the gel precursor was tested, and the results are shown in Table 1.

[0024] Table 1: Gelation ability of sodium alginate-gelatin gel precursors at different concentrations

[0025]

[0026] Note: The gelling ability of gel precursor is divided into five levels: poor, poor, general, good and excellent according to the curing time and fluidity.

[0027] As can be seen from Table 1, when the concentration of sodium alginate in the gel precursor is 1.0-1.5% (w / v) and the concentration of gelatin in the gel precursor is 0.2-0.3% (w / v), the gelling ability of the obtained gel precursor is better; among them, when the concentration of sodium alginate is 1.0% (w / v) and the concentration of gelatin is 0.25% (w / v), the gelling ability of the obtained gel precursor is the best.

[0028] (2) Addition of laminin: As shown in Table 1, the precursor of sodium alginate (1%)-gelatin (0.25%) has the best gelling ability. Use an electronic analytical balance to weigh 0.100g of sodium alginate (viscosity 200-500 mPa•s) and 0.025g of gelatin (viscosity 25-45mPa•s), add to 10ml PBS (pH = 7.4), stir, and heat in a 60℃ water bath for 30 min. After cooling naturally to room temperature, sterilize with high-pressure steam to obtain a gel precursor of sodium alginate (mass fraction 1%) and gelatin (mass fraction range 0.25%). The gel precursor was stored in a 4°C refrigerator. Before use, laminin 521, 411, 111, 511, 311, 211, 421, 221 were added with a concentration gradient of 0.05 mg / ml, 0.10 mg / ml, and 0.15 mg / ml to obtain a new hydrogel; laminin 322 was added with a concentration gradient of 0.02 mg / ml, 0.04 mg / ml, and 0.06 mg / ml to obtain a new hydrogel. A total of 27 new hydrogels containing different types and concentrations of laminin were obtained (see Table 2). The morphology of the new hydrogel is shown in Figure 1 .

[0029] Example 2: Extraction of tumor cells

[0030] (1) Clinical sample collection and pretreatment: Specimens can be obtained through surgery and biopsy. Fresh tumor samples (gastric cancer samples) are immediately immersed in tissue preservation fluid after being removed from the body, placed on ice, and sent to the laboratory for relevant processing within 30 minutes. The tissue transport fluid is Advanced DMEM / F12 basal culture medium, supplemented with 1% (V / V%) Glutamax, 10mmol / L HEPES, and 2% (V / V%) penicillin / streptomycin solution. The sample is repeatedly shaken and rinsed 5-10 times with sterile saline or sterile PBS to ensure that foreign matter, blood, bacteria and other impurities are reduced as much as possible. Then, use high-pressure sterilized instruments (scissors and tweezers) to cut the tumor tissue into 1mm. 3 Left and right volume.

[0031] (2) Preparation of tissue digestion solution: Use an electronic analytical balance to weigh 2.5 mg / mL type IV collagenase and 0.1 mg / mL deoxyribonuclease I into a 50 ml centrifuge tube, then add 50 ml Advanced DMEM / F12 culture medium, 10 mmol / L HEPES buffer and 1% (V / V%) Glutamax additive, shake well to mix and place in a 4°C refrigerator for 30 min. Then use a 50 ml syringe and a 0.22 μm filter to sterilize and filter, and store in a -20°C refrigerator. Before use, place in a room temperature water bath to melt into liquid.

[0032] (3) Digestion of tissue fragments: Place the cut tissue fragments in a 10ml centrifuge tube containing an appropriate amount of PBS, use a Pasteur pipette to blow the tissue fragments up and down, let them settle for about 1 minute, discard the turbid supernatant after 1 minute, and repeat the above steps for about 2-3 times until the supernatant is clear. Centrifuge at 300g for 5 minutes, discard the supernatant, add the pre-prepared and thawed liquid digestion solution to the 10ml centrifuge tube, use a Pasteur pipette to mix the digestion solution and tissue fragments, and then place on a constant temperature shaker for shaking, set the temperature to 37℃, and shake for about 50 minutes.

[0033] (4) Collect tumor cells: Add an equal volume of pre-cooled digestion stop solution to terminate digestion. The digestion stop solution consists of complete cell culture medium (basal culture medium Advanced DMEM / F12 + 10% fetal bovine serum), 10µmol / L cell apoptosis protectant Y27632, and 1% (V / V%) penicillin / streptomycin solution. Filter the cell suspension through a 100µm filter and centrifuge at 300g for 10 minutes at 4℃. Discard the supernatant and wash twice with pre-cooled saline. The precipitate collected is the tumor cell.

[0034] Example 3: Construction of organoids

[0035] The tumor cells extracted in Example 2 were resuspended in a mixture of pre-cooled gastric cancer organoid culture medium and 27 novel hydrogels prepared in Example 1 at a volume ratio of 1:1.5, and the cell concentration was adjusted to 2500-4000 cells / 50μL, and inoculated on a 12-well low-adhesion cell culture plate with 1ml of 75mM CaCl2 solution attached to the bottom, and the volume of each well was 100μL. The plate was placed in a cell culture incubator at 37°C and 5% carbon dioxide for 30 minutes of solidification. Then the CaCl2 solution at the bottom of the plate was aspirated, and the gastric cancer organoid culture medium was added. The corresponding culture medium was replaced every 2-3 days, and the organoid size on the 1st and 7th days was observed and recorded under a light microscope. After 7 days of culture, the relative growth rate of gastric cancer organoids in different groups was calculated. The results are shown in Table 2.

[0036] Among them, the gastric cancer organoid culture medium is based on Advanced DMEM / F12, and a final concentration of 1% Glutamax, 1.25mmol / L N-acetyl-l-cysteine, 100ng / mL Wnt-3a, 100ng / mL R-Spondin 1, 100ng / mL Noggin, 10mmol / L HEPES, 1× N2 and B27, 10mmol / L Nicotinamide, 10nmol / L Gastrin, 50ng / mL EGF, 100ng / mL FGF10, 5µmol / L A8301, 3µmol / L CHIR99021 and 10µmol / L Y27632 are added and mixed.

[0037] According to the different new hydrogels used, 10 groups were set up (control group, laminin 521 group, laminin 332 group, laminin 411 group, laminin 111 group, laminin 511 group, laminin 311 group, laminin 211 group, laminin 421 group, laminin 221 group), and each experimental group was divided into 3 groups according to the different laminin concentrations, for a total of 27 experimental groups. In summary, a total of 29 groups of organoids were set up, including the control group and the precursor group. Each group had 3 wells.

[0038] Control group: Matrigel was used instead of the novel hydrogel to culture the tumor cells obtained in Example 2; precursor group: a gel precursor containing 1% sodium alginate and 0.25% gelatin was used instead of the novel hydrogel to culture the tumor cells obtained in Example 2; laminin 521 group: (i) the tumor cells obtained above were cultured using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.05 mg / ml laminin 521; (ii) a hydrogel containing 1 The tumor cells obtained above were cultured using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.10 mg / ml laminin 521; (iii) the tumor cells obtained above were cultured using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.15 mg / ml laminin 521; Laminin 332 group: (i) the tumor cells obtained above were cultured using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.02 mg / ml laminin 322; (ii) using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.04 mg / ml laminin 322 to culture the above-obtained tumor cells; (iii) using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.06 mg / ml laminin 322 to culture the above-obtained tumor cells; Laminin 411 group: (i) using a novel hydrogel containing 1% sodium alginate, 0. (ii) using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.10 mg / ml laminin 411 to culture the above-obtained tumor cells; (iii) using a novel hydrogel containing 1% sodium alginate, 0.25% gelatin and 0.15 mg / ml laminin 411 to culture the above-obtained tumor cells. The same applies to the other experimental groups.

[0039] Table 2: Relative growth rates of gastric cancer organoids in different gels

[0040]

[0041] Note: “--” means a decrease of 50-100%, “-” means a decrease of 0-50%, “ / ” means no significant change, “+” means an increase of 0-50%, “++” means an increase of 50-100%, “+++” means an increase of 100-150%, and “++++” means an increase of 150-200%.

[0042] As can be seen from Table 2, compared with the control group and the precursor group, the effects of different subtypes and concentrations of laminin on the growth rate of gastric cancer organoids are very different. Among them, the growth rate of tumor organoids in the laminin 521 group is significantly different from that in the precursor group, and the experimental group with 0.10 mg / ml laminin 521 showed the fastest organoid growth rate among all groups, with a growth rate increase of 150-250% compared with the precursor group.

[0043] Example 4: Cultivation of tumor organoids

[0044] Fresh tumor specimens from different patients were collected, including gastric cancer, colorectal cancer, lung cancer, and osteosarcoma (reference example 2). The optimal new hydrogel (1% sodium alginate, 0.25% gelatin, and 0.10 mg / ml laminin 521) and Matrigel were used to culture tumor organoids (reference example 3). The corresponding organoid culture medium was replaced every 2-3 days. The growth of the organoids was observed under a light microscope and the size of the organoids on the 7th day was recorded. The steps for culturing the organoids of each tumor type were the same, but the components of the culture medium were different. The results are shown in Figure 2 .

[0045] Among them, gastric cancer organoid culture medium: Advanced DMEM / F12 as the basic culture medium, added with a final concentration of 1% Glutamax, 1.25mmol / L N-acetyl-l-cysteine, 100ng / mL Wnt-3a, 100ng / mL R-Spondin 1, 100ng / mL Noggin, 10mmol / L HEPES, 1× N2 and B27, 10mmol / L Nicotinamide, 10nmol / L Gastrin, 50ng / mL EGF, 100ng / mL FGF10, 5µmol / L A8301, 3µmol / L CHIR99021 and 10µmol / L Y27632 and mixed well.

[0046] Among them, colorectal cancer organoid culture medium: Advanced DMEM / F12 as the basic culture medium, added with a final concentration of 1% Glutamax, 1.25mmol / L N-acetyl-l-cysteine, 20ng / mL Wnt-3a, 100ng / mL R-Spondin 1, 100ng / mL Noggin, 10mmol / L HEPES, 1× N2 and B27, 10mmol / L Nicotinamide, 10nmol / L Gastrin, 50ng / mL EGF, 100ng / mL FGF10, 5µmol / L A8301, 3µmol / L CHIR99021 and 10µmol / L Y27632 and mixed well.

[0047] Among them, lung cancer organoid culture medium: Advanced DMEM / F12 is used as the basic culture medium, and Glutamax and N-acetyl-l-cysteine ​​with a final concentration of 2mM, Noggin of 50ng / mL, HEPES of 10mM, N2, B27, Primicin of 1×, EGF of 50ng / mL, CHIR99021 of 1µM and Y27632 and SB 431542 of 10µM are added and mixed.

[0048] Among them, osteosarcoma organoid culture medium: Advanced DMEM / F12 as the basic culture medium, add HEPES and N-Acety-l-cysteine ​​with a final concentration of 1mM, 1× Glutamax, Pen-Strep Glutamine, B27, 10 mM Nico, 0.5 μM A8301, 50 ng / mL EGF, 10nM Gastrin, 100 ng / ml Noggin, 500 ng / mL RSPO1, 10 μM SB-202190 and mix well.

[0049] Table 3: Average diameter of organoids cultured with novel hydrogels and Matrigel

[0050]

[0051] Figure 2 The results showed that tumor cells of four different tumor types could successfully grow into well-functioning organoids in the novel hydrogel with defined composition. The diameters of the organoids in the two groups (novel hydrogel group and Matrigel hydrogel group) were measured using imageJ analysis software. The results are shown in Table 3, indicating that there was no significant difference in the diameter of the organoids cultured in the novel hydrogel and the Matrigel group.

[0052] In summary, the novel hydrogel provided by the present invention has clear components, and its performance in culturing tumor organoids is comparable to that of Matrigel, overcoming the technical problems of Matrigel's complex components, batch-to-batch differences, and high prices, which lead to limited transformation and application of tumor patient-derived organoid models. In addition, the novel hydrogel component provided by the present invention has a high degree of industrialization, good stability, and is easy to obtain, and can achieve standardized and economical production, providing support for the construction of tumor organoid models.

[0053] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A hydrogel for culturing tumor organoids with a clear composition, characterized in that: The invention comprises a gel precursor and laminin 521; the gel precursor is prepared by dissolving sodium alginate and gelatin in PBS; The concentration of sodium alginate in the gel precursor was 1% (w / v), the concentration of gelatin in the gel precursor was 0.25% (w / v), and the concentration of laminin 521 was 0.10 mg / ml.

2. A method for preparing a hydrogel for culturing tumor organoids with clear components, characterized in that: The method comprises the following steps: weighing 0.1 g sodium alginate and 0.025 g gelatin, adding them into 10 ml PBS and stirring, heating them in a 60° C. water bath for 30 min, and sterilizing them after natural cooling to obtain a gel precursor; adding 1 mg laminin 521 into the gel precursor to obtain the tumor organoid culture hydrogel with clear components.

3. The method for preparing a tumor organoid culture hydrogel with a clear composition according to claim 2, characterized in that: The viscosity of sodium alginate is 200-500mPa·s, and the viscosity of gelatin is 25-45mPa·s.

4. An application of a hydrogel with a clear composition for culturing tumor organoids, characterized in that: The tumor organoid culture hydrogel with a clear composition as described in claim 1 is used for the culture of tumor organoids.

5. The use of the tumor organoid culture hydrogel with a clear composition according to claim 4, characterized in that: The tumor organoids include gastric cancer organoids, colorectal cancer organoids, lung cancer organoids, and osteosarcoma organoids.

6. The use of the tumor organoid culture hydrogel with a clear composition according to claim 4, characterized in that: The tumor organoid culture method comprises the following steps: resuspending tumor cells in a mixture consisting of a pre-cooled tumor organoid culture medium and a tumor organoid culture hydrogel with a clear composition in a volume ratio of 1:1.5, inoculating them on a 12-well low-adhesion cell culture plate with 1 ml of 75 mM CaCl2 solution attached to the bottom, and curing them in a cell culture incubator at 37°C and 5% carbon dioxide for 30 minutes; removing the CaCl2 solution in the well plate, adding 1 ml of the corresponding tumor organoid culture medium to each well for culture, and obtaining tumor organoids; replacing the corresponding tumor organoid culture medium every 2-3 days, and observing the growth morphology of the organoids under a light microscope.

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