Screening method of anti-cancer drug

Abstract

The invention belongs to the technical field of tumor drug screening, and provides an anti-cancer drug screening method which comprises the following steps: S1, preparing tumor cell 3D microspheres with vesicle structures by utilizing a microfluidic technology; s2, putting the tumor cell 3D microspheres and a to-be-screened anti-cancer drug with a certain concentration into a culture medium for culture; s3, carrying out bright field photographing and dead and living cell AO / PI dyeing on the tumor cell 3D microspheres before culture and after culture is finished, detecting the cell activity of the tumor cell 3D microspheres by using a kit, and calculating an IC50 value; and S4, if the IC50 value is less than 50 [mu] M and the cell dissociation is complete, indicating that the to-be-screened anti-cancer drug with a certain concentration has the anti-tumor effect, otherwise, indicating that the to-be-screened anti-cancer drug with a certain concentration does not have the anti-tumor effect. Compared with a traditional 2D cell model, the tumor cell 3D microspheres prepared by the method reserve the mutual structure between cells and the genetic characteristics of tumor cells, can significantly improve the accuracy of drug screening, and have wide application prospects in the research and development process of new drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor drug screening, and specifically relates to a method for screening anticancer drugs. Background Art

[0002] Establishing a preclinical tumor model that can accurately reflect drug response is the key to promoting the development of precision tumor treatment. The 2D cultured tumor cell lines used in traditional research are easy to manage, fast to culture, and low in cost, so a large number of tumor studies use cell line models. However, there are significant differences between the physiological structure of 2D cell lines and human cells. The contact area between cells is small, only a single surface is in contact with the culture medium, the monolayer cell structure is single, and the cell line is highly homogeneous. As a result, the cell line cannot truly and accurately reflect the characteristics of the tumor itself and its response to drugs. These shortcomings limit the application and value of cell lines in tumor drug development research. Summary of the invention

[0003] In order to solve the problem in the prior art that the cell line cannot truly and accurately reflect the characteristics of the tumor itself and the drug response, the present invention provides a method for screening anticancer drugs.

[0004] The anticancer drug screening method comprises the following steps:

[0005] S1: Preparation of tumor cell 3D microspheres with vesicle-like structure using microfluidic technology;

[0006] S2: placing the tumor cell 3D microspheres and a certain concentration of the anticancer drug to be screened into a culture medium for cultivation;

[0007] S3: After the culture is completed, the cell viability of the tumor cell 3D microspheres is detected using a kit and the IC 50 value;

[0008] S4: If IC 50 If the value is less than 50 μM, it means that the anticancer drug to be screened at a certain concentration has an anti-tumor effect. Otherwise, it means that the anticancer drug to be screened at a certain concentration does not have an anti-tumor effect.

[0009] The certain concentration may be any concentration.

[0010] Further, step S1 includes:

[0011] S11: Using microfluidics technology, the cell-matrix gel mixture was sheared into monodisperse droplets, which were then solidified to obtain tumor cell 3D microsphere precursors;

[0012] S12: placing the tumor cell 3D microsphere precursor in a culture medium and culturing it until it has a vesicle-like structure to obtain the tumor cell 3D microsphere.

[0013] Further, step S1 includes:

[0014] S11: Add 3 × 10 4 cancer cells, mixing them evenly to obtain a cell-matrix gel mixture, shearing the cell-matrix gel mixture into monodisperse droplets using microfluidics technology, and solidifying the monodisperse droplets at 37° C. for 15 minutes to obtain tumor cell 3D microsphere precursors after solidification;

[0015] S12: The cell 3D microsphere precursor is inoculated into a DMEM culture medium containing 10% FBS, and cultured in a 37° C., 5% CO 2 incubator for 7-10 days to obtain the tumor cell 3D microsphere.

[0016] Furthermore, the tumor cell 3D microspheres were inoculated in a DMEM culture medium containing 10% FBS and cultured in a light-proof 96-well plate, with 4-5 tumor cell 3D microspheres inoculated in each well.

[0017] Furthermore, in step S1, the size of the vesicle-like structure is between 400-500 μm.

[0018] Furthermore, the drug culture time in step S2 is 6 days, and the drug-containing culture medium is replaced on the 3rd day.

[0019] Furthermore, in step S3, in the process of using the kit to detect the cell viability of the tumor cell 3D microspheres, the 3D microspheres are lysed using a cell lysis solution, wherein the cell microspheres need to be blown 10-15 times with a gun, and whether the cell microsphere structure is completely dissociated is observed under a microscope.

[0020] Furthermore, the certain concentration of anticancer drugs to be screened is selected from one or more of 50nM, 0.2μM, 1μM, 5μM, 25μM, 125μM gefitinib, 60nM, 0.3μM, 1.5μM, 7.5μM, 37.5μM, 187.5μM gemcitabine, 44.8nM, 0.224μM, 1.12μM, 5.6μM, 22.8μM, 140μM oxaliplatin, 44.8nM, 200nM, 1.02μM, 5.1μM, 25.5μM, 127.5μM floxuridine.

[0021] Beneficial effects:

[0022] Compared with traditional 2D cell models, the tumor cell 3D microspheres prepared by this method retain the mutual structure between cells and the genetic characteristics of tumor cells, can significantly improve the accuracy of drug screening, and have broad application prospects in the process of new drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Cell viability detection, bright field and live and dead cell staining of 3D microspheres and 2D cells derived from the lung cancer A549 cell line after treatment with gefitinib;

[0024] Figure 2 Cell viability detection, bright field and live and dead cell staining of 3D microspheres and 2D cells derived from lung cancer A549 cell line after gemcitabine treatment;

[0025] Figure 3 Cell viability detection, bright field and live-dead cell staining of 3D microspheres and 2D cells derived from lung cancer PC-9 cell line after treatment with gefitinib;

[0026] Figure 4 Cell viability detection, bright field and live / dead cell staining of 3D microspheres and 2D cells derived from lung cancer PC-9 cell line after gemcitabine treatment;

[0027] Figure 5 Cell viability detection, bright field and live / dead cell staining of 3D microspheres and 2D cells derived from colon cancer HCT-116 cell line after treatment with oxaliplatin;

[0028] Figure 6 Cell viability detection, bright field and live / dead cell staining of 3D microspheres and 2D cells derived from the breast cancer MBA-MB-231 cell line after treatment with fluorouridine. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0030] Tumor cell 3D microspheres refer to tumor cell microspheres with certain structures and functions formed by encapsulating cancer cell lines with matrix gel. Compared with 2D cells, it can simulate the in vivo tissue structure and function to the greatest extent in terms of structure and function, and has the characteristics of short construction cycle, easy preservation, and retention of cell heterogeneity. Similar to 2D cell lines, tumor cell 3D microspheres can be used for large-scale drug screening. At the same time, compared with in vivo models, the development and maintenance costs of tumor cell 3D microspheres are reduced, the construction cycle is shortened, and experimental data can be obtained quickly. This patent uses microfluidic technology to construct tumor cell 3D microspheres, involving lung cancer cells, colon cancer cells and breast cancer cells, which are used to screen tumor anti-cancer drugs such as gefitinib, gemcitabine, oxaliplatin and floxuridine. Compared with traditional 2D cells, tumor cell 3D microspheres are more sensitive to drugs, and the resulting IC 50 The value is closer to clinical data.

[0031] Example 1: Preparation of 3D microspheres of lung cancer A549 cells

[0032] Step 1) Cell digestion and counting

[0033] A549 cells can be digested when they grow to a confluency of 80%-90%. Remove the original culture medium, add 2mL PBS to wash once, aspirate PBS, add 2mL trypsin to digest at room temperature for 2min. Under the microscope, the cells are round and the adhesion is reduced. Immediately add 2mL of culture medium containing 10% FBS by volume to terminate the digestion. Gently mix the cells with a pipette, centrifuge at 300g, 4℃ for 5min, and discard the supernatant. Add 1mL of complete culture medium to resuspend the cell pellet, take 10μL of cell suspension and add it to 10μL of AOPI. After thorough mixing, inject 20μL of the mixture into the counting plate for cell counting.

[0034] Step 2) Preparation and culture of A549 cell 3D microspheres

[0035] Take a small amount of dry ice in advance and spread it inside the pre-cooled freezer to ensure that the temperature is kept low to prevent the matrix gel from solidifying. Thaw the matrix gel in a 4°C refrigerator or on ice. Draw 3 mL of fluorinated oil into a 10 mL syringe, expel the air, connect a 23G needle and about 25 cm of polytetrafluoroethylene (PTFE) tube, install it in the right hole of the tee, install about 10 m of PTFE coil in the left hole of the tee, and install it in the injection pump outside the refrigerator (flow rate: 110 μL / min), adjust the injection pump so that the fluorinated oil in the 10 mL syringe fills the 25 cm PTFE tube connected to its 23G needle.

[0036] The cell suspension was transferred to a 1.5 mL centrifuge tube according to the required cell amount, centrifuged at 360 g for 3 min at room temperature, and the supernatant was discarded. The required volume of matrix gel was calculated at a concentration of 10,000 cells / μL, and matrix gel was added to the cell pellet. After gently stirring on ice for several times, it was blown to ensure that no bubbles were generated. After mixing until there was no cell block, the matrix gel was injected into a pre-cooled 1 mL syringe containing fluorinated oil, connected to a 23G needle and a 10 cm PTFE tube, and then installed in a syringe pump (flow rate: 15 μL / min) in the refrigerator and installed in the middle hole of the three-way. The syringe pump was adjusted to make the matrix gel appear in the 10 cm PTFE tube until the matrix gel moved to 1-2 cm away from the three-way tube. The syringe pump outside the refrigerator was started first, and then the syringe pump inside the refrigerator was started. The matrix gel was sheared into monodisperse droplets through the three-way tube, and then transferred to the PTFE tube of about 10 m. After the matrix gel of the PTFE tube connected to the 1 mL syringe was completely discharged, the two syringe pumps were stopped. Place the PTFE tube carrying the matrix gel droplets in a 37°C incubator to solidify for 20-30 minutes, use a 10mL syringe to blow the cell microspheres into a 96-well plate containing 10% FBSDMEM culture medium, and use a 1mL syringe to gently suck out the fluorine oil on the surface of the culture dish. The well plate is placed in a 37°C, 5.0% CO2 incubator for culture.

[0037] Example 2: A549 cell 3D microsphere drug screening and cell viability detection

[0038] After the tumor cell 3D microspheres grow out of the structure, they are spread in a light-proof 96-well plate, with 4-5 microspheres per well. Each cell type needs to be inoculated in 28 wells. 100 μL of culture medium containing different concentrations of gefitinib or gemcitabine is added to each well, and the culture is terminated on the 6th day. After drug treatment, the 3D microspheres are photographed in the bright field and then stained with AO / PI dye: the original culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the microspheres are incubated in a 37°C incubator for 20-30 minutes, and photographed under a fluorescence microscope.

[0039] The CellTiter-Glo (CTG) Luminescent Cell Viability Assay Kit was used to detect the viability of tumor cell 3D microspheres. Thaw the CTG buffer at room temperature in advance, and balance the temperature of the CTG substrate to room temperature; mix the CTG buffer and CTG substrate in a light-proof bottle, and gently vortex to mix to form the CTG reagent; the light-proof 96-well plate was equilibrated at room temperature for 30 minutes; 100 μL of CTG reagent was added to each well, and the plate was shaken on a shaker for 2 minutes to lyse the cells; incubate at room temperature for 10 minutes, and after the light signal is stabilized, the LUM light signal intensity is recorded by the microplate reader to make IC 50 curve.

[0040] Comparative Example 1: Drug screening and cell viability detection in 2D cell model derived from A549

[0041] When the cells grow to a confluence of 80%-90%, trypsin is used to digest the cells, and the cells are counted. 8,000 cells are inoculated in each well of a 96-well plate, and 29 wells are required for each type of cell. After 24 hours of culture, the density is about 70%, and the culture medium is replaced, and 100 μL of culture medium containing different concentrations of gefitinib or gemcitabine is added to each well. After 24 hours, the cell death is observed and bright field photos are taken, and then AO / PI dye is used for staining: the old culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the cells are incubated in a 37°C incubator for 20-30 minutes, and photos are taken under a fluorescence microscope.

[0042] CCK-8 assay was performed 24 hours after drug treatment. 10 μL of CCK-8 solution was added to each well (be careful not to generate bubbles), and the culture plate was placed in an incubator for 1-4 hours; the absorbance at 450 nm was measured with an ELISA reader (if the absorbance value is too large, the absorbance at 500 nm can be used), and the cell IC was calculated. 50 value.

[0043] The bright field results and live and dead cell staining experiments showed that the 3D microspheres prepared from the lung cancer cell line A549 in Example 2 had obvious cell apoptosis after adding 5 μM gefitinib ( Figure 1 A), while the A549 cell line in comparative example 1 showed apoptosis only after the addition of 125 μM gefitinib ( Figure 1 B). In Example 2, the A549 cell 3D microspheres began to undergo apoptosis after the addition of 7.5 μM gemcitabine ( Figure 2 A), while the A549 cells in Comparative Example 1 were tolerant to gemcitabine and the cell morphology did not show obvious changes ( Figure 2 B).

[0044] The results of cell viability test showed that after adding different concentrations of the drug gefitinib in Example 2, the lung cancer cell line A549 cell 3D microsphere IC 50 The value was 4.65 μM, and the IC value of A549 cells in Comparative Example 1 was 50 The value is 36.23μM ( Figure 1 C) After adding different concentrations of gemcitabine in Example 2, the lung cancer cell line A549 cell 3D microsphere IC 50 The value is 9.19 μM, and the IC of A549 cells to gemcitabine in Comparative Example 1 is 50 The value is 3101μM ( Figure 2 C).

[0045] Example 3 Preparation of 3D microspheres of lung cancer cell line PC-9 cells

[0046] Step 1) Cell digestion and counting

[0047] PC-9 cells can be digested when they grow to a confluence of 80%-90%. Remove the original culture medium, add 2mL PBS to wash once, aspirate PBS, add 2mL trypsin to digest at room temperature for 2min. Under the microscope, the cells are round and the adhesion is reduced. Immediately add 2mL of culture medium containing 10% FBS by volume to terminate the digestion. Gently mix the cells with a pipette, centrifuge at 300g, 4℃ for 5min, and discard the supernatant. Add 1mL of complete culture medium to resuspend the cell pellet, take 10μL of cell suspension and add it to 10μL of AOPI. After thorough mixing, inject 20μL of the mixture into the counting plate for cell counting.

[0048] Step 2) Preparation and culture of PC-9 cell 3D microspheres

[0049] Take a small amount of dry ice in advance and spread it inside the pre-cooled freezer to ensure that the temperature is kept low to prevent the matrix gel from solidifying. Thaw the matrix gel in a 4°C refrigerator or on ice. Draw 3 mL of fluorinated oil into a 10 mL syringe, expel the air, connect a 23G needle and about 25 cm of polytetrafluoroethylene (PTFE) tube, install it in the right hole of the tee, install about 10 m of PTFE coil in the left hole of the tee, and install it in the injection pump outside the refrigerator (flow rate: 110 μL / min), adjust the injection pump so that the fluorinated oil in the 10 mL syringe fills the 25 cm PTFE tube connected to its 23G needle.

[0050] The cell suspension was transferred to a 1.5 mL centrifuge tube according to the required cell amount, centrifuged at 360 g for 3 min at room temperature, and the supernatant was discarded. The required volume of matrix gel was calculated at a concentration of 10,000 cells / μL, and matrix gel was added to the cell pellet. After gently stirring on ice for several times, it was blown to ensure that no bubbles were generated. After mixing until there was no cell block, the matrix gel was injected into a pre-cooled 1 mL syringe containing fluorinated oil, connected to a 23G needle and a 10 cm PTFE tube, and then installed in a syringe pump (flow rate: 15 μL / min) in the refrigerator and installed in the middle hole of the three-way. The syringe pump was adjusted to make the matrix gel appear in the 10 cm PTFE tube until the matrix gel moved to 1-2 cm away from the three-way tube. The syringe pump outside the refrigerator was started first, and then the syringe pump inside the refrigerator was started. The matrix gel was sheared into monodisperse droplets through the three-way tube, and then transferred to the PTFE tube of about 10 m. After the matrix gel of the PTFE tube connected to the 1 mL syringe was completely discharged, the two syringe pumps were stopped. Place the PTFE tube carrying the matrix gel droplets in a 37°C incubator to solidify for 20-30 minutes, use a 10mL syringe to blow the cell microspheres into a 96-well plate containing 10% FBSDMEM culture medium, and use a 1mL syringe to gently suck out the fluorine oil on the surface of the culture dish. The well plate is placed in a 37°C, 5.0% CO2 incubator for culture.

[0051] Example 4: PC-9 cell 3D microsphere drug screening and cell viability detection

[0052] After the tumor cell 3D microspheres grow out of the structure, they are spread in a light-proof 96-well plate, with 4-5 microspheres per well. Each cell type needs to be inoculated in 28 wells. 100 μL of culture medium containing different concentrations of gefitinib or gemcitabine is added to each well, and the culture is terminated on the 6th day. After drug treatment, the 3D microspheres are photographed in the bright field and then stained with AO / PI dye: the original culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the microspheres are incubated in a 37°C incubator for 20-30 minutes, and photographed under a fluorescence microscope.

[0053] The CellTiter-Glo (CTG) Luminescent Cell Viability Assay Kit was used to detect the viability of tumor cell 3D microspheres. Thaw the CTG buffer at room temperature in advance, and balance the temperature of the CTG substrate to room temperature; mix the CTG buffer and CTG substrate in a light-proof bottle, and gently vortex to mix to form the CTG reagent; the light-proof 96-well plate was equilibrated at room temperature for 30 minutes; 100 μL of CTG reagent was added to each well, and the plate was shaken on a shaker for 2 minutes to lyse the cells; incubate at room temperature for 10 minutes, and after the light signal is stabilized, the LUM light signal intensity is recorded by the microplate reader to make IC 50 curve.

[0054] Comparative Example 2: Drug screening and cell viability detection in 2D cell model derived from PC-9

[0055] When the cells grow to a confluence of 80%-90%, trypsin is used to digest the cells, and the cells are counted. 8,000 cells are inoculated in each well of a 96-well plate, and 29 wells are required for each type of cell. After 24 hours of culture, the density is about 70%, and the culture medium is replaced, and 100 μL of culture medium containing different concentrations of gefitinib or gemcitabine is added to each well. After 24 hours, the cell death is observed and bright field photos are taken, and then AO / PI dye is used for staining: the old culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the cells are incubated in a 37°C incubator for 20-30 minutes, and photos are taken under a fluorescence microscope.

[0056] CCK-8 assay was performed 24 hours after drug treatment. 10 μL of CCK-8 solution was added to each well (be careful not to generate bubbles), and the culture plate was placed in an incubator for 1-4 hours; the absorbance at 450 nm was measured with an ELISA reader (if the absorbance value is too large, the absorbance at 500 nm can be used), and the cell IC was calculated. 50 value.

[0057] The bright field results and live and dead cell staining experiments showed that the concentration of 5 μM ( Figure 3 A), while the concentration of gefitinib in PC-9 cells in comparative example 2 that caused apoptosis was 25 μM ( Figure 3 B). The sensitive concentration of PC-9 cell 3D microspheres to gemcitabine in Example 4 is 37.5 μM ( Figure 4 A), while the PC-9 cells in Comparative Example 2 were sensitive to gemcitabine at a concentration of 187.5 μM ( Figure 4 B).

[0058] The results of cell viability test showed that after adding different concentrations of gefitinib in Example 4, the IC50 value of PC-9 cell 3D microspheres for gefitinib was 2.296 μM, while the IC50 value of PC-9 cells in Comparative Example 2 was 50 The value is 52.28μM ( Figure 3 C) After adding different concentrations of gemcitabine in Example 4, the lung cancer cell line PC-9 cell 3D microsphere IC 50 The value was 36.26 μM, and the IC value of PC-9 cells in Comparative Example 2 was 50 The value is 451.9μM ( Figure 4 C).

[0059] Example 5 Preparation of 3D Microspheres of Colon Cancer Cell Line HCT-116 Cells

[0060] Step 1) Cell digestion and counting

[0061] HCT-116 cells can be digested when they grow to a confluence of 80%-90%. Remove the original culture medium, add 2mL PBS to wash once, aspirate PBS, add 2mL trypsin to digest at room temperature for 2min. Under the microscope, the cells are round and the adhesion is reduced. Immediately add 2mL of culture medium containing 10% FBS by volume to terminate the digestion. Gently mix the cells with a pipette, centrifuge at 300g, 4℃ for 5min, and discard the supernatant. Add 1mL of complete culture medium to resuspend the cell pellet, take 10μL of cell suspension and add it to 10μL of AOPI. After thorough mixing, inject 20μL of the mixture into the counting plate for cell counting.

[0062] Step 2) Preparation and culture of HCT-116 cell 3D microspheres

[0063] Take a small amount of dry ice in advance and spread it inside the pre-cooled freezer to ensure that the temperature is kept low to prevent the matrix gel from solidifying. Thaw the matrix gel in a 4°C refrigerator or on ice. Draw 3 mL of fluorinated oil into a 10 mL syringe, expel the air, connect a 23G needle and about 25 cm of polytetrafluoroethylene (PTFE) tube, install it in the right hole of the tee, install about 10 m of PTFE coil in the left hole of the tee, and install it in the injection pump outside the refrigerator (flow rate: 110 μL / min), adjust the injection pump so that the fluorinated oil in the 10 mL syringe fills the 25 cm PTFE tube connected to its 23G needle.

[0064] The cell suspension was transferred to a 1.5 mL centrifuge tube according to the required cell amount, centrifuged at 360 g for 3 min at room temperature, and the supernatant was discarded. The required volume of matrix gel was calculated at a concentration of 10,000 cells / μL, and matrix gel was added to the cell pellet. After gently stirring on ice for several times, it was blown to ensure that no bubbles were generated. After mixing until there was no cell block, the matrix gel was injected into a pre-cooled 1 mL syringe containing fluorinated oil, connected to a 23G needle and a 10 cm PTFE tube, and then installed in a syringe pump (flow rate: 15 μL / min) in the refrigerator and installed in the middle hole of the three-way. The syringe pump was adjusted to make the matrix gel appear in the 10 cm PTFE tube until the matrix gel moved to 1-2 cm away from the three-way tube. The syringe pump outside the refrigerator was started first, and then the syringe pump inside the refrigerator was started. The matrix gel was sheared into monodisperse droplets through the three-way tube, and then transferred to the PTFE tube of about 10 m. After the matrix gel of the PTFE tube connected to the 1 mL syringe was completely discharged, the two syringe pumps were stopped. Place the PTFE tube carrying the matrix gel droplets in a 37°C incubator to solidify for 20-30 minutes, use a 10mL syringe to blow the cell microspheres into a 96-well plate containing 10% FBSDMEM culture medium, and use a 1mL syringe to gently suck out the fluorine oil on the surface of the culture dish. The well plate is placed in a 37°C, 5.0% CO2 incubator for culture.

[0065] Example 6: HCT-116 cell 3D microsphere drug screening and cell viability detection

[0066] After the tumor cell 3D microspheres grow out of the structure, they are spread in a light-proof 96-well plate, with 4-5 microspheres per well. Each cell type needs to be inoculated in 28 wells, and 100 μL of culture medium containing different concentrations of oxaliplatin is added to each well. The culture is terminated on the 6th day. After drug treatment, the 3D microspheres are photographed in the bright field and then stained with AO / PI dye: the original culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the microspheres are incubated in a 37°C incubator for 20-30 minutes, and photographed under a fluorescence microscope.

[0067] The CellTiter-Glo (CTG) Luminescent Cell Viability Assay Kit was used to detect the viability of tumor cell 3D microspheres. Thaw the CTG buffer at room temperature in advance, and balance the temperature of the CTG substrate to room temperature; mix the CTG buffer and CTG substrate in a light-proof bottle, and gently vortex to mix to form the CTG reagent; the light-proof 96-well plate was equilibrated at room temperature for 30 minutes; 100 μL of CTG reagent was added to each well, and the plate was shaken on a shaker for 2 minutes to lyse the cells; incubate at room temperature for 10 minutes, and after the light signal is stabilized, the LUM light signal intensity is recorded by the microplate reader to make IC 50 curve.

[0068] Comparative Example 3: Drug screening and cell viability detection in 2D cell models derived from HCT-116

[0069] When the cells grow to a confluence of 80%-90%, trypsin is used to digest the cells, and the cells are counted. 8,000 cells are inoculated in each well of a 96-well plate, and 29 wells are required for each type of cell. After 24 hours of culture, the density is about 70%, and the culture medium is replaced, and 100 μL of culture medium containing different concentrations of oxaliplatin is added to each well. After 24 hours, the cell death is observed and bright field photos are taken, and then AO / PI dye is used for staining: the old culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the cells are incubated in a 37°C incubator for 20-30 minutes, and photos are taken under a fluorescence microscope.

[0070] CCK-8 assay was performed 24 hours after drug treatment. 10 μL of CCK-8 solution was added to each well (be careful not to generate bubbles), and the culture plate was placed in an incubator for 1-4 hours; the absorbance at 450 nm was measured with an ELISA reader (if the absorbance value is too large, the absorbance at 500 nm can be used), and the cell IC was calculated. 50 value.

[0071] The bright field results and live and dead cell staining experiments showed that the 3D microspheres prepared from the colon cancer cell line HCT-116 cells in Example 6 were sensitive to oxaliplatin at a concentration of 1.12 μM ( Figure 5 A), while the sensitive concentration of HCT-116 cells to oxaliplatin in Comparative Example 3 was 22.8 μM ( Figure 5 B).

[0072] The results of cell viability detection showed that after adding different concentrations of the drug oxaliplatin in Example 6, the IC50 value of the 3D cell microspheres prepared by HCT-116 for the drug oxaliplatin was 1.91 μM, and the IC50 value of the HCT-116 cells for oxaliplatin in Comparative Example 3 was 12.17 μM ( Figure 5 C).

[0073] Example 7 Preparation of 3D microspheres of breast cancer cell line MBA-MB-231 cells

[0074] Step 1) Cell digestion and counting

[0075] When the MBA-MB-231 cells grow to a confluence of 80%-90%, digestion can be performed. Remove the original culture medium, add 2mL PBS to wash once, aspirate PBS, and add 2mL trypsin to digest at room temperature for 3min. Under the microscope, the cells are round and the adhesion is reduced. Immediately add 2mL of culture medium containing 10% FBS by volume to terminate digestion, gently mix the cells with a pipette, centrifuge at 300g, 4℃ for 5min, and discard the supernatant. Add 1mL of complete culture medium to resuspend the cell pellet, take 10μL of cell suspension and add it to 10μL of AOPI. After thorough mixing, inject 20μL of the mixture into the counting plate for cell counting.

[0076] Step 2) Preparation and culture of MBA-MB-231 cell 3D microspheres

[0077] Take a small amount of dry ice in advance and spread it inside the pre-cooled freezer to ensure that the temperature is kept low to prevent the matrix gel from solidifying. Thaw the matrix gel in a 4°C refrigerator or on ice. Draw 3 mL of fluorinated oil into a 10 mL syringe, expel the air, connect a 23G needle and about 25 cm of polytetrafluoroethylene (PTFE) tube, install it in the right hole of the tee, install about 10 m of PTFE coil in the left hole of the tee, and install it in the injection pump outside the refrigerator (flow rate: 110 μL / min), adjust the injection pump so that the fluorinated oil in the 10 mL syringe fills the 25 cm PTFE tube connected to its 23G needle.

[0078] The cell suspension was transferred to a 1.5 mL centrifuge tube according to the required cell amount, centrifuged at 360 g for 3 min at room temperature, and the supernatant was discarded. The required volume of matrix gel was calculated at a concentration of 10,000 cells / μL, and matrix gel was added to the cell pellet. After gently stirring on ice for several times, it was blown to ensure that no bubbles were generated. After mixing until there was no cell block, the matrix gel was injected into a pre-cooled 1 mL syringe containing fluorinated oil, connected to a 23G needle and a 10 cm PTFE tube, and then installed in a syringe pump (flow rate: 15 μL / min) in the refrigerator and installed in the middle hole of the three-way. The syringe pump was adjusted to make the matrix gel appear in the 10 cm PTFE tube until the matrix gel moved to 1-2 cm away from the three-way tube. The syringe pump outside the refrigerator was started first, and then the syringe pump inside the refrigerator was started. The matrix gel was sheared into monodisperse droplets through the three-way tube, and then transferred to the PTFE tube of about 10 m. After the matrix gel of the PTFE tube connected to the 1 mL syringe was completely discharged, the two syringe pumps were stopped. Place the PTFE tube carrying the matrix gel droplets in a 37°C incubator to solidify for 20-30 minutes, use a 10mL syringe to blow the cell microspheres into a 96-well plate containing 10% FBSDMEM culture medium, and use a 1mL syringe to gently suck out the fluorine oil on the surface of the culture dish. The well plate is placed in a 37°C, 5.0% CO2 incubator for culture.

[0079] Example 8 MBA-MB-231 cell 3D microsphere drug screening and cell viability detection

[0080] After the tumor cell 3D microspheres grow out of the structure, they are spread in a light-proof 96-well plate, with 4-5 microspheres per well. Each cell type needs to be inoculated in 28 wells, and 100 μL of culture medium containing different concentrations of the drug fluorouridine is added to each well. The culture is terminated on the 6th day. After drug treatment, the 3D microspheres are photographed in the bright field and then stained with AO / PI dye: the original culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the microspheres are incubated in a 37°C incubator for 20-30 minutes, and photographed under a fluorescence microscope.

[0081] The CellTiter-Glo (CTG) Luminescent Cell Viability Assay Kit was used to detect the viability of tumor cell 3D microspheres. Thaw the CTG buffer at room temperature in advance, and balance the temperature of the CTG substrate to room temperature; mix the CTG buffer and CTG substrate in a light-proof bottle, and gently vortex to mix to form the CTG reagent; the light-proof 96-well plate was equilibrated at room temperature for 30 minutes; 100 μL of CTG reagent was added to each well, and the plate was shaken on a shaker for 2 minutes to lyse the cells; incubate at room temperature for 10 minutes, and after the light signal is stabilized, the LUM light signal intensity is recorded by the microplate reader to make IC 50 curve.

[0082] Comparative Example 4: Drug screening and cell viability detection in 2D cell model derived from MBA-MB-231

[0083] When the cells grow to a confluency of 80%-90%, trypsin is used to digest the cells and count the cells. 8,000 cells are inoculated in each well of a 96-well plate, and 29 wells are required for each type of cell. After 24 hours of culture, the density is about 70%, the culture medium is replaced, and 100 μL of culture medium containing different concentrations of the drug fluorouridine is added to each well. After 24 hours, the cell death is observed and bright field photos are taken, and then AO / PI dye is used for staining: the old culture medium is removed, 100 μL of the mixed AO / PI dye is added, and the cells are incubated in a 37°C incubator for 20-30 minutes, and photos are taken under a fluorescence microscope.

[0084] CCK-8 assay was performed 24 hours after drug treatment. 10 μL of CCK-8 solution was added to each well (be careful not to generate bubbles), and the culture plate was placed in an incubator for 1-4 hours; the absorbance at 450 nm was measured with an ELISA reader (if the absorbance value is too large, the absorbance at 500 nm can be used), and the cell IC was calculated. 50 value.

[0085] The bright field results and live and dead cell staining experiments showed that the breast cancer cell line MBA-MB-231 cell 3D microspheres in Example 8 began to undergo apoptosis after being treated with 5.1 μM of the drug floxuridine ( Figure 6 A), while the sensitive concentration of MBA-MB-231 cells to floxuridine in Comparative Example 4 was 25.5 μM ( Figure 6 B).

[0086] The results of cell viability test showed that after adding different concentrations of the drug floxuridine in Example 8, the IC values ​​of the breast cancer cell line MBA-MB-231 cell 3D microspheres to the drug floxuridine were 50 The value is 6.09 μM, and the IC value of MBA-MB-231 to fluorouridine in Comparative Example 4 is 50 The value is 22.46μM ( Figure 6 C).

[0087] The above results show that the sensitivity of tumor cell lines and corresponding tumor cell 3D microspheres to the same drug is significantly different, and the effective concentration that induces a decrease in cell viability in tumor cell 3D microspheres is usually lower. Tumor cell 3D microspheres are more sensitive to drugs, and their IC 50 The value is more informative.

Claims

1. A method for screening anticancer drugs, characterized in that: The following steps are involved: S1: Preparation of tumor cell 3D microspheres with vesicle-like structure using microfluidic technology; S2: placing the tumor cell 3D microspheres and a certain concentration of the anticancer drug to be screened in a culture medium for cultivation; S3: After the culture is completed, the cell viability of the tumor cell 3D microspheres is detected using a kit and the IC 50 value; S4: If IC 50 If the value is less than 50 μM, it means that the anticancer drug to be screened at a certain concentration has an anti-tumor effect. Otherwise, it means that the anticancer drug to be screened at a certain concentration does not have an anti-tumor effect.

2. The method for screening anticancer drugs according to claim 1, characterized in that: Step S1 includes: S11: Using microfluidics technology, the cell-matrix gel mixture was sheared into monodisperse droplets, which were then solidified to obtain tumor cell 3D microsphere precursors; S12: placing the tumor cell 3D microsphere precursor in a culture medium and culturing it until it has a vesicle-like structure to obtain the tumor cell 3D microsphere.

3. The method for screening anticancer drugs according to claim 2, characterized in that: Step S1 includes: S11: Add 3 × 10 4 cancer cells, mixing them evenly to obtain a cell-matrix gel mixture, shearing the cell-matrix gel mixture into monodisperse droplets using microfluidics technology, and solidifying the monodisperse droplets at 37° C. for 15 minutes to obtain tumor cell 3D microsphere precursors after solidification; S12: The cell 3D microsphere precursor is inoculated into a DMEM culture medium containing 10% FBS, and cultured in a 37° C., 5% CO 2 incubator for 7-10 days to obtain the tumor cell 3D microsphere.

4. The method for screening anticancer drugs according to claim 3, characterized in that: The tumor cell 3D microspheres were inoculated in a DMEM culture medium containing 10% FBS and cultured in a light-proof 96-well plate, with 4-5 tumor cell 3D microspheres inoculated in each well.

5. The method for screening anticancer drugs according to claim 1, characterized in that: In step S1, the size of the vesicle-like structure is between 400-500 μM.

6. The method for screening anticancer drugs according to claim 1, characterized in that: The drug culture time in step S2 is 6 days, and the drug-containing culture medium is replaced on the 3rd day.

7. The method for screening anticancer drugs according to claim 1, characterized in that: In step S3, during the process of using the kit to detect the cell viability of the tumor cell 3D microspheres, the 3D microspheres are lysed using a cell lysis solution, wherein the cell microspheres need to be blown 10-15 times with a gun, and whether the cell microsphere structure is completely dissociated is observed under a microscope.

8. The method for screening anticancer drugs according to claim 1, characterized in that: The certain concentration of anticancer drugs to be screened is selected from one or more of 50nM, 0.2μM, 1μM, 5μM, 25μM, 125μM gefitinib, 60nM, 0.3μM, 1.5μM, 7.5μM, 37.5μM, 187.5μM gemcitabine, 44.8nM, 0.224μM, 1.12μM, 5.6μM, 22.8μM, 140μM oxaliplatin, 44.8nM, 200nM, 1.02μM, 5.1μM, 25.5μM, 127.5μM floxuridine.

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