CN111996168A - Construction method and application of in-vitro three-dimensional tumor cell drug-resistant model - Google Patents

Abstract

The invention relates to a construction method and application of an in-vitro three-dimensional tumor cell drug-resistant model, wherein the construction method comprises the following steps: (1) preparing a three-dimensional space fabric with a gradient aperture structure, wherein the arrangement rule of fibers or yarns of a space layer of the three-dimensional space fabric is V-shaped, X-shaped or flexible changing; (2) preparing a bioactive material solution, compounding the bioactive material solution and the three-dimensional spacer fabric in a mould, and then pre-freezing and freezing to prepare a scaffold pre-structure body; (3) vacuum drying the scaffold preformed body to prepare a composite scaffold preformed body; (4) performing cross-linking treatment on the composite scaffold pre-structure to prepare a composite porous scaffold; (5) co-culturing the composite porous scaffold and tumor cells to prepare an in-vitro three-dimensional tumor cell drug-resistant model; the application is as follows: used for drug sensitivity detection. The establishment method is simple, and the three-dimensional culture model with stable mechanics and uniform size can be prepared to meet the requirements of in vitro cell culture, antitumor drug screening and the like.

Description

Construction method and application of an in vitro three-dimensional tumor cell drug resistance model

technical field

The invention belongs to the field of three-dimensional culture of tumor cells, and relates to a construction method and application of an in vitro three-dimensional tumor cell drug resistance model.

Background technique

The main disadvantage of using traditional two-dimensional culture to simulate life systems is that cells can only be cultured in monolayer. Under this condition, the distribution of oxygen, nutrients and signal molecules in the culture environment does not conform to the real physiological environment, and cannot accurately reflect the cell-extracellular environment. Matrix, cell-cell interactions. In the past few decades, with the advent of 3D cell culture technology, there has been a lot of research on 3D cell culture models in the field of cancer and regenerative medicine. The development and utilization of 3D culture models are indispensable when studying the interaction of tumor cells with the extracellular matrix and other cells in the microenvironment.

The commonly used three-dimensional culture models of tumor cells are mainly multicellular tumor spheroids (multicellular tumor spheroids, MCTS) and three-dimensional scaffold culture models. Multicellular tumor spheroid models are produced in a variety of ways, usually by aggregation and compaction of cell suspensions cultured under conditions of low or non-adherent surfaces. This model has been commercially produced semi-automatically and is widely used in the field of high-throughput drug screening. However, the size of multicellular spheroids prepared by this technology is not uniform, which limits the application of this model to a certain extent. In order to achieve real cell interactions, there are also studies using biomimetic materials and structures to construct a three-dimensional scaffold similar to the tumor cell growth microenvironment, and use the scaffold to culture cells to form tumor cell colonies. Researchers have developed a variety of scaffold materials, including natural biomaterials (such as cellulose, collagen, fibrin, hyaluronic acid, or alginate), synthetic biomaterials (such as polycaprolactone, polyethylene), and inorganic Materials (eg ceramic, titanium).

In application, the above materials are usually processed into a hydrogel type, or 3D printed and freeze-dried into a single scaffold type. For example, Chinese patent CN109022342A introduces a three-dimensional culture system using chiral hydrogel as adherent cell culture, using sodium alginate, sodium hyaluronate, chitosan and other substances as coagulants to establish a three-dimensional culture system for adherent cells. The three-dimensional culture system has good biocompatibility and can simulate the biological environment in vivo to the greatest extent. However, because the cells are fixed in the chiral hydrogel scaffold, the cell activity is not high, and the cell growth is only increased by 20～ 40%. Scaffolding is also a commonly used method for constructing a three-dimensional culture model of tumor cells. For example, Chinese patent CN104312975B introduces a three-dimensional collagen scaffold. The three-dimensional collagen scaffold is soaked in a medium for pretreatment, and then the spherical glioma stem cells are planted. In the pretreated three-dimensional collagen scaffold, after adding endothelial medium for co-cultivation for several days, an in vitro three-dimensional culture model for studying the vascular mimicry of glioma stem cells was obtained. However, due to the poor mechanical properties of the collagen scaffold and degradation, it cannot maintain a high degree of cell activity for a long time.

Some researchers have paid attention to the mechanical properties of scaffolds and introduced scaffolds with improved mechanical properties. For example, Chinese patent CN106676074A discloses a porous cellulose scaffold with a stiffness of 5-100 kPa, which can induce liver cancer cells to transform into liver cancer stem cells. It has a certain anti-sensitivity to tumor drugs, but the shape and size of the cellulose scaffold cannot be kept highly uniform, and the size is not consistent, and there will be data differences during application. Therefore, it is urgent to develop a new technology that can effectively prepare a three-dimensional culture model with mechanical stability and uniform size to meet the needs of in vitro cell culture, antitumor drug screening and regenerative medicine.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problems existing in the prior art, and to provide a construction method and application of an in vitro three-dimensional tumor cell drug resistance model.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

A method for constructing an in vitro three-dimensional tumor cell drug resistance model, the steps are as follows:

(1) Prepare a three-dimensional spacer fabric with a gradient pore size structure. The gradient pore size structure, that is, the pore size of the upper layer and the lower layer of the three-dimensional spacer fabric is different, and there is a trend of "from large to small" or "from small to large". The average of the upper layer The pore size is 2-5 mm, and the average pore size of the lower layer is 1-4 mm; if the pore size is too small, the porosity of the composite scaffold prepared is not high, and the cell growth effect is not ideal; if the pore size is too large, the mechanical properties of the scaffold will be unstable and the compression resistance will change Weak; the arrangement of fibers or yarns in the spacer layer of the three-dimensional spacer fabric is V-shaped, X-shaped or flexible. Signs";

(2) preparing a solution of biologically active material, pre-freezing and freezing after compounding it with a three-dimensional spacer fabric in a mold, to obtain a scaffold preform;

(3) vacuum drying the stent preform to obtain a composite stent preform;

(4) Cross-linking the composite scaffold preform to obtain a composite porous scaffold; the composite porous scaffold has a three-dimensional through-hole structure with a porosity of more than 90% and an average pore diameter of 10-50 μm (measured by a porosity measuring instrument). obtained), the thickness is 3-15mm, the length is 5-20mm, and the width is 5-20mm; if the average pore size is too small, it is difficult for cells to grow in the scaffold, and it has been reported in the literature that the average pore size is too large and is not conducive to cell proliferation and growth; too thick It is difficult to process small cells, and it is difficult for cells with too large thickness to penetrate into the scaffold to proliferate; the length and width can be processed into different sizes according to different needs, and the optional range can be changed according to the required size; the mechanical properties of the composite porous scaffold are stable, and the deformation is When its own thickness is 50%, the maximum compressive strength is 200-1300cN, which can keep the shape unchanged during application. The composite porous scaffold is suitable for the growth and proliferation of tumor cells, and provides three-dimensional growth space for cells, with excellent mass transfer performance;

The "average pore diameter of the upper layer is 2-5mm, and the average pore diameter of the lower layer is 1-4mm" of the three-dimensional spacer fabric enables it to carry biologically active materials. A composite porous scaffold with a large number of pore structures, a porosity of more than 90%, and an average pore diameter of 10-50 μm, the arrangement of the spacer yarns and the stiffness of the yarn itself make the composite porous scaffold excellent in mechanical properties;

(5) The composite porous scaffold was co-cultured with tumor cells to obtain a three-dimensional tumor cell drug resistance model in vitro.

In the present invention, a three-dimensional spacer fabric with a gradient pore size structure and a biologically active material are used to composite a composite porous scaffold, and then co-culture it with tumor cells to obtain a three-dimensional tumor cell drug resistance model in vitro. All three-dimensional spacer fabrics have a sandwich structure. , the sandwich structure is specifically the upper layer - the spacer layer - the lower layer, the spacer layer connects the upper layer and the lower layer, this connection makes the three-dimensional spacer fabric have a three-layer structure and is not easy to loosen, so that the three-dimensional spacer fabric has good mechanical stability;

In addition, the arrangement of fibers or yarns in the spacer layer is V-shaped, X-shaped or flexible. This arrangement makes the three-dimensional spacer fabric have good mechanical properties. It can quickly restore the shape and state before deformation, which further improves the mechanical stability of the three-dimensional spacer fabric;

In addition, a large number of holes are distributed in the upper and lower layers of the sandwich-type structure, and there are certain gaps between the fibers. The sandwich-type structure causes the three-dimensional spacer fabric to have extremely high porosity. The present invention also controls the three-dimensional spacer fabric to have a gradient pore size structure. , the gradient pore structure can simulate the material gradient distribution of the tumor microenvironment in vivo, and the extremely high porosity and gradient pore structure provide an environment for the circulation of media (nutrients and metabolic waste) for the three-dimensional growth of cells;

In addition, the three-dimensional spacer fabric itself has the characteristics of uniform size. Since its raw material is fiber or yarn, it can be easily cut into various shapes and sizes using scissors and other tools. Specifically, culture plates of different shapes and sizes are required for cell culture. .

As the preferred technical solution:

A method for constructing an in vitro three-dimensional tumor cell drug resistance model as described above, in step (1), in the three-dimensional spacer fabric, the fibers or yarns of the upper layer and the lower layer are of the same or different types, and are selected from polypropylene and polyester, and the thickness is different. The same or different; polypropylene and polyester are two kinds of high molecular polymers that can be used for biomedical materials, limited by the processing requirements of the machine, the best types of yarns that can be used to be processed into spacer fabrics are these two.

The fiber or yarn of the spacer layer is nylon or polyester, and the fiber or yarn of the spacer layer needs to have a certain strength to support the two surfaces of the fabric and the external pressure, so that the bracket has a certain thickness and elasticity;

The three-dimensional spacer fabric is processed by an integrated double needle bed Raschel warp knitting machine, and the size is uniform and controllable;

In the weaving process of the three-dimensional spacer fabric, the air-piercing bar process is used (the air-piercing bar process is a process for processing warp-knitted spacer fabrics, the purpose is to form different apertures of the spacer fabrics, which are manifested in aperture shape, aperture size, pore size gradient distribution).

In the above-mentioned construction method of a three-dimensional tumor cell drug resistance model in vitro, in step (2), the preparation process of the bioactive material solution is as follows:

(2.1) According to the mass ratio of 1:99～399 (the mass ratio of bioactive material and acetic acid solution determines the concentration of bioactive material, if the concentration of bioactive material is too low, the effect of compounding will be unsatisfactory, less biomaterial is compounded to the spacer fabric; too high concentration will result in lower porosity of the composite porous scaffold, and if the porosity is too low, the material transport of cells and the proliferation of cells will be affected) The bioactive material is mixed with the acetic acid solution in the glass bottle (As a solvent that can dissolve most biologically active materials, acetic acid is not only fast in dissolving, but also volatile, so that it can be completely volatilized through freeze-drying and other processes after dissolving, and will not be toxic to cells. Other alternative solvents can be used according to biological The different choices of material solubility include the following but not limited solvents: deionized water, ethanol or PBS), at normal temperature, use a magnetic stirrer at a speed of 400-800r/min (in the present invention, considering that the speed is too slow, Make the dissolution time longer, because the bioactive material will degrade in the solvent, and the longer the dissolution time, the more the degradation will affect the subsequent use effect; the same is true for too fast rotation speed, and there will be a larger centrifugal force at high rotation speed. Large centrifugal force will accelerate the degradation of the material or destroy the performance of the material, so set the rotation speed to 400-800r/min) and stir to dissolve;

(2.2) Invert the glass bottle and stir for 4-48 hours to fully dissolve the bioactive material; if the stirring time is too short, the material will not be fully dissolved, and if the dissolution time is too long, the material will degrade too much;

(2.3) centrifuge the mixture in the glass bottle and take the supernatant to prepare a biologically active material solution;

The mold is a sterile petri dish, or a 4-well, 6-, 12-, 24- or 48-well cell culture plate;

The composite method is dipping method or coating method;

The proportional relationship between the bioactive material solution and the three-dimensional spacer fabric satisfies: when using the dipping method for compounding, the prepared bioactive material solution just covers the three-dimensional spacer fabric; when using the coating method for compounding, so that the prepared bioactive material solution The active material solution can be repeatedly coated for 3 to 5 times;

The compounding time is 4-48 hours, the pre-freezing time is 4-48 hours, and the freezing time is 4-48 hours. If the compounding time is too short, the compounding effect is not good, and the bioactive material cannot fully contact the three-dimensional spacer fabric; Long time will cause the material to degrade too much, and the same is true for other time options.

A method for constructing an in vitro three-dimensional tumor cell drug resistance model as described above, the biologically active materials are collagen I-I and its derivatives, alginate and its derivatives, hyaluronic acid and its derivatives, polypeptides, gelatin , chitosan or chitin, and other materials that have biocompatibility and can promote the growth of tumor cells, such as polyethylene glycol hydrogel, polylactic acid hydrogel, can replace the above materials.

In the above-mentioned method for constructing an in vitro three-dimensional tumor cell drug resistance model, in step (3), the vacuum drying time is 4-48 hours. If the drying time is too short, the drying will be insufficient, and a large amount of water molecules will remain, which will affect the porous composite The porosity of the stent; if the drying time is too long, the material will be degraded too much, and the drying effect can be achieved, and it does not need to be dried for a long time.

In the above-mentioned construction method of an in vitro three-dimensional tumor cell drug resistance model, in step (4), the cross-linking method is photo-cross-linking method, radiation cross-linking method, thermogravimetric cross-linking method or chemical reagent cross-linking method; The time of crosslinking is 2～48h.

In the above-mentioned method for constructing an in vitro three-dimensional tumor cell drug resistance model, in step (5), the co-cultivation process is as follows: firstly, the tumor cells are cultured on a plane for 1 to 7 days, and then digested and resuspended in a medium to obtain a tumor cell suspension. Then use a syringe or a pipette to implant the tumor cell suspension into the composite porous scaffold for culture;

The implantation density of tumor cells is 1～5×10 ⁶ /200μl; if the implantation density is too high, the cells will suffer from contact inhibition and the growth will be hindered, resulting in waste. Prolonged, resulting in excess consumption of time, energy and material resources; tumor cells are prostate cancer cells, breast cancer cells, ovarian cancer cells, renal cancer cells, erythroleukemia K562 cells, human promyelocytic leukemia cells, liver cancer cells or cervical cancer cells, including but not limited to Not limited to this; the culture medium is the culture medium corresponding to the corresponding cells well known to those skilled in the art.

The present invention also provides the application of the in vitro three-dimensional tumor cell drug resistance model prepared by the method for constructing an in vitro three-dimensional tumor cell drug resistance model as described in any one of the above, for drug sensitivity detection. The specific process is: first Determine the target drug and its corresponding drug sensitivity test method, and then test the in vitro three-dimensional tumor cell drug resistance model according to the drug sensitivity test method corresponding to the target drug, and calculate the cell proliferation ability and growth of the in vitro three-dimensional tumor cell drug resistance model. factor expression levels.

Beneficial effects:

(1) The composite porous scaffold provided by the present invention has a large number of three-dimensional through-hole structures, and at the same time has the advantages of high porosity and a pore size suitable for the growth and proliferation of tumor cells, providing a three-dimensional growth space for cells, and excellent mass transfer performance. It can make up for the deficiency of two-dimensional plane culture, which is conducive to the interaction between cells and cells and cells and matrix, and better simulates the tumor microenvironment of cell growth;

(2) In the present invention, the composite porous scaffold with a sandwich structure has good mechanical properties. Compared with other single hydrogel scaffolds, this model has a certain mechanical support force, and can keep the shape unchanged during application. It does not impair the viability of co-cultured cells, which is an important condition for cells to grow in this model for a long time;

(3) The composite porous scaffold of the present invention is easy to manufacture and has a controllable size (it can be processed into different sizes and shapes according to different needs), and can be used for culturing tumor cells in reactors of different shapes;

(4) The in vitro three-dimensional tumor cell drug resistance model provided by the present invention is green and non-toxic in the preparation process, the in vitro cultured cells are easy to operate, the cells proliferate rapidly, and have good drug resistance to tumor drugs, and can be used for anti-tumor drug sensitivity testing.

Description of drawings

1 is a schematic diagram of the composite porous scaffold of Example 1;

Fig. 2 is the yarn-laying motion diagram of the three-dimensional spacer fabric of Design Example 1. It is intended to make the three-dimensional spacer fabric obtain a structure with a diamond-shaped hole structure on the surface, wherein F and B represent the two needle beds on the double needle-bed warp knitting machine, and GB represents the comb comb;

Fig. 3 is the surface structure diagram of the three-dimensional spacer fabric of Example 1, as can be seen from the figure, the surface of the prepared three-dimensional spacer fabric has a diamond-shaped hole structure;

Fig. 4 is the porosity map of composite porous scaffold and pure collagen scaffold;

Fig. 5 is a confocal microscope overlay image obtained in Example 1 of fluorescent staining of cell nuclei during 5 days of cell culture. The first day, the third day, and the fifth day respectively represent the number of days that the cells were cultured in the scaffold. The node stains the cell nucleus, and the dye is DAPI. In order to observe the growth of cells inside the scaffold, the surface and inside of the scaffold are scanned and imaged by confocal microscope at different depths of culture layers, and the fluorescence images of each layer of cells are superimposed to obtain images. The scanning depth was 500 μm.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

A method for constructing an in vitro three-dimensional tumor cell drug resistance model, the steps are as follows:

(1) preparing a three-dimensional spacer fabric with a gradient aperture structure, the gradient aperture structure is that the apertures of the upper layer and the lower layer of the three-dimensional spacer fabric are different, and the arrangement rule of the yarns of the spacer layer of the three-dimensional spacer fabric is V-shaped;

In the three-dimensional spacer fabric, the yarns of the upper layer and the lower layer are of the same type and the same thickness, specifically polyester with a fineness of 100D (D: denier);

The average pore diameter of the upper layer is 2mm, and the average pore diameter of the lower layer is 1mm;

The type of yarn of the spacer layer is polyester with a diameter of 0.15mm;

The three-dimensional spacer fabric is processed by an integrated double-needle-bed Raschel warp knitting machine; the movement diagram of the laying yarn is shown in Figure 2;

In the weaving process of the three-dimensional spacer fabric, the air-through bar technology is adopted;

The finally obtained three-dimensional spacer fabric is shown in Figure 3, the upper surface is a checkered hole structure, and the lower surface is a diamond-shaped hole;

(2) preparing a collagen (type I collagen) solution with a mass fraction of 0.2%, compounding it with a three-dimensional spacer fabric in a mold, and pre-freezing and freezing to obtain a scaffold preform;

The preparation process of the collagen solution is as follows:

(2.1) The collagen is mixed in the acetic acid solution in the glass bottle according to the mass ratio of 1:300, and at normal temperature, use a magnetic stirrer to stir at a rotating speed of 500r/min to dissolve;

(2.2) Invert the glass bottle and stir for 4h;

(2.3) centrifuge the mixture in the glass bottle and take the supernatant to prepare a collagen solution;

The mold is a sterile petri dish;

The composite method is the dipping method;

The compounding time is 4 hours, the pre-freezing time is 4 hours, and the freezing time is 4 hours;

(3) vacuum-drying the scaffold prefab for 24 hours to obtain a composite scaffold prefab;

(4) cross-linking the composite scaffold preform to obtain a composite porous scaffold, as shown in Figure 1;

The cross-linking method is the thermogravimetric cross-linking method; the cross-linking time is 24 hours; the composite porous scaffold has a three-dimensional through-hole structure with a porosity of 93% (as shown in Figure 4), the average pore size is 34 μm, and the thickness is 5 mm. , the length is 10mm, the width is 10mm, and the maximum compressive strength is 1000cN when it is deformed to 50% of its own thickness;

The pure collagen scaffolds were prepared according to the operations similar to steps (2) to (4), without adding three-dimensional spacer fabrics during the preparation process, and other parameters were the same as those of steps (2) to (4). It can be seen from Figure 4 that the composite porous scaffold has higher porosity than the same collagen scaffold, which is more conducive to the transport of nutrients and oxygen required for cell adhesion and growth;

(5) Co-culturing the composite porous scaffold with tumor cells to obtain a three-dimensional tumor cell drug resistance model in vitro;

The co-cultivation process is as follows: firstly, the tumor cells are cultured on a flat surface for 5 days, then digested and resuspended in the medium to obtain a tumor cell suspension, and then the tumor cell suspension is implanted into a composite porous scaffold with a syringe for culture (gas phase: air, 95%; Carbon dioxide, 5%; temperature: 37 degrees Celsius);

The implantation density of tumor cells was 2×10 ⁶ /200 μl; the tumor cells were DU145 human prostate cancer cells (purchased from the Cell Bank of the Chinese Academy of Sciences, for the culture and passage methods, please refer to the cell culture instructions provided by the cell bank);

The culture results were observed using a confocal microscope, and the results are shown in Figure 5. Figure 5 shows fluorescent staining of cells on the first, third, and fifth days of culture in the scaffold, and the surface of the scaffold is 500 μm deep. The number of cells was observed and superimposed into a graph. As shown in the figure, the cells cultured on the scaffold for 5 days could effectively carry out cell adhesion and proliferation, and the number of cells on the surface and inside of the scaffold increased.

The finally prepared in vitro three-dimensional tumor cell drug resistance model is used for drug sensitivity detection. The specific process is: first determine the target drug (enzalutamide) and its corresponding drug sensitivity test method (refer to the drug sensitivity test of patent CN109735496A). Step), then according to the drug sensitivity test method corresponding to the target drug, the plane cultured cells and the three-dimensional scaffold cultured cells (that is, the in vitro three-dimensional tumor cell drug resistance model) are tested, and the cell proliferation ability after drug action is calculated. It was observed that under different drug concentrations, the proliferation ability of cells cultured on three-dimensional scaffolds was higher than that of cells cultured on planes, and the expression level of angiogenic growth factor IL-8 secreted by cells cultured on three-dimensional scaffolds was also higher than that of cells cultured on planes. In addition, the half-inhibitory concentration of enzalutamide in cells cultured on three-dimensional scaffolds was higher than that of cells cultured in planes, indicating that the drug resistance of cells cultured in three-dimensional scaffolds was better than that of cells cultured in planes, and it was closer to the actual situation in vivo, in the field of drug screening. There are great application prospects.

Example 2

A method for constructing an in vitro three-dimensional tumor cell drug resistance model, the steps are as follows:

(1) preparing a three-dimensional spacer fabric with a gradient aperture structure, the gradient aperture structure is that the apertures of the upper layer and the lower layer of the three-dimensional spacer fabric are different, and the arrangement rule of the yarns of the spacer layer of the three-dimensional spacer fabric is V-shaped;

In the three-dimensional spacer fabric, the yarns of the upper layer and the lower layer are of the same type and the same thickness, specifically polyester with a fineness of 100D (D: denier);

The average pore diameter of the upper layer is 2mm, and the average pore diameter of the lower layer is 1mm;

The type of yarn of the spacer layer is polyester with a diameter of 0.15mm;

The three-dimensional spacer fabric is processed by an integrated double-needle-bed Raschel warp knitting machine;

In the weaving process of the three-dimensional spacer fabric, the air-through bar technology is adopted;

The upper surface of the finally prepared three-dimensional spacer fabric has a checkered hole structure, and the lower surface is a diamond-shaped hole;

(2) preparing a collagen (type II) solution with a mass fraction of 1%, compounding it with a three-dimensional spacer fabric in a mold, and pre-freezing and freezing to obtain a scaffold preform;

The preparation process of the collagen solution is as follows:

(2.1) The collagen is mixed in the acetic acid solution in the glass bottle according to the mass ratio of 1:200, and at normal temperature, use a magnetic stirrer to stir at a rotating speed of 500r/min to dissolve;

(2.2) Invert the glass bottle and stir for 4h;

(2.3) centrifuge the mixture in the glass bottle and take the supernatant to prepare a collagen solution;

The mold is a cell culture plate with a 24-well plate specification;

The composite method is the coating method;

The number of repetitions of the coating is 3 times, the compounding time is 4 hours, the pre-freezing time is 4 hours, and the freezing time is 4 hours;

(3) vacuum-drying the scaffold prefab for 24 hours to obtain a composite scaffold prefab;

(4) cross-linking the composite scaffold preform to obtain a composite porous scaffold;

The cross-linking method is EDC/NHS cross-linking method; the cross-linking time is 4 h; the composite porous scaffold has a three-dimensional through-hole structure with a porosity of 91%, an average pore size of 30 μm, a thickness of 6 mm, a length of 10 mm, and a width of 10 mm. is 10mm, and the maximum compressive strength is 1100cN when it is deformed to 50% of its own thickness;

(5) Co-culturing the composite porous scaffold with tumor cells to obtain a three-dimensional tumor cell drug resistance model in vitro;

The process of co-cultivation is as follows: firstly, the tumor cells are cultured on a flat surface for 5 days, then digested and resuspended in the medium to obtain a tumor cell suspension, and then the tumor cell suspension is implanted into a composite porous scaffold with a pipette gun for culture (gas phase: air, 95 ℃). %; carbon dioxide, 5%; temperature: 37 degrees Celsius);

The implantation density of tumor cells was 2×10 ⁶ /200 μl; the tumor cells were MCF-7 human breast cancer cells (purchased from the Cell Bank of the Chinese Academy of Sciences, and the culture and passage methods refer to the instructions of the cell bank);

The culture results were observed with a confocal microscope. On the 1st, 3rd, and 5th days, the cells cultured in the scaffolds were fluorescently stained, and the number of cells in the surface of the scaffolds 500 μm deep was observed. Human breast cancer cells cultured on the scaffold for 5 days can effectively adhere and proliferate, and the number of cells on the surface and inside of the scaffold increases.

The finally prepared in vitro three-dimensional tumor cell drug resistance model is used for drug sensitivity detection. The specific process is as follows: first determine the target drug (doxorubicin) and its corresponding drug sensitivity test method (refer to the drug sensitivity test steps of patent CN109735496A). ), and then test the plane cultured cells and the three-dimensional scaffold-cultured cells (ie, the in vitro three-dimensional tumor cell drug resistance model) according to the drug sensitivity test method corresponding to the target drug, and calculate the cell activity. It can be observed that under different drug concentrations, The cell viability of the cells cultured on the three-dimensional scaffolds was higher than that of the cells cultured on the plane, and the expression level of the angiogenic growth factor IL-8 secreted by the cells cultured on the three-dimensional scaffolds was also higher than that of the cells cultured on the plane. The half-inhibitory concentration of mycin was 7.3 times that of cells cultured in plane, indicating that the drug resistance of cells cultured on three-dimensional scaffolds is better than that of cells cultured in planes, and it is closer to the actual situation in vivo, and has great application prospects in the field of drug screening.

Claims (8)

1. a construction method of an in vitro three-dimensional tumor cell drug resistance model, is characterized in that, the steps are as follows: (1) Prepare a three-dimensional spacer fabric with a gradient pore size structure. The gradient pore size structure, that is, the pore size of the upper layer and the lower layer of the three-dimensional spacer fabric is different. The arrangement of fibers or yarns of the spacer layer is V-shaped, X-shaped or flexible; (2) preparing a solution of biologically active material, pre-freezing and freezing after compounding it with a three-dimensional spacer fabric in a mold, to obtain a scaffold preform; (3) vacuum drying the stent preform to obtain a composite stent preform; (4) Cross-linking the composite scaffold preform to obtain a composite porous scaffold; the composite porous scaffold has a three-dimensional through-hole structure with a porosity of more than 90%, an average pore diameter of 10-50 μm, and a thickness of 3-15 mm , when the deformation becomes 50% of its own thickness, the maximum compressive strength is 200 ~ 1300cN; (5) The composite porous scaffold was co-cultured with tumor cells to obtain a three-dimensional tumor cell drug resistance model in vitro. 2. The method for constructing an in vitro three-dimensional tumor cell drug resistance model according to claim 1, wherein in step (1), in the three-dimensional spacer fabric, the fibers or yarns of the upper layer and the lower layer are of the same or different types , selected from polypropylene and polyester, with the same or different thickness; The type of fiber or yarn of the spacer layer is nylon or polyester; The three-dimensional spacer fabric is processed by an integrated double-needle-bed Raschel warp knitting machine; In the weaving process of the three-dimensional spacer fabric, the air-through bar process is adopted. 3. The method for constructing an in vitro three-dimensional tumor cell drug resistance model according to claim 1, wherein in step (2), the preparation process of the bioactive material solution is as follows: (2.1) Mix the bioactive material in the acetic acid solution in the glass bottle according to the mass ratio of 1:99～399, and at normal temperature, use a magnetic stirrer to stir at a rotating speed of 400～800r/min to dissolve; (2.2) Invert the glass bottle and stir for 4-48h; (2.3) centrifuge the mixture in the glass bottle and take the supernatant to prepare a biologically active material solution; The mold is a sterile petri dish, or a 4-well, 6-, 12-, 24- or 48-well cell culture plate; The composite method is dipping method or coating method; The compounding time is 4-48 hours, the pre-freezing time is 4-48 hours, and the freezing time is 4-48 hours. 4 . The method for constructing an in vitro three-dimensional tumor cell drug resistance model according to claim 3 , wherein the bioactive materials are I-I collagen and its derivatives, alginate and its derivatives, hyaluronan Acids and their derivatives, polypeptides, gelatin, chitosan or chitin. 5 . The method for constructing an in vitro three-dimensional tumor cell drug resistance model according to claim 1 , wherein, in step (3), the vacuum drying time is 4-48 hours. 6 . 6. The method for constructing an in vitro three-dimensional tumor cell drug resistance model according to claim 1, wherein in step (4), the cross-linking method is photocross-linking, radiation cross-linking, thermal re-crosslinking Cross-linking method or chemical reagent cross-linking method; cross-linking time is 2 ~ 48h. 7 . The method for constructing an in vitro three-dimensional tumor cell drug resistance model according to claim 1 , wherein in step (5), the co-cultivation process is: firstly culturing the tumor cells on a plane for 1 to 7 days, and then undergoing The culture medium is digested and resuspended to obtain a tumor cell suspension, and then the tumor cell suspension is implanted into the composite porous scaffold with a syringe or pipette for culture; The implantation density of tumor cells is 1～5×10 ⁶ /200μl; tumor cells are prostate cancer cells, breast cancer cells, ovarian cancer cells, renal cancer cells, erythroleukemia K562 cells, human promyelocytic leukemia cells, liver cancer cells or Cervical cancer cells. 8. The application of the in vitro three-dimensional tumor cell drug resistance model prepared by the method for constructing an in vitro three-dimensional tumor cell drug resistance model according to any one of claims 1 to 7, characterized in that it is used for drug sensitivity detection The specific process is: first determine the target drug and its corresponding drug sensitivity test method, and then test the in vitro three-dimensional tumor cell drug resistance model according to the drug sensitivity test method corresponding to the target drug, and calculate the in vitro three-dimensional tumor cell drug resistance model. cell proliferation and growth factor expression levels.

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