CN116004540A - A Lung Tumor Organoid Model Based on Sodium Alginate Cryogel Scaffold - Google Patents

Abstract

The invention belongs to the field of biological materials, and discloses a lung tumor organoid model based on a sodium alginate cryogel bracket. According to the invention, alginate is used as a matrix material, and a micro porous structure is obtained by freeze drying to form a bracket; the scaffold is used for constructing a tumor organoid three-dimensional culture model, is low in cost, has controllable physicochemical properties and is similar to human alveolar tissues; and the tumor microenvironment can be better simulated by adding cytokines required by tumor culture and co-culturing other stromal cells contained in the tumor microenvironment with tumor cells. The invention can provide an excellent model for the field of lung tumor organoids and has great application potential.

Description

A Lung Tumor Organoid Model Based on Sodium Alginate Cryogel Scaffold

technical field

The invention belongs to the field of biological materials, and more specifically relates to a lung tumor organoid model based on a sodium alginate cryogel scaffold.

Background technique

Tumor cell culture is one of the basic technologies to study its physiological and pathological mechanisms, while traditional monolayer cell culture technology cannot simulate the environment of tumors in vivo. Organoid technology can highly simulate the physiological structure and function of in situ tissue, has been recognized as an important tool for biomedical research, and has become a new type of in vitro research model that is superior to traditional ones. This technology provides a convenient research platform for cancer disease modeling, drug screening, and regenerative medicine.

At present, the tumor organoid models on the market are represented by imported products such as Matrigel, which are expensive and not suitable for matrix materials for large-scale culture of tumor organoids. Not only that, but due to its unknown composition, it is difficult to use in drug screening experiments . Moreover, there are few reports on the construction of lung tumor organoids, and the culture methods are not mature. Alginate is a natural polysaccharide extracted from brown algae. It is easy to obtain, low in price, and has good biocompatibility and parameter controllability. However, it is rare to use alginate to construct lung tumor organoids in the prior art. .

Contents of the invention

In view of the above defects or improvement needs of the prior art, the object of the present invention is to provide a lung tumor organoid model based on sodium alginate cryogel scaffold, by using sodium alginate cryogel scaffold to establish lung tumor organoid Model, the scaffold is based on alginate as a matrix material, which is obtained by freeze-drying to obtain a microscopic porous structure (ie, a sponge structure), and then a cross-linking agent containing calcium ions is added to obtain a scaffold through cross-linking; the scaffold is used to construct tumor organoids Three-dimensional culture model, the resulting tumor organoid three-dimensional culture model is cheap, with controllable physical and chemical properties and similar to human alveolar tissue (the average alveolar size of normal adults is 200-450 μm, and the average size of alveolar alveoli in children is 100-170 μm); Further, by adding cytokines required for tumor culture and other stromal cells contained in the tumor microenvironment to co-culture with tumor cells, the tumor microenvironment can be better simulated. The invention can provide an excellent model for the field of lung tumor organoids, and has great application potential.

In order to achieve the above object, according to one aspect of the present invention, an application of a sodium alginate cryogel scaffold in establishing a lung tumor organoid model is provided, which is characterized in that the scaffold is first added to the mold with an aqueous solution of sodium alginate Freeze-drying in a medium to obtain a sponge structure, and then adding a cross-linking agent containing calcium ions to the sponge structure for cross-linking.

As a further preference of the present invention, the mass fraction of the sodium alginate aqueous solution is 0.5-5%;

The microscopic pore size of the support is 20-300 μm, and the porosity is 10%-80%.

As a further preference of the present invention, the sodium alginate aqueous solution is formed by dispersing sodium alginate powder with a viscosity of 5-40 mPa·s in water.

As a further preference of the present invention, the cross-linking agent is calcium chloride; the cross-linking condition is preferably an aqueous calcium chloride solution with a mass fraction of 0.5-5%, and the cross-linking time is 5 min-4h.

As a further preference of the present invention, the scaffold is also modified after cross-linking to load bioactive molecules;

The bioactive molecule is preferably selected from RGD peptide, FGF2, VEGF, IL-6, IFN-γ, CSF1, TGF-β;

Preferably, the modification of the scaffold is as follows:

The cross-linked scaffold is sterilized with 75% alcohol for 1-4 hours, washed with sterile water, and then added with complete cell culture medium containing biologically active molecules to incubate for 12-24 hours.

As a further preference of the present invention, specifically, using the scaffold modified with bioactive molecules, the tumor microenvironment stromal cells are first cultured on the scaffold, and then the tumor cells are cultured, thereby obtaining a lung tumor organoid model.

As a further preference of the present invention, the tumor microenvironment stromal cells are selected from at least one of the following cells: tumor-associated macrophages, tumor-associated fibroblasts, endothelial cells, and T lymphocytes;

The tumor cells are selected from at least one of the following cells: lung cancer cells, breast cancer cells, gastric cancer cells, prostate cancer cells, osteosarcoma cells, colorectal cancer cells, kidney cancer cells, liver cancer cells, ovarian cancer cells, pancreatic cancer cells and lymphoma cells; the tumor cells are preferably lung cancer cells.

As a further preference of the present invention, the number of tumor microenvironment stromal cells and tumor cells loaded on the scaffold is 5×10 ³ -5×10 ⁵ /scaffold, and the ratio of the number of tumor microenvironment stromal cells to tumor cells is 1: (0.1-10);

Preferably, the inner cavity of the mold is a cylinder with a diameter of 0.5-2 cm and a thickness of 0.1-1 cm; preferably a cylinder with a diameter of 0.8 cm and a thickness of 0.3 cm.

As a further preference of the present invention, it specifically includes the following steps:

(1) Place the scaffold modified with bioactive molecules in the cell culture plate to reduce the remaining moisture in the scaffold;

(2) Add the cell suspension of the tumor microenvironment stromal cells to be cultured dropwise on the support, let stand for 10 minutes to 1 hour until the cells are adsorbed, then add the cell culture medium to cover the support, and carry out cell culture for 1-2 days;

(3) Discard the cell culture medium used for culturing tumor microenvironment stromal cells, then drop the cell suspension of the tumor cells to be cultured on the support, let it stand for 10 minutes to 1 hour until the cells are adsorbed, and then add The cell culture medium was submerged in the scaffold, and the cells were cultured for 7-10 days until the tumor spheres were formed.

According to another aspect of the present invention, the present invention provides a lung tumor organoid model obtained by using the above application.

Through the above technical scheme conceived by the present invention, compared with the prior art, in the present invention, a specific scaffold is used to construct a lung tumor organoid model, and the scaffold is firstly added into a mold with sodium alginate aqueous solution for freeze-drying A sponge structure is obtained, and then a cross-linking agent containing calcium ions is added to the sponge structure and obtained through cross-linking. The scaffold uses sodium alginate cryogel as a matrix material, and can be further modified by bioactive molecules; when using this scaffold to cultivate lung tumor cells and construct lung tumor organoid models, other tumor-related tumors can be cultured on the scaffold first. Cells, simulating the tumor microenvironment in the body, and then culturing tumor cells; the scaffold is loose and porous inside, and the pore size is similar to the size of alveoli. The scaffold has good water absorption and can maintain the original shape and hardness under wet conditions. The physiological structure of the alveoli is similar, and it is suitable for the construction of lung tumor organoid models (it can be tumor in situ for lung cancer cells, or it can be for breast cancer cells, gastric cancer cells, prostate cancer cells, osteosarcoma cells, colorectal cancer cells , kidney cancer cells, liver cancer cells, ovarian cancer cells, pancreatic cancer cells, lymphoma cell metastases), and further through bioactive molecular modification and co-culture with other tumor-related cells, it can simulate lung tumor in situ and metastasis in vivo The tumor microenvironment of tumor can be further used for basic research of lung tumor in situ and metastases, as well as anti-tumor drug screening.

The tumor organoid scaffold in the present invention uses a sodium alginate aqueous solution and uses freeze-drying technology to remove water in the sodium alginate to form a network porous structure, and then cross-links with Ca ²⁺ to form a stable and uniform sponge. The present invention preferably uses 0.5-5% mass fraction as the cryogel prepared by sodium alginate aqueous solution, and the viscosity of the raw material sodium alginate powder used for preparing sodium alginate aqueous solution can be particularly preferably 5-40mPa·s, and the corresponding obtained The pore size of the scaffold is similar to the size of alveoli, which can simulate the matrix environment that tumor cells face in lung tissue, which can make the research results more realistic and accurate. At the same time, the modulus of the scaffold can be adjusted by cross-linking conditions (eg, the concentration of cross-linking agent, cross-linking time) to meet different needs.

The culture model based on the above-mentioned scaffold can make cancer cells grow and develop in three-dimensional space, and make the cancer cell-matrix interact to form a three-dimensional overall structure, which highly simulates the microenvironment in vivo. More and more evidence shows that the micro-spatial structure of the stromal environment is also an influencing factor of the tumor microenvironment, which can change the growth process of the tumor. form. The scaffold of the present invention can simulate the growth environment of tumor cells or tumor microenvironment stromal cells, and achieve better culture effects. At the same time, according to actual needs, growth factors and inflammatory factors contained in the tumor microenvironment in the body can be simulated by grafting different bioactive molecules, so that tumor cells can grow healthier in the matrix environment. For example, RGD peptide (belonging to Cell adhesion peptide) grafted onto the scaffold can improve cell adhesion and facilitate the growth of tumor cells on the scaffold. The model provided by the present invention can simulate the tumor microenvironment in vivo from aspects such as microscopic physical structure and biochemical factors, and provide a good growth and development platform for tumor cells.

Based on the present invention, by first cultivating tumor microenvironment stromal cells (denoted as cell A) and then culturing tumor cells (denoted as cell B) on the scaffold, it is possible to construct a multicellular common tumor that can simulate lung tumor in situ and metastatic tumor. growth microenvironment. In addition to tumor cells and cytokines, the tumor microenvironment also includes many stromal cells in the environment, such as immune cells, fibroblasts, and vascular endothelial cells, which jointly participate in the regulation of tumor biological functions. In order to simulate the tumor microenvironment in the body as much as possible, the model obtained by the present invention can especially utilize the porous characteristics of the scaffold for multi-cell co-culture, and through multi-cellular cell-cell interactions, various physiological functions of the tumor can be regulated, such as Growth and development, metastasis, angiogenesis, etc., are convenient for follow-up research. Taking the following examples as an example, the model obtained in the present invention successfully cultured macrophages on the scaffold, and selectively polarized the cells into M2 macrophages (which can promote the growth of cancer cells). In the presence of M2 macrophages, Tumor cells were replanted to construct a microenvironment conducive to the growth and development of tumor cells. The results showed that the growth, development and metastasis of tumor cells could be enhanced under this model culture compared with tumor cells cultured alone. Moreover, the scaffold is translucent, which is convenient for observation during cultivation.

Moreover, in the present invention, by first cultivating tumor microenvironment stromal cells (denoted as cell A) and then culturing tumor cells (denoted as cell B) on the scaffold, compared with traditional monolayer cell culture, tumor cells The physical and chemical properties and physiological functions of the cells are closer to the tumor cells in the body, and the research on the cells is more accurate and credible. The construction process is simple and can be operated at room temperature. Tumor cells are evenly distributed in each pore of the scaffold. Since the porous scaffold can effectively transport nutrients and cytokines, it can effectively reduce the generation of tumor necrosis centers. Not only that, the organoid model has high reproducibility and can replace many tedious animal experiments, ensuring economical and labor cost savings while improving efficiency. Taking the commonly used Matrigel organoids on the market as an example, the cost of Matrigel organoids is too high, the culture conditions are harsh, and the ingredients contained in the product are unknown, which may affect the experimental results. However, the model provided by the present invention can It is a good substitute for Matrigel and can provide great convenience for basic research and drug development and screening of lung tumors in situ and metastatic tumors.

In summary, the lung tumor organoid model provided by the present invention can well simulate the physical and chemical environment in the body, and can be used as a research platform for lung tumors in situ and metastatic tumors and a screening platform for anti-tumor drugs.

Description of drawings

Fig. 1 is the scanning electron micrograph and the pore size measurement statistical diagram of the bracket made of sodium alginate with different viscosities in Example 1; wherein, (a) in Fig. 1 corresponds to the use of sodium alginate powder of 5-40mPa·s (scale in the figure Represents 500μm), (b) in Figure 1 corresponds to the use of sodium alginate powder of 180-220mPa s (the scale in the figure represents 500μm), (c) in Figure 1 corresponds to the use of sodium alginate powder of 350-550mPa s (The scale bar in the figure represents 500 μm), (d) in Fig. 1 corresponds to the pore size measurement statistical chart of the scaffolds made of sodium alginate with different viscosities.

FIG. 2 is a bright field image (DAY7) of MCF-7 cell culture in Example 1. FIG.

Fig. 3 is the effect comparison diagram of control group BK, experimental group i, and experimental group D in embodiment 2; Wherein, A in Fig. 3 corresponds to observing the cell state diagram when cultivating 4, 7, and 10 days under the same magnification; Fig. 3 B in the figure corresponds to the statistical graph of the number of tumor sphere cells.

Fig. 4 is KI-67 protein fluorescence staining and data statistical figure among the embodiment 3; Wherein, (a) among Fig. 4 is the fluorescent staining figure (Fig. , the blue area corresponds to DAPI, and the green area corresponds to KI-67; the scales in the figure represent 50 μm); (b) in Figure 4 is a statistical histogram of the fluorescence positive rate of KI-67.

Fig. 5 is the staining fluorescence diagram of living and dead cells of Spheroid (traditional group) and Model (model group) in Example 4 (in the figure, the green area corresponds to living cells, and the red area corresponds to dead cells).

Fig. 6 is a statistical diagram of protein expression levels of ALCAM, LAMA3 and MMP9 in Example 5.

Fig. 7 is the actual picture of the sodium alginate cryogel prepared in the embodiment of the present invention (the cavity of the mold is cylindrical).

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The scaffold used for culturing tumor organoids that simulate alveolar tissue in the present invention is to add sodium alginate aqueous solution to the mold for freeze-drying, and then add calcium chloride aqueous solution to cross-link to form a sponge scaffold with a porous and loose structure, which better simulates The structure of the alveoli in the body, and then through surface modification, make it better as a growth matrix for tumor organoids.

For example, the preparation method of the lung tumor organoid scaffold may include the following steps:

(1) adding the sodium alginate powder into an aqueous solution and then freeze-drying it in the mold;

(2) add a cross-linking agent to cross-link for 1 hour, and wash with deionized water;

(3) Mix and disinfect with alcohol for 1 hour, wash with deionized water and set aside;

(4) Before culturing the cells, dissolve and dilute the bioactive molecules to the storage concentration, mix with the complete medium of the cells to the final concentration, and finally mix with the scaffold overnight.

Wherein, the mass fraction of sodium alginate aqueous solution can be between 0.1%-5%;

The inner cavity of the mold can be a cylinder with a diameter of 0.5-2cm and a thickness of 0.1-1cm. Correspondingly, the geometric shape of the final stent molding is a disc with a diameter of 0.5-2cm and a thickness of 0.1-1cm;

The microscopic pore size of the scaffold can be 20-300 μm, the porosity can be 10%-80%, the Young's modulus can be 50-200kPa, and the specific parameters can be adjusted according to actual needs. For example, the pore size and porosity are mainly affected by the viscosity of the raw material sodium alginate powder itself and the concentration of the sodium alginate aqueous solution. The greater the viscosity and the greater the concentration of the sodium alginate aqueous solution, the smaller the pore size and the higher the porosity of the obtained scaffold. large; the hardness (Young's modulus) of the scaffold is mainly affected by the concentration of the crosslinking agent and the crosslinking time, the greater the concentration of the crosslinking agent and the longer the crosslinking time, the greater the hardness; the specific preparation process used The size of parameter conditions can be flexibly adjusted according to actual needs. Considering the alveolar size of the human body (the average alveolar size of a normal adult is 200-450 μm, and the average alveolar size of a child is 100-170 μm), the pore size of the scaffold is 20-300 μm, which can cover most of the physiological size of the alveoli, and can simulate cells in the In the physical matrix environment faced by the body, the interior of the scaffold is loose and porous, and the pores are connected to each other, which is conducive to the exchange of nutrients, and the scaffold has good water absorption, and can maintain the original shape and hardness under wet conditions. Not only that, but the scaffold can be stretched and compressed to a certain extent and recovered, which is closer to the actual situation when used to simulate human alveoli.

The crosslinking agent can be an inorganic salt or an organic salt containing calcium ions;

Bioactive molecules are generally immune factors and cytokines contained in the tumor microenvironment.

For example, the cross-linking agent can be calcium chloride with a concentration of 2wt% (of course, other concentrations of calcium chloride aqueous solution, such as 0.5-5wt%) can also be used; according to actual needs, biologically active molecules can contain One or several that are commonly found in tumor microenvironment, for example, can be selected from: RGD peptide, fibroblast growth factor (FGF2), vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), interferon- γ (γ-IFN), colony-stimulating factor-1 (CSF-1), etc. In a specific embodiment of the present invention, the bioactive molecule is RGD peptide, and the final concentration is 10-200ng/ml.

Further, using the above-mentioned scaffold, a method for constructing a lung tumor organoid model can be obtained, which may include the following steps:

(1) Place the modified scaffold in a cell culture plate, and try to remove the remaining water of the scaffold.

(2) Drop the cell suspension of cell A (tumor microenvironment stromal cells) to be cultured on the scaffold, let it stand for 10 minutes to 1 hour until the cells are adsorbed, then slowly add conventional cell culture medium to cover the scaffold, and conventionally culture the cells 1-2 days.

(3) Discard the scaffold medium loaded with cell A, and load the tumor cells to be cultured (denoted as cell B) on the scaffold according to the treatment method in step (2), and the cells are routinely cultured for 7-10 days until the tumor spheres are formed .

Among them, cell A can be selected from one or several types contained in the tumor microenvironment: tumor-associated macrophages, tumor-associated fibroblasts, endothelial cells, etc.; cell B can be selected from any one of the following tumors: lung cancer , breast cancer, gastric cancer, prostate cancer, osteosarcoma, colorectal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer and lymphoma cells.

The cell numbers of the cultured cells A and B are 5×10 ³ -5×10 ⁵ /support, and the ratio of the cell numbers of the cells A and B is controlled between 1:0.1-10.

Further, in a specific embodiment of the present invention, the cell A is a human monocyte (THP-1), and the cell B is a human breast cancer cell (MCF-7).

In addition, the above-mentioned scaffold can be used for culturing tumor organoids; the above-mentioned construction method can be applied to culturing lung tumor organoids in vitro, and can also be applied to culturing normal tissue organoids in vitro.

The following are specific examples:

Example 1: Preparation of sodium alginate sponge scaffold and construction of lung tumor organoid model

The preparation of the stent in this embodiment includes the following specific steps: (1) dissolving sodium alginate powder (with viscosities of 5-40mPa·s, 180-220mPa·s, and 350-550mPa·s) in water to form a mass fraction of 2 % solution; (2) Add 400 μl of sodium alginate solution into a cylindrical mold with a diameter of 1 cm, place it in a refrigerator at 4°C to remove air bubbles, and then freeze it at -80°C for later use; (3) Put the sodium alginate solution in step (2) The sodium alginate solution was placed in a freeze dryer and freeze-dried for 12 hours to obtain a sodium alginate sponge (dry); (4) prepare a 2% calcium chloride solution for subsequent use; (5) add the calcium chloride solution in step (4) The sodium alginate sponge in step (3) was cross-linked for 1 hour, washed with sterile water for later use, the sample was freeze-dried again to remove water, and scanning electron microscope images were taken and the pore size and porosity were counted.

Because sodium alginate aqueous solution can be added to any ideal mold, by controlling the shape of the mold and the concentration of sodium alginate solution, the size and microscopic shape of the scaffold can be precisely controlled, so that it can better simulate the microscopic structure of alveoli in vivo . The appearance of the scaffold is a disc with a diameter of 0.8cm and a height of 0.3cm. The scaffold has strong water absorption and can be used as a basic environment for the growth and development of tumor cells. According to the microscopic topography of the scanning electron microscope (Figure 1), the figure shows the relationship between different viscosities and pore diameters. The greater the viscosity, the smaller the pore diameter. Based on this embodiment, 5-40 mPa·s can be preferably used, and the corresponding internal pore diameter of the prepared scaffold is 73.6-177.2 μm, and the porosity is 57.8±3.7%, which is close to the diameter of alveoli. The porous scaffold can well accommodate the growth of tumor cells. With its high porosity, it can enhance the transfer of nutrients, oxygen and waste, and facilitate the exchange of information between cells.

The method for modifying the tumor organoid culture scaffold in this example specifically includes the following steps: (1) place the cleaned scaffold in a 48-well ordinary cell culture plate, and add 500 μl of alcohol to disinfect it for 4 hours; (2) use a sterile (3) RGD peptide (purchased from Dalian Meilun Biological Co., Ltd.) was first dissolved in DMSO to form a 5 mg/ml mother solution, and then DMEM cell complete medium (89% DMEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin solution) are diluted to 50ng/ml, obtain the cell culture medium containing RGD peptide; (4) the cell culture medium that step (3) contains RGD peptide is added to step (2 ) in a sterilized rack (400 μl/well), and incubated in a cell culture incubator for 12 hours.

After being modified by RGD peptide, the content of N atoms introduced on the surface is 0.5% (atomic percentage, that is, the atomic percentage of N atoms to total atoms to total atoms).

The method for constructing lung tumor organoids in this example specifically includes the following steps:

S1: Preparation of solution for polarized THP-1 cells

-M0 medium: Dilute the PMA stock solution with a concentration of 200mg/ml (purchased from Dalian Meilun Biological Co., Ltd.) with DMEM cell complete medium to a PMA concentration of 20ng/ml;

-M2 medium: IL-4 and IL-13 (purchased from peprotech company) were prepared into 1 mg/ml stock solution with 0.1% bovine serum albumin aqueous solution, and then diluted to IL-4 with DMEM cell complete medium , IL-13 concentration is 20ng/ml.

S2: THP-1 cells (ie, human monocytes, which can be induced to differentiate into macrophages)

- Take out the incubated scaffold and put it into a new 48-well plate. Adjust the concentration of THP-1 cell suspension (100×10 ⁴ /ml) and add 100 μl of cell suspension to each well, let it stand for 15 minutes, and add 500 μl of medium containing M0. After 24 hours, the medium was discarded, and 500ul of M2 medium was added. After 24 hours, 500ul of regular cell culture medium was added and cultured for 24 hours.

S3: MCF-7 cells

-Concentration of cell suspension (50×10 ⁴ /ml) 100 μl of cell suspension was added to each well and allowed to stand for 15 minutes, then 500 μl of medium was added. Add 500ul DMEM cell complete medium 24 hours later, transfer to a 24-well plate after 24 hours, and change the medium two days later. After 10 days of culture, cell aggregate growth can be observed ( FIG. 2 ), indicating that the scaffolds provided by the present invention are used to culture cells, and the cells grow in clusters, which have certain organoid characteristics and are more in line with the growth state of tumor cells in vivo.

Example 2: Studying the effect of tumor-associated macrophages on the morphology of breast cancer lung metastases

M2 culture supernatant was obtained: THP-1 cells were seeded into 24-well plates at 50,000/well, and 500 μl of M0-containing medium was added. After 24 hours, discard the medium, add 500ul of M2 medium, and add 500ul of regular cell culture medium after 24h. Cell medium was changed once a day, and cell culture supernatant was collected.

Control group BK: Different from the construction method of lung tumor organoids in Example 1, which required three steps of S1, S2, and S3, the control group BK only adopted the S3 step when constructing organoids, that is, cultured MCF- 7 cells.

Experimental group i: Different from the construction method of lung tumor organoids in Example 1, which required three steps of S1, S2, and S3, experimental group i only adopted step S3 when constructing organoids, and at the same time, the DMEM cells in step S3 were completely The medium was replaced with M2 culture supernatant.

Experimental group D: organoids were constructed according to the method for constructing lung tumor organoids in Example 1.

The culture status of tumor cells in the organoid model was observed at days 4, 7, and 10 of organoid culture, and the number of tumor cells in a single tumorsphere was counted. The results are shown in Figure 3. It can be seen from the figure that the tumor mass in the experimental group D is larger, and the tumor cell count results show that the experimental group D is more than the other control groups. The results show that in the presence of tumor-associated macrophages, the growth and development of tumor cell clusters can be significantly enhanced through direct cell-cell contact and mutual influence, and the proliferation of breast cancer cells can be enhanced. It can be seen that by using the method for culturing the sodium alginate cryogel scaffold model of the present invention, a breast cancer lung metastases model can be constructed more quickly and efficiently. This organoid model not only provides cells with a similar physical matrix environment as in vivo, but also includes Multicellular environments and biomolecules.

Example 3: Ki-67 protein staining to explore the proliferation of breast cancer lung metastases model

For the organoid scaffolds cultured for 10 days, the medium was removed, and carefully washed with PBS; each scaffold was fixed with 500 μl of 4% paraformaldehyde for 15 minutes, and then washed with PBS; Wash with PBS; add 500 μl of 5% BSA to each bracket to block for 30 min; add 500 μl of Ki-67 antibody (diluted with antibody diluent) to each bracket and wash with PBS overnight at 4°C; add 500 μl of fluorescent secondary antibody to each bracket to block light for 30 min, and wash with PBS; A few drops of anti-fluorescence quenching agent were added to each scaffold and observed under a fluorescence microscope. The results are shown in Figure 4.

Ki-67 protein is a protein related to the proliferation of cancer cells, and the positive rate of its fluorescent staining can directly reflect the proliferation ability of cancer cells. As shown in Figure 4, the positive rate of group D is significantly higher than that of the control group and group i; the results show that the tumor organoids constructed by the construction method provided by the present invention have stronger proliferation ability. In the presence of relevant tumor-associated macrophages, the expression of Ki-67 protein in tumorspheres was significantly increased compared with the control group and group i, indicating that tumor-associated macrophages (M2 type) can promote the proliferation of cancer cells. Methods The cellular functions of tumor-associated macrophages can be used to construct tumor organoids more quickly, and the morphology and physical and chemical properties of the resulting cancer cells are closer to those of cancer cells in vivo.

Example 4: Staining of living and dead cells to explore the growth and development status of cancer cells

Experimental group Spheroid: Traditional tumor sphere culture method, cells are seeded on agarose, and the tumor spheres are formed. Model: The organoid model obtained as described in Example 1.

Use Calcein-AM/PI Live Cell/Dead Cell Double Staining Reagent (Beijing Suo Laibao Biological Co., Ltd.), operate according to the instructions of the reagent, take out the Ca, PI, and 10X buffer in the kit from the refrigerator in advance and thaw them with Prepare 1Xbuffer with sterile water. The fluorescent dye ratio is 7.5μl Calcein-AM, 15μl PI, 5ml 1Xbuffer. In the dark, discard the medium in the culture plate, add PBS to carefully wash the scaffold once, transfer the scaffold to a 48-well plate, add 400 μl of the prepared fluorescent dye to each well, and incubate for 30 minutes. After staining, discard the dye, add 500 μl/well of 1X buffer to wash the dye (gently) once, discard the 1X buffer (absorb clean), add 1 drop of anti-fluorescence quencher (Biyuntian) to each well, and observe under a fluorescence microscope. As shown in Figure 5.

Tumor necrosis refers to the large area of cell death inside a well-cultured tumor sphere, which is a common phenomenon in traditional tumor sphere culture, which is not conducive to subsequent scientific research, and is also a technical difficulty that is difficult to break through in 3D tumor cell culture. The main reason for the formation is the rapid growth of the tumor, the relative lack of blood vessel formation, and the abnormal structure and function of the new blood vessels, which lead to the lack of oxygen and nutrients inside the tumor. It can be seen from Figure 5 that the tumor necrosis center of the traditional tumor sphere culture has been formed, but the tumor necrosis center of the tumor sphere cultured in this model is much smaller than that of the traditional group, and the statistics of the fluorescence results show that the mortality rate of the model group is also much lower than that of the traditional group ( The statistically obtained mortality rates of these two groups are shown in Table 1 below), the results show that the tumor organoids obtained by the method provided by the present invention have significantly weakened tumor necrosis, high cell survival rate, and good growth and development status. Since the culture environment is loose and porous, which is conducive to the delivery of oxygen and nutrients, the organoid construction method provided by the present invention can construct an organoid model with a low tumor necrosis rate, which is beneficial to subsequent research.

Table 1: Mortality statistical results of Spheroid (traditional group) and Model (model group)

Example 5: Using fluorescent quantitative PCR to explore the expression of tumor cell metastasis and adhesion-related genes in this model

The cells on the scaffold of Example 1 were carefully blown off with the medium, collected by centrifugation,

The specific steps of RNA extraction using the Trizol method are as follows:

-1.5ml EP tubes, absolute ethanol, Trizol, isopropanol, and chloroform are all pre-cooled in advance to prevent RNA degradation;

-Centrifuge the cell suspension (1000r/3min), discard the supernatant to obtain the cell pellet, blow the cell pellet with 1ml Trizol solution and let it stand for 15min;

-Add 200μl of chloroform, shake for 15s, let stand for 10min, and centrifuge at 12000r/15min/4℃;

- Transfer 400 μl supernatant to a new EP tube, add an equal volume of isopropanol, shake for 15 seconds, let stand for 10 minutes, and centrifuge again;

- Discard the supernatant, wash the precipitate with 1ml of absolute ethanol, and centrifuge at 7500/15min/4°C;

- Discard the supernatant, suck it off carefully, let it dry for about 15 minutes, and dissolve in DPEC water (~20 μl).

reverse transcription:

A 10 μl reaction system was prepared according to the instructions of the cDNA kit (TAKARA Company), and reverse transcription was performed with a fixed reverse transcription program in a PCR machine, and the synthesized samples were stored in a -80°C refrigerator.

qPCR

Prepare a 10 μl reaction system according to the qPCR reagent instructions, and run q-PCR.

The experimental results are processed as shown in Figure 6.

ALCAM is a cell adhesion-related protein, and LAMA3 and MMP9 are cell metastasis-related proteins. As shown in the figure, the gene expression levels of the three proteins in group D are higher than those of the control group and group i, indicating that the tumor obtained by the culture method provided by the present invention The level of adhesion and metastasis of the cells increased significantly, indicating that the growth and development of the cells were more similar to the growth of malignant tumors in the body, which could provide a reliable theoretical basis for subsequent scientific research.

The above-mentioned embodiments are only examples, and the above-mentioned construction method can be applied to culturing various lung tumor organoids.

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (10)

1. The application of the sodium alginate cryogel stent in establishing lung tumor organoid models is characterized in that the stent is obtained by adding sodium alginate aqueous solution into a mold to carry out freeze drying to obtain a sponge structure, and then adding a crosslinking agent containing calcium ions into the sponge structure to crosslink.

2. The use according to claim 1, wherein the mass fraction of the aqueous sodium alginate solution is 0.5-5%;

the microscopic pore size of the scaffold is 20-300 mu m, and the porosity is 10% -80%.

3. The use according to claim 1, wherein the aqueous sodium alginate solution is formed by dispersing sodium alginate powder having a viscosity of 5-40 mPa-s in water.

4. The use according to claim 1, wherein the cross-linking agent is calcium chloride; the crosslinking condition is preferably that a calcium chloride aqueous solution with the mass fraction of 0.5-5% is adopted, and the crosslinking time is 5min-4h.

5. The use according to claim 1, wherein the scaffold is further modified after crosslinking is completed to support bioactive molecules;

the bioactive molecule is preferably selected from RGD peptide, FGF2, VEGF, IL-6, IFN-gamma, CSF1, TGF-beta;

preferably, the modification treatment of the bracket is specifically as follows:

sterilizing the crosslinked stent with 75% alcohol for 1-4 hr, washing with sterile water, and incubating with cell complete culture medium containing bioactive molecules for 12-24 hr.

6. The use according to claim 5, wherein the lung tumor organoid model is obtained by culturing tumor microenvironment stromal cells on a scaffold, followed by culturing the tumor cells, in particular using a scaffold modified with bioactive molecules.

7. The use of claim 6, wherein the tumor microenvironment stromal cells are selected from at least one of the following: tumor-associated macrophages, tumor-associated fibroblasts, endothelial cells, T lymphocytes;

the tumor cell is selected from at least one of the following: lung cancer cells, breast cancer cells, stomach cancer cells, prostate cancer cells, osteosarcoma cells, colorectal cancer cells, kidney cancer cells, liver cancer cells, ovarian cancer cells, pancreatic cancer cells, and lymphoma cells; the tumor cells are preferably lung cancer cells.

8. The method of claim 6, wherein the cell numbers of the tumor microenvironment stromal cells and tumor cells supported on the scaffold are 5X 10 ³ -5×10 ⁵ Scaffold, the ratio of the cell number of tumor microenvironment stromal cells to tumor cells is 1: (0.1-10);

preferably, the inner cavity of the die is cylindrical with the diameter of 0.5-2cm and the thickness of 0.1-1 cm; preferably a cylinder with a diameter of 0.8cm and a thickness of 0.3 cm.

9. The use according to claim 6, characterized in that it comprises in particular the following steps:

(1) Placing the scaffold modified with the bioactive molecules in a cell culture plate, and reducing residual moisture in the scaffold;

(2) Dripping a cell suspension of the tumor microenvironment stromal cells to be cultured on a bracket, standing for 10 minutes to 1 hour for cell adsorption, adding a cell culture medium until the cell culture medium is over the bracket, and culturing the cells for 1 to 2 days;

(3) Discarding the cell culture medium used when culturing the tumor microenvironment stromal cells, then dripping the cell suspension of the tumor cells to be cultured on a bracket, standing for 10 minutes to 1 hour for cell adsorption, adding the cell culture medium, and performing cell culture for 7 to 10 days until tumor balls are formed.

10. A lung tumor organoid model obtainable by the use of any of claims 1-9.

Images