CN112300979A

CN112300979A - Method for in-vitro construction of disease model based on porous microspheres and application thereof

Info

Publication number: CN112300979A
Application number: CN202011079051.XA
Authority: CN
Inventors: 陈爱政; 王颖; 王士斌
Original assignee: Huaqiao University
Current assignee: Huaqiao University
Priority date: 2020-10-10
Filing date: 2020-10-10
Publication date: 2021-02-02

Abstract

The invention provides a method for constructing a disease model in vitro based on porous microspheres and application thereof. Then, taking the liver cancer disease model as an example, a 3D liver cancer tumor micro-tissue model was formed by filling PLGA PMs with human liver cancer cells (HepG2) and Human Umbilical Vein Endothelial Cells (HUVECs) using a dynamic culture method. In addition, the tumor tissue was used to evaluate various chemotherapeutic anti-cancer drugs, including cellular responses to Doxorubicin (DOX) and Cisplatin (CIS). In addition, in the arthritis disease model, for example, in vitro culture of disease microtissue was performed by dynamically culturing chondrocytes (C518) and preosteoblasts (MC3T 3). The disease model of the invention can be used for screening drugs.

Description

Method for in-vitro construction of disease model based on porous microspheres and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a method for constructing a disease tissue model in vitro based on porous microspheres and application thereof, wherein the technical method is the combination of a microfluidic coaxial technology and a dynamic culture method.

Background

Over the past few decades, there has been a continuing effort to explore effective clinical drug screening methods to clearly demonstrate the pharmacological and toxicological properties of various chemotherapeutic drugs. Although the conventional two-dimensional (2D) cell culture method based on a cell monolayer, which is widely used, has a certain limitation in simulating a highly complex extracellular matrix (ECM) microenvironment, despite its simple operation, and cannot accurately summarize in vivo cell-cell and cell-environment interactions. To overcome these limitations and to obtain tissue substitutes that can highly mimic the characteristics of tumors in vivo, three-dimensional (3D) tumor models based on cell aggregates have become a promising alternative.

Compared with the traditional 2D cell culture method, the 3D tissue model can reflect more accurate tumor microenvironment. The 3D tumor model can not only simulate the complex spatial arrangement of cells, but also can more accurately predict the drug resistance and drug resistance of tissues when the antitumor drugs are used for drug evaluation and cancer research. Therefore, the study of the efficacy and intercellular interactions of 3D scaffold-based microarchitectures is crucial for the development and screening of drugs. Due to their high porosity and good biocompatibility, the large number of porous microspheres have shown great potential in the in vitro preparation of cell-loaded tissue models.

The porous microspheres have an internal porous structure and high porosity, so that a larger surface area can be provided for cell growth, adhesion sites of cells in the scaffold can be increased, and a sufficient number of cells can be obtained; and the porous microsphere is favorable for mass transfer, provides a good microenvironment for the growth of cells in the porous microsphere, maintains the cell differentiation phenotype and is convenient for adjusting and monitoring the cell culture environment. In addition, the porous microspheres provide better mechanical properties than microgel or tissue engineering cell sheets while maintaining the special components and structures of the microcells, and are more favorable for screening the tissue-forming drugs after long-term clinical culture.

However, in the conventional preparation method of porous microspheres, most of the pores in the microspheres are concentrated on the surface, cells can only adhere to the surface of the microspheres to grow, and cannot enter the inside of the scaffold, and the number of the cells capable of adhering is very limited, so that the cell density and number requirements of tissues formed in vitro cannot be met. In addition, the traditional in vitro microsphere disease model preparation method has the defects that the adhesion quantity of cells is low, the proliferation condition of the cells is limited, and intercellular force is difficult to form between the cells to generate enough ECM so as to support the growth of the cells, particularly simulated tissues.

Disclosure of Invention

The invention mainly aims to provide a method for constructing a disease model in vitro based on porous microspheres.

In order to achieve the purpose of the invention, one of the technical schemes of the invention is as follows:

a method for in vitro construction of a disease model based on porous microspheres comprises the following steps:

1) adopting a microfluidic self-assembly coaxial nozzle, wherein the nozzle comprises an outer-layer thin tube and an inner-layer needle head arranged in the thin tube; the outer layer is filled with a dispersed phase containing a pore-foaming agent, and the inner layer is filled with a continuous phase; the dispersed phase comprises one or more polymer emulsions of PLGA (polylactic-co-glycolic acid), PCL (polycaprolactone), PLLA (L-polylactic acid) and other high polymer, and the pore-forming agent comprises but is not limited to gelatin, ammonium bicarbonate and menthol; under the action of the dispersed phase and the continuous phase, micro liquid drops are generated, wherein the flow rate of the dispersed phase is 1.8-2.2ml/min, and the flow rate of the continuous phase is 0.03-0.08 ml/min;

2) standing the micro-droplets at room temperature for 2-24h, and curing and forming after the solvent is completely volatilized; placing the collected product in ultrapure water at 37-60 ℃ and heating to dissolve out the pore-forming agent, and cleaning for several times to remove the pore-forming agent to obtain the polymer porous microspheres; the average particle size of the polymer porous microsphere is 100-900 mu m, the interior of the polymer porous microsphere is provided with through holes, and the size of the inner pore diameter is 5-100 mu m;

3) the obtained porous microspheres are collected and,

a. HUVECs (human umbilical vein endothelial cells) and HepG2 (human liver cancer cells) live cells are added,adjusting the cell density of both cells to 2X10^5-7Performing dynamic culture for 18-30h to obtain a liver cancer model per mL; or

b. C518 (chondrocyte) and MC3T3-E1 (preosteoblasts) were added to adjust the cell density of both cells to 2X10^5-7Culturing dynamically for 18-30h per mL, and treating with 0.4-0.6 μ g/mL LPS (lipopolysaccharide) for 40-60h to obtain arthritis model.

In one embodiment: the outer layer of the nozzle is a glass capillary tube (0.5-2mm), and the inner layer is a needle head (18-28G)

In one embodiment: the continuous phase comprises at least one of polyvinyl alcohol, silicone oil and edible oil.

In one embodiment: in the step 3) a, the dynamic culture condition is 36.5-37.5 ℃ and the rotating speed is 30-300 rpm.

In one embodiment: in the step 3), a step of adding a medicament is also included, the medicament is directly added into the culture medium and acts on cells along with the dynamic culture process.

In one embodiment: in step 3) a, the drug includes but is not limited to one or more of DOX, CIS, paclitaxel and docetaxel.

In one embodiment: in the step 3) b, the dynamic culture condition is 36.5-37.5 ℃ and the rotating speed is 30-300 rpm.

In one embodiment: in the step 3) b, a step of adding a medicament is also included, wherein the medicament is directly added into the culture medium and acts on cells along with the dynamic culture process.

In one embodiment: the medicine comprises one of curcumin, ibuprofen, aspirin and penicillamine.

In one embodiment: the dynamic culture method is one of a shaking table, a three-dimensional cell culture instrument, an oscillator, a rotator and other dynamic culture devices.

The invention provides a construction method of a three-dimensional disease model based on porous microspheres, which comprises a liver cancer model and an arthritis model and provides application of different disease models in drug screening. First, using microfluidic technology, self-assembled coaxial nozzles fabricated polylactic-co-glycolic acid (PLGA PMs) macroporous microspheres. Then, taking the liver cancer disease model as an example, a 3D liver cancer tumor micro-tissue model was formed by filling PLGA PMs with human liver cancer cells (HepG2) and Human Umbilical Vein Endothelial Cells (HUVECs) using a dynamic culture method. The tumor tissue can be used to evaluate the cellular response to various chemotherapeutic anti-cancer drugs, including Doxorubicin (DOX) and Cisplatin (CIS). In addition, in the case of an arthritis disease model, study of in vitro culture of disease microtissue can be carried out by dynamically culturing chondrocytes (C518) and preosteoblasts (MC3T 3).

The invention has the advantages that:

(1) the preparation method of the macroporous microspheres provided by the invention has the advantages of simple and feasible process, short preparation period, green and environment-friendly preparation process, strong practicability and wide application prospect. And the cell can be used for culturing different disease tissues by loading different cells, and has wider application range and higher drug screening efficiency.

(2) According to the liver cancer model provided by the invention, HepG2 and HUVECs are loaded at the same time, cells among different microspheres can aggregate mutually to form vascularized liver cancer tissues through in vitro culture, so that the construction of a liver cancer model with a complex vascular network and high bionics is facilitated, and compared with 2D culture, the drug resistance of cells to DOX and CIS can be obviously improved.

(3) The arthritis model provided by the invention is loaded with C518 and MC3T3-E1, cells among different microspheres can aggregate mutually, the joint model with the lower layer being osteoblast/endothelial cell tissue and the upper layer being cartilage tissue can be constructed, and the arthritis model is constructed by using LPS to induce the joint model and is used for screening and researching drugs such as curcumin and the like.

Drawings

FIG. 1 is an electron micrograph of PLGA PMs constructed according to the present invention.

FIG. 2 is a confocal fluorescence diagram of HUVECs and HepG2 loaded laser. Fig. 2a is an external view, and fig. 2b is an internal view.

FIG. 3 is a confocal fluorescence image of the aggregation and intercellular binding between microspheres according to a first embodiment of the present invention.

FIG. 4 is IC of DOX effect on cells in 3D culture compared to 2D culture₅₀And (5) analyzing the change.

FIG. 5 is IC of CIS effect on cells in 3D culture compared to 2D culture₅₀And (5) analyzing the change.

FIG. 6 is a confocal laser photograph (scale: 100 μm) of MC3T3-E1 cells co-cultured with multi-well microspheres for 5 days.

FIG. 7 is a confocal image of laser light (scale: 100 μm) of C518 cells co-cultured with porous microspheres for 5 days.

FIG. 8 is a CCK-8 method for detecting the influence of curcumin with different concentrations on cell activity after 48 hours of action.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. It should also be understood that various modifications may occur to those skilled in the art upon reading the teachings herein, and that such equivalents are within the scope of the appended claims.

The first embodiment is as follows:

dissolving 0.0048g of PLGA in 2.4mL of dichloromethane to prepare 2% PLGA solution, dissolving 0.0075g of gelatin in 1mL of ultrapure water to prepare 1% gelatin aqueous solution, mixing the PLGA solution and the gelatin aqueous solution, and performing ultrasonic treatment for 90s to prepare PLGA-gelatin emulsion as a dispersion phase; 1% polyvinyl alcohol is used as a continuous phase, the two phases are respectively arranged in two disposable syringes, the disposable syringes are arranged on a microfluidic injection pump and connected with a guide pipe, and the other end of the guide pipe is inserted into a self-assembly coaxial nozzle. The outer layer of the self-assembly coaxial nozzle is a glass capillary tube with the diameter of 2mm, and the inner layer of the self-assembly coaxial nozzle is a needle head of 18-28G; the outer layer is filled with polyvinyl alcohol with the continuous phase of 1 percent, and the inner layer is filled with dispersed phase PLGA-gelatin emulsion. The flow rate of polyvinyl alcohol is 2mL/min, the flow rate of a dispersion phase is 0.05mL/min, 1% of polyvinyl alcohol is used as a collection phase to collect a sample, and after dichloromethane is completely volatilized, ultrapure water and PBS are used for washing to prepare the PLGA porous microsphere. The prepared PLGA porous microspheres were lyophilized and photographed with an electron microscope, see FIG. 1.

Cell Trace Using Cell markerr respectively performing cell nucleus staining on HUVECs and HepG2 live cells, wherein the yellow is HUVECs, the purple is HepG2, and the cell density of the two cells is adjusted to be 2x10⁶Adding 50mL of a centrifuge tube, adding the sterilized PLGA porous microspheres, and sucking a culture Medium by using the sterile centrifuge tube, wherein the culture Medium of Hpeg2 is Dulbecco's Modified Eagle Medium (DMEM) containing serum, and the culture Medium of HUVECs is F12K containing serum. Respectively blowing and uniformly beating the two cells, then placing the two cells into a shaking table, setting the shaking condition to be 37 ℃ and 110rpm under the aseptic condition, observing the cell adhesion and distribution condition after 1D, wherein a figure 2a is a 3D scanning superposition graph, and a figure 2b is an internal graph of the bracket, so that the cells are mostly gathered on the surface of the bracket after 1D of culture, and a little cell adhesion exists in the bracket; the aggregation and intercellular binding between microspheres are shown in FIG. 3;

and (3) taking the prepared porous microspheres loaded with HepG2 and HUVECs, dynamically culturing for 24 hours, adding DOX (Dox) of 15 mu g/mL, taking 2D culture as an experimental control group, and detecting the cell survival rate by using a CCK-8 method. The results are shown in fig. 4, and it can be seen that the IC50 value of the cells under the 3D culture condition is significantly higher under the same culture time, and the results have significant difference, which indicates that the cells under the 3D culture have better drug resistance, and the constructed tumor model is closer to the real tumor tissue.

And (3) taking the prepared porous microspheres loaded with HepG2 and HUVECs, replacing the model drug DOX with CIS, observing the growth and survival conditions of cells, and further verifying the drug resistance of the constructed tumor model. The results are shown in fig. 5, and it can be seen that the IC50 value of the cells under 3D culture conditions is significantly higher during CIS action under the same culture time, and the results have significant differences. The liver cancer model has universality and can be used for screening and developing various medicines.

Example two:

this example is essentially the same as the first example, except that C518 and MC3T3-E1 were loaded separately and the growth and distribution of cells were observed. The culture medium was aspirated by a sterile centrifuge tube, and the culture medium of C518 and MC3T3-E1 was DMEM medium containing serum. The two cells were each shaken with medium and placed in a shaker under sterile conditions at 37 ℃ and 110 rpm. In addition, the tissue structure can simulate a joint model, and then a arthritis model is constructed through the occurrence of pathological changes induced by LPS. After different concentrations of LPS act on the constructed microtissue for different time periods, the kit is used for investigating and measuring the contents of Interleukin 1 beta (Interleukin-1 beta, IL-1 beta) and Tumor necrosis factor alpha (Tumor necrosis factor alpha, TNF-alpha) in the microtissue supernatant, and the results are shown in fig. 6 and fig. 7, and the experimental result shows that the contents of IL-1 beta and TNF-alpha in the microtissue supernatant are the highest after the LPS with the concentration of 0.5 mug/mL acts for 48 hours. Therefore, the optimal concentration of the lipopolysaccharide for causing the arthropathy is 0.5 mug/mL, and the optimal action time is 48 h. Finally, the influence of curcumin on the content of IL-1 beta and TNF-alpha in the supernatant of the constructed arthritis model is researched. The CCK-8 method is adopted to detect the toxicity of the curcumin with different concentrations on cells, the result is shown in figure 8, the cytotoxicity result shows that the curcumin with the concentration of 10 mug/mL acts on the cells for 48 hours, and the curcumin with the concentration of 10 mug/mL has toxicity on the cells from the change of the cell morphology and the relative proliferation rate of the cells. When the curcumin concentration is 8 mug/mL, the morphology of the cells is not changed, and the growth of the cells is not influenced. Therefore, 8 μ g/mL curcumin was used as the optimum administration concentration. Curcumin was phagocytized by cells after pretreatment of the joint model with CLSM to observe 8 μ g/mL curcumin for 4 h. The detection result of the kit shows that: the curcumin can reduce the level of inflammatory factors IL-1 beta and TNF-alpha of arthritis model tissues induced by LPS after pretreatment, and the inhibition of the release of proinflammatory factors is a protection mechanism for articular cartilage, and further proves that the joint unit model constructed in the invention can be used for the research of drug screening.

Claims

1. A method for in vitro construction of a disease model based on porous microspheres comprises the following steps:

1) adopting a microfluidic self-assembly coaxial nozzle, wherein the nozzle comprises an outer-layer thin tube and an inner-layer needle head arranged in the thin tube; the outer layer is filled with a dispersed phase containing a pore-foaming agent, and the inner layer is filled with a continuous phase; the dispersed phase comprises at least one of PLGA, PCL and PLLA; the pore-forming agent comprises at least one of gelatin, ammonium bicarbonate and menthol; under the action of the dispersed phase and the continuous phase, micro liquid drops are generated, wherein the flow rate of the dispersed phase is 1.8-2.2ml/min, and the flow rate of the continuous phase is 0.03-0.08 ml/min;

3) the obtained porous microspheres are collected and,

a. HUVECs and HepG2 live cells were added to adjust the cell density of both cells to 2X10^5-7Performing dynamic culture for 18-30h to obtain a liver cancer model per mL; or

b. Adding C518 and MC3T3-E1 cells, adjusting cell density of both cells to 2x10^5-7Culturing dynamically for 18-30h per mL, and treating with 0.4-0.6 μ g/mL LPS for 40-60h to obtain arthritis model.

2. The method for in vitro construction of a disease model based on porous microspheres of claim 1, wherein the outer layer of the nozzle is a glass capillary tube with a diameter of 0.5-2mm, and the inner layer is a needle with a diameter of 18-28G.

3. The method of claim 1, wherein the continuous phase comprises at least one of polyvinyl alcohol, silicone oil, and edible oil.

4. The method for in vitro construction of a disease model based on porous microspheres of claim 1, wherein in step 3) a or b, the dynamic culture conditions are 36.5-37.5 ℃ and 30-300 rpm.

5. The method for in vitro construction of a disease model based on porous microspheres of claim 1, wherein step 3) a further comprises the step of adding a drug directly into the culture medium, wherein the drug acts on the cells during the dynamic culture process.

6. The method for in vitro construction of disease model based on porous microspheres of claim 5, wherein in step 3) a, the drugs include but are not limited to one or more of DOX, CIS, paclitaxel, and docetaxel.

7. The method for in vitro construction of a disease model based on porous microspheres of claim 1, wherein in step 3) b, the dynamic culture conditions are 36.5-37.5 ℃ and 30-300 rpm.

8. The method for in vitro construction of a disease model based on porous microspheres of claim 1, wherein step 3) b further comprises the step of adding a drug directly into the culture medium, wherein the drug acts on the cells during the dynamic culture process.

9. The method of claim 8, wherein the drug comprises one of curcumin, ibuprofen, aspirin, and penicillamine.

10. Use of the method of any one of claims 1 to 9 for the in vitro construction of a disease model based on porous microspheres in drug screening.