Osteosarcoma cell microcapsule for drug evaluation as well as preparation method and application of osteosarcoma cell microcapsule

Abstract

The invention discloses an osteosarcoma cell microcapsule for drug evaluation and a preparation method and application thereof, and belongs to the technical field of biological materials.The preparation method comprises the steps that 1, the osteosarcoma cell microcapsule is prepared through a microfluidic electronic injection device; the micro-fluidic electronic injection device comprises a micro-fluidic chip and an electric field; a sodium alginate solution is used as an external phase, a mixed solution of a carboxymethyl cellulose solution and a human osteosarcoma cell line is used as an internal phase, the internal phase and the external phase are injected from an inlet end of a micro-fluidic chip and then sprayed out from an outlet end, the external phase wraps the internal phase, the micro-droplets are separated under the action of an electric field, and then the osteosarcoma cell microcapsules are formed in a collection phase; and 2, culturing the osteosarcoma cell microcapsule, and after cells dispersed in the osteosarcoma cell microcapsule converge and grow to form cell spheres, obtaining the osteosarcoma cell microcapsule for drug evaluation. The method can be used for evaluating the curative effect of clinical anti-tumor drugs and improving the accuracy and safety of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and relates to an osteosarcoma microenvironment simulation product, and in particular to an osteosarcoma cell microcapsule for drug evaluation, a preparation method and an application thereof. Background Art

[0002] Osteosarcoma is an extremely aggressive primary bone malignant tumor that not only grows rapidly but also easily metastasizes to other parts of the body, especially the lungs, through the blood. As the most common malignant bone tumor in adolescents and children, osteosarcoma poses a serious threat to the life and health of patients. Although medical technology has made significant progress in recent years, traditional treatments such as surgical resection combined with chemotherapy have improved patients' prognosis and survival rates to a certain extent, but these therapies still face many challenges. For example, tumor cells may develop resistance to chemotherapy drugs, which not only reduces the effectiveness of treatment, but may also lead to disease recurrence. In addition, there is a high degree of heterogeneity within osteosarcoma, that is, tumor cells in different regions differ in genetic background, biological behavior, etc. This heterogeneity further increases the difficulty of treatment.

[0003] In view of the above problems, researchers are actively exploring new methods to improve the treatment of osteosarcoma. Among them, building a more realistic in vitro model to simulate the in vivo microenvironment of osteosarcoma has become one of the research hotspots. In this way, it is hoped that the performance of drugs in actual clinical applications can be more accurately predicted, thereby screening out more effective treatment options. At the same time, this method can also help to deeply understand the development mechanism of osteosarcoma and provide a theoretical basis for the development of new drugs targeting specific molecular targets. Although Summary of the invention

[0004] The present invention provides an osteosarcoma cell microcapsule for drug evaluation and a preparation method and application thereof, so as to overcome the defects of the prior art.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing osteosarcoma cell microcapsules for drug evaluation, comprising the following steps: step 1, preparing osteosarcoma cell microcapsules using a microfluidic electrospray device; the microfluidic electrospray device comprises a microfluidic chip consisting of an inner tube and an outer tube nested in each other and an electric field formed by applying a voltage at the outlet end of the microfluidic chip; using a sodium alginate solution as an outer phase and a mixed solution of a carboxymethyl cellulose solution and a human osteosarcoma cell line as an inner phase, the inner phase and the outer phase are respectively injected into the inner tube and between the inner tube and the outer tube from the inlet end of the microfluidic chip, and then sprayed out from the outlet end, the outer phase is wrapped around the inner phase, separated into micro droplets under the action of the electric field, and then osteosarcoma cell microcapsules are formed in the collection phase; step 2, culturing the osteosarcoma cell microcapsules, and waiting for the dispersed cells in the osteosarcoma cell microcapsules to converge and grow to form cell spheres, so as to obtain osteosarcoma cell microcapsules that can be used for drug evaluation.

[0007] Optionally, in step 1, the mass fraction of the sodium alginate solution is 0.5-5%.

[0008] Optionally, in step 1, the preparation method of the internal phase is: culture a human osteosarcoma cell line, digest and centrifuge the cells when the cell confluence reaches more than 80%; mix the carboxymethyl cellulose solution and the cell culture medium at a volume ratio of 1:1, and then use the mixed solution to resuspend the centrifuged cells to obtain the internal phase; the mass fraction of the carboxymethyl cellulose solution is 0.5-2%; the cell culture medium includes MEM culture medium, fetal bovine serum, penicillin-streptomycin mixed solution (100X), sodium bicarbonate solution and sodium pyruvate solution; the concentration of the sodium bicarbonate solution is 100mM, the mass fraction of the sodium pyruvate solution is 7.5%, and the volume ratio of the MEM culture medium, fetal bovine serum, penicillin-streptomycin mixed solution (100X), sodium bicarbonate solution and sodium pyruvate solution is 86:10:1:2:1.

[0009] Optionally, in step 1, the collecting phase is a calcium chloride solution; the mass fraction of the calcium chloride solution is 1 to 5%.

[0010] Optionally, in step 1, the diameters of the inner tube and the outer tube of the microfluidic chip are 100 to 500 μm; and the flow rate ratio of the outer phase to the inner phase is 30 to 50:8.

[0011] Optionally, in step 1, the voltage is 3 to 10 kV.

[0012] Optionally, in step 2, the culturing method of the osteosarcoma cell microcapsules is: placing the osteosarcoma cell microcapsules in a cell culture medium, culturing in an incubator at 37° C. and 5% CO2, and replacing the cell culture medium every 1 to 2 days; the cell culture medium includes MEM culture medium, fetal bovine serum, a penicillin-streptomycin mixture (100X), a sodium bicarbonate solution, and a sodium pyruvate solution; the concentration of the sodium bicarbonate solution is 100 mM, the mass fraction of the sodium pyruvate solution is 7.5%, and the volume ratio of the MEM culture medium, fetal bovine serum, a penicillin-streptomycin mixture (100X), a sodium bicarbonate solution, and a sodium pyruvate solution is 86:10:1:2:1.

[0013] Optionally, in step 2, the size of the cell sphere is 100 to 300 μm.

[0014] In a second aspect, the present invention also provides osteosarcoma cell microcapsules prepared by the above-mentioned preparation method for drug evaluation.

[0015] In a third aspect, the present invention also provides the use of the above-mentioned osteosarcoma cell microcapsules in the analysis of the efficacy of anti-tumor drugs.

[0016] The beneficial effects of the present invention are as follows: the present invention provides an osteosarcoma cell microcapsule for drug evaluation, a preparation method, and an application thereof. The hydrogel microcapsule prepared by the method of the present invention has high uniformity, stability, and precise size controllability. The cell spheres in the hydrogel microcapsule can rapidly proliferate to form 3D tumor spheres with uniform size and good cell activity. The hydrogel microcapsule has a certain mechanical strength and is not easy to disintegrate, thereby helping to maintain the long-term stability of the tumor spheres in drug screening. This 3D tumor model can provide a reliable and accurate drug evaluation platform and provide guidance for the use of clinical anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the preparation process of osteosarcoma cell microcapsules;

[0018] Figure 2 This is a microscope image of a microcapsule;

[0019] Figure 3 is a scanning electron microscope image of the microcapsules;

[0020] Figure 4 is the size distribution diagram of the microcapsules, where a is the size distribution diagram of the microcapsules, b is the size distribution diagram of the core layer, and c is the size distribution diagram of the shell layer;

[0021] Figure 5 These are microscopic images of the cell sphere formation process in osteosarcoma cell microcapsules, at 1, 3, 5, 7, and 9 days;

[0022] Figure 6is a diagram of the formation process of cell spheres in osteosarcoma cell microcapsules, where a is a fluorescent image of cell spheres with live and dead staining, b and c are the corresponding cell viability statistics, and d is the distribution statistics of cell sphere diameters on day 9;

[0023] Figure 7 These are the drug evaluation results of osteosarcoma cell microcapsules, where a is a fluorescent image of live-dead staining of cell spheres after doxorubicin treatment, b is a statistical graph of cell viability after doxorubicin treatment, and c is a statistical graph of cell viability after combined treatment of doxorubicin and cisplatin. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments.

[0025] Example 1

[0026] This embodiment provides an osteosarcoma cell microcapsule for drug evaluation. The preparation process is as follows: Figure 1 As shown, the following steps are included:

[0027] Step 1: Preparation of osteosarcoma cell microcapsules using a microfluidic electrospray device:

[0028] The microfluidic electrospray device comprises a microfluidic chip composed of mutually nested inner tubes and outer tubes and an electric field formed by applying voltage to the outlet end of the microfluidic chip.

[0029] Specifically, the preparation method of the microfluidic chip is as follows: capillaries with inner diameters of 100 μm and 500 μm respectively are used as inner and outer tubes, both ends are polished and smoothed, and then the two capillaries are nested and combined, and the two are adjusted to be on the same central axis under a microscope, and then the two capillaries are fixed above 30 cm on the glass slide with quick-drying glue, and then a groove matching the capillary is processed at the bottom of the dispensing needle, and quick-drying glue is applied, and it is stuck into the capillary connection part, and finally the bottom of the dispensing needle is sealed with quick-drying glue to ensure the sealing of the microfluidic chip.

[0030] The preparation method of osteosarcoma cell microcapsules is as follows: sodium alginate solution is used as the external phase, and a mixture of carboxymethyl cellulose solution and human osteosarcoma cell line is used as the internal phase. The internal phase and the external phase are respectively injected into the inner tube and between the inner tube and the outer tube from the inlet end of the microfluidic chip, and then sprayed out from the outlet end. The external phase is wrapped around the outside of the internal phase, separated into microdroplets under the action of the electric field, and then osteosarcoma cell microcapsules are formed in the collection phase.

[0031] The structure of the microcapsules is explained by preparing microcapsules without adding osteosarcoma cells: prepare 1.5wt% sodium alginate solution, 2wt% carboxymethyl cellulose solution and 2wt% calcium chloride solution. Fix the microfluidic chip 10cm above the collection phase container, and then connect a high-voltage power supply between the microfluidic chip and the collection phase to apply voltage. The above sodium alginate solution is sucked into a 10mL syringe as the external phase, the carboxymethyl cellulose solution is sucked into a 2.5mL syringe as the internal phase, and the calcium chloride solution is used as the collection phase. The two syringes of the external phase and the internal phase are then fixed on the microfluidic pump, and the flow rate parameters of the microfluidic pump are adjusted to 30μL / min and 8μL / min, respectively. Microcapsules with uniform size and core-shell structure can be collected in the collection phase, such as Figures 2 to 4 shown.

[0032] In this embodiment, human osteosarcoma cell lines are cultured in a culture bottle, and when the cell confluence reaches more than 80%, they are digested and centrifuged. The above carboxymethyl cellulose solution and the cell culture medium are mixed at a volume ratio of 1:1, and then the centrifuged cells are resuspended with the mixed solution and sucked into the inner phase syringe. The cell culture medium includes 86% (v / v) MEM culture medium, 10% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin mixed solution (100X), 2% (v / v) sodium bicarbonate solution (100mM) and 1% (v / v) sodium pyruvate solution (7.5wt%). The remaining steps and parameters are the same as those for the preparation of the above microcapsules, and microcapsules encapsulating osteosarcoma cells can be obtained in the collection phase.

[0033] Step 2: Transfer the collected osteosarcoma cell microcapsules to a culture dish, replace the calcium chloride solution with the above cell culture medium, and continue to culture in a 37°C, 5% CO2 incubator for 9 days, replacing the cell culture medium every day. The cells dispersed in the microcapsules gather and grow to form cell spheres, thus obtaining osteosarcoma cell microcapsules that can be used for drug evaluation. The growth process of osteosarcoma cell spheres in microcapsules is as follows Figure 5 As shown, live-dead staining was performed on osteosarcoma cell spheres during growth to assess their viability. Figure 6 shown.

[0034] Osteosarcoma cell spheroid microcapsules can be used to analyze the efficacy of anti-tumor drugs:

[0035] Study on the sensitivity analysis of osteosarcoma cell spheroid microcapsules to doxorubicin: Osteosarcoma cell spheroid microcapsules were transferred to the cell culture chamber on the drug concentration microfluidic chip for culture for 2 days. Doxorubicin was dissolved in the cell culture fluid and prepared into a mixed solution of 20μg / mL. The cell culture fluid and doxorubicin solution were pumped into the two inlets of the chip by a microfluidic pump, and then the cell spheroid viability was observed at three time points of 12h, 24h, and 48h. Figure 7Figures a and b show the effect of the microfluidic chip in successfully establishing a drug concentration gradient. It can be observed that with the increase of doxorubicin concentration, the cell spheroid viability decreased significantly. In addition, with the extension of culture time, the cell spheroid viability further decreased, indicating that doxorubicin has a significant apoptosis-inducing effect on tumor cells, and exhibits dose-dependent and time-dependent characteristics.

[0036] Study on the sensitivity analysis of osteosarcoma cell spheroid microcapsules to the combined treatment of doxorubicin and cisplatin: The osteosarcoma cell spheroid microcapsules were transferred to the cell culture chamber on the drug concentration microfluidic chip for culture for 2 days. Doxorubicin and cisplatin were dissolved in the cell culture medium and prepared into a mixed solution of 20μg / mL. The cisplatin solution and doxorubicin solution were pumped into the two inlets of the chip by a microfluidic pump, and then the cell spheroid viability was observed at three time points of 12h, 24h, and 48h. Figure 7 As shown in middle c, the experimental results show that the microfluidic chip successfully constructed a combination of two tumor drugs at different concentration ratios. Compared with single drug treatment, the combined treatment of doxorubicin and cisplatin resulted in a significant decrease in cell spheroid viability and a more significant inhibitory effect. These results show that osteosarcoma cell spheroid microcapsules can be used as an effective 3D model for in vitro drug evaluation.

[0037] Example 2

[0038] This embodiment provides an osteosarcoma cell microcapsule for drug evaluation. The preparation method is basically the same as that of Example 1, except that the mass fraction of the sodium alginate solution is 2%.

[0039] Example 3

[0040] This embodiment provides an osteosarcoma cell microcapsule for drug evaluation. The preparation method is basically the same as that of Example 1, except that the mass fraction of the carboxymethyl cellulose solution is 1%.

[0041] Example 4

[0042] This embodiment provides an osteosarcoma cell microcapsule for drug evaluation. The preparation method is basically the same as that of Example 1, except that the flow rates of the external phase and the internal phase of the microfluidic pump are 50 μL / min and 8 μL / min, respectively.

[0043] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operating procedures used herein are reagents, materials and conventional procedures widely used in the corresponding fields.

[0044] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing osteosarcoma cell microcapsules for drug evaluation, characterized in that: The following steps are involved: Step 1: preparing osteosarcoma cell microcapsules using a microfluidic electrospray device; the microfluidic electrospray device includes a microfluidic chip consisting of an inner tube and an outer tube nested with each other and an electric field formed by applying a voltage at the outlet of the microfluidic chip; Using sodium alginate solution as the external phase and carboxymethyl cellulose solution and a mixture of human osteosarcoma cell lines as the internal phase, the internal phase and the external phase are injected into the inner tube and between the inner tube and the outer tube from the inlet end of the microfluidic chip, respectively, and then ejected from the outlet end. The external phase is wrapped around the internal phase and separated into microdroplets under the action of the electric field, and then osteosarcoma cell microcapsules are formed in the collection phase. Step 2: culturing the osteosarcoma cell microcapsules, and waiting for the dispersed cells in the osteosarcoma cell microcapsules to aggregate and grow to form cell spheres, thereby obtaining osteosarcoma cell microcapsules that can be used for drug evaluation.

2. The method for preparing osteosarcoma cell microcapsules for drug evaluation according to claim 1, characterized in that: In step 1, the mass fraction of the sodium alginate solution is 0.5-5%.

3. The method for preparing osteosarcoma cell microcapsules for drug evaluation according to claim 1, characterized in that: In step 1, the preparation method of the inner phase is as follows: culturing a human osteosarcoma cell line, digesting and centrifuging the cells when the cell confluence reaches more than 80%; mixing a carboxymethyl cellulose solution and a cell culture medium at a volume ratio of 1:1, and then resuspending the centrifuged cells with the mixed solution to obtain the inner phase; The mass fraction of carboxymethyl cellulose solution is 0.5-2%; The cell culture medium includes MEM culture medium, fetal bovine serum, penicillin-streptomycin mixed solution, sodium bicarbonate solution and sodium pyruvate solution; the concentration of the sodium bicarbonate solution is 100mM, the mass fraction of the sodium pyruvate solution is 7.5%, and the volume ratio of the MEM culture medium, fetal bovine serum, penicillin-streptomycin mixed solution, sodium bicarbonate solution and sodium pyruvate solution is 86:10:1:2:

1.

4. The method for preparing osteosarcoma cell microcapsules for drug evaluation according to claim 1, characterized in that: In step 1, the collecting phase is a calcium chloride solution; The mass fraction of the calcium chloride solution is 1 to 5%.

5. The method for preparing osteosarcoma cell microcapsules for drug evaluation according to claim 1, characterized in that: In step 1, the diameters of the inner tube and the outer tube of the microfluidic chip are 100 to 500 μm; The flow rate ratio of the outer phase to the inner phase is 30-50:

8.

6. The method for preparing osteosarcoma cell microcapsules for drug evaluation according to claim 1, characterized in that: In step 1, the voltage is 3-10 kV.

7. The method for preparing osteosarcoma cell microcapsules for drug evaluation according to claim 1, characterized in that: In step 2, the osteosarcoma cell microcapsule culture method is: placing the osteosarcoma cell microcapsule in a cell culture medium, culturing in an incubator at 37° C. and 5% CO 2 , and replacing the cell culture medium every 1 to 2 days; The cell culture medium includes MEM culture medium, fetal bovine serum, penicillin-streptomycin mixed solution, sodium bicarbonate solution and sodium pyruvate solution; the concentration of the sodium bicarbonate solution is 100mM, the mass fraction of the sodium pyruvate solution is 7.5%, and the volume ratio of the MEM culture medium, fetal bovine serum, penicillin-streptomycin mixed solution, sodium bicarbonate solution and sodium pyruvate solution is 86:10:1:2:

1.

8. The method for preparing osteosarcoma cell microcapsules for drug evaluation according to claim 1, characterized in that: In step 2, the size of the cell sphere is 100-300 μm.

9. Osteosarcoma cell microcapsules for drug evaluation prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the osteosarcoma cell microcapsule as claimed in claim 9 in the analysis of the efficacy of anti-tumor drugs.

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