CN114457019A - Lung cancer organoid model constructed based on porous microspheres and culture method and application thereof - Google Patents
Lung cancer organoid model constructed based on porous microspheres and culture method and application thereof Download PDFInfo
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- CN114457019A CN114457019A CN202210144648.0A CN202210144648A CN114457019A CN 114457019 A CN114457019 A CN 114457019A CN 202210144648 A CN202210144648 A CN 202210144648A CN 114457019 A CN114457019 A CN 114457019A
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- polylactic acid
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- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 57
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- 210000002220 organoid Anatomy 0.000 title claims abstract description 38
- 238000012136 culture method Methods 0.000 title claims abstract description 19
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Abstract
The invention discloses a lung cancer organoid model constructed based on porous microspheres and a culture method and application thereof, wherein hydrophilic gel is used as a load body to load growth promoting factors, and the hydrophilic gel is used as a water phase to be mixed and emulsified with polylactic acid oil phase to form water-in-oil emulsion so as to obtain the polylactic acid microspheres coated with gel particles, so that the growth promoting factors are coated inside the porous polylactic acid microspheres, and the proliferation and differentiation of internal tumor cells are facilitated; meanwhile, ammonium bicarbonate is used as a pore-forming agent, the emulsification process of the water phase and the oil phase is controlled, and the porous polylactic acid microspheres with uniform pores inside and on the surface are prepared, so that the tumor cells can be favorably adhered and grown on the inner pores of the porous polylactic acid microsphere support, and the requirements on the cell density and the number of the lung cancer organoid tissues in vitro are met.
Description
Technical Field
The invention relates to the technical field of tumor immunology, in particular to a lung cancer organoid model constructed based on porous microspheres and a culture method and application thereof.
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.
Porous microspheres show great potential in the preparation of cell-loaded tissue models in vitro due to their high porosity and good biocompatibility. 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.
Disclosure of Invention
In view of the above problems in the background art, an object of the present invention is to provide a culture method for constructing a lung cancer organoid model based on porous microspheres, wherein a hydrophilic gel is used as a loading body to load a growth promoting factor, and the growth promoting factor is used as a water phase to be mixed and emulsified with a polylactic acid oil phase to form a water-in-oil emulsion, so as to obtain a polylactic acid microsphere coated with gel particles, such that the growth promoting factor is coated inside the porous polylactic acid microsphere, thereby facilitating proliferation and differentiation of internal tumor cells.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a culture method for constructing a lung cancer organoid model based on porous microspheres specifically comprises the following steps:
(1) preparing gel particles loaded with growth promoting factors:
adding the growth promoting factor into the hydrophilic gel material, centrifuging, cleaning, and freeze-drying to obtain gel particles loaded with the growth promoting factor;
(2) preparing polylactic acid W/O emulsion:
dissolving polylactic acid and levorotatory polylactic acid in an organic solvent to be fully dissolved to be used as an oil phase; preparing an ammonium bicarbonate aqueous solution with the concentration of 2.0-6.0%, adding the gel particles loaded with the growth promoting factors obtained in the step (1), and uniformly mixing to obtain a water phase;
adding the water phase into the oil phase, mixing the two phases, and performing ultrasonic emulsification for 5-30 min by using a cell disruption instrument with the power of 200W in a mode of ultrasonic for 1s at intervals of 1s to form a polylactic acid W/O emulsion;
(3) preparing porous polylactic acid microspheres coated with growth promoting factors:
stirring the polylactic acid W/O emulsion prepared in the step (2) at 600-1000 rpm, volatilizing an organic solvent in the polylactic acid W/O emulsion in a low-temperature water bath to solidify polylactic acid to separate out a microsphere material, centrifugally collecting microspheres at 3000-6000 rpm, cleaning the microspheres with deionized water, and freeze-drying the microspheres at-45 to-55 ℃ for 12-24 hours to obtain porous polylactic acid microspheres coated with growth promoting factors;
(4) culturing a lung cancer organoid model:
dissociating the lung cancer solid tumor tissue by using the sample dissociation liquid to obtain primary lung cancer solid tumor cells; culturing the separated tumor cells by using a special culture medium to obtain a lung cancer tumor cell suspension; and (4) adding the porous polylactic acid microspheres coated with the growth promoting factors obtained in the step (3) to perform three-dimensional culture to obtain the lung cancer organoid model.
Further, the growth promoting factor in step (1) is selected from Bovine Serum Albumin (BSA); BMP 4; BMP 2; b27; ITS-X; y-27632; EGF; one of Human FGF 4.
Further, the hydrophilic gel material is selected from one of gelatin, chitosan or sodium alginate.
Further, in the step (1), the concentration of the polylactic acid and the L-polylactic acid in the oil phase in the organic solvent is 10-30%, and the organic solvent is dichloromethane, ethyl acetate or chloroform.
Further, the volume ratio of the water phase to the oil phase in the step (1) is 1 (10-40).
Further, the average diameter of the porous polylactic acid microspheres coated with the growth promoting factor is 2 +/-1 μm.
Further, the special culture medium comprises 70-90% of basal culture medium and 10-30% of fetal bovine serum.
Further, the basal medium is selected from one of RPMI-1640 medium, DMEM medium, F-12 medium or DMEM/F-12 medium.
The second purpose of the invention is to provide the lung cancer organoid model constructed by the culture method for constructing the lung cancer organoid model based on the porous microspheres.
The third purpose of the invention is to provide the application of the lung cancer organoid model in the development of lung cancer tumor vaccines and medicaments.
Compared with the prior art, the invention has the following advantages:
firstly, hydrophilic gel is used as a loading body to load a growth promoting factor, and the growth promoting factor is used as a water phase to be mixed and emulsified with polylactic acid oil phase to form water-in-oil emulsion so as to obtain the polylactic acid microspheres coated with gel particles, so that the growth promoting factor is coated inside the porous polylactic acid microspheres, and the proliferation and differentiation of internal tumor cells are facilitated; meanwhile, ammonium bicarbonate is used as a pore-forming agent, the emulsification process of the water phase and the oil phase is controlled, and the porous polylactic acid microspheres with uniform pores inside and on the surface are prepared, so that the tumor cells can be favorably adhered and grown on the inner pores of the porous polylactic acid microsphere support, and the requirements on the cell density and the number of the lung cancer organoid tissues in vitro are met.
Secondly, the hydrophilic gel is used as a loading body for promoting growth factors, so that the biocompatibility of the hydrophilic gel and the polylactic acid microsphere scaffold is improved, meanwhile, the growth factor components in the hydrophilic gel can be regulated and controlled, and the factors for promoting the growth of lung cancer tumor cells are added in a targeted manner, so that the culture of in vitro lung cancer organoids is facilitated, the preparation process is simple, the reaction conditions are mature, and the operability is high.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
Example 1
A culture method for constructing a lung cancer organoid model based on porous microspheres specifically comprises the following steps:
(1) preparing Y-27632 gelatin-loaded microspheres:
adding 1mL of 0.01% (W/V) Y-27632 solution into 20mg of gelatin microspheres, incubating at 4 ℃ for 6h, centrifuging to remove supernatant, and freeze-drying to obtain Y-27632-loaded gelatin microspheres;
(2) preparing polylactic acid W/O emulsion:
dissolving 11mg of polylactic acid and 3mg of levorotatory polylactic acid in dichloromethane, adjusting the concentration to be 20% and fully dissolving the polylactic acid and the levorotatory polylactic acid to be used as an oil phase; preparing 5mL of 2.0% ammonium bicarbonate aqueous solution, adding the Y-27632-loaded gelatin microspheres obtained in the step (1), and uniformly mixing to obtain a water phase;
adding the water phase into the oil phase, wherein the volume ratio of the water phase to the oil phase is 1: 20; mixing the two materials, and performing ultrasonic emulsification for 6min by a cell crusher with the power of 200W in a mode of ultrasonic for 1s at an interval of 1s to form polylactic acid W/O emulsion;
(3) preparing porous polylactic acid microspheres coated with Y-27632:
stirring the polylactic acid W/O emulsion prepared in the step (2) at 8000 revolutions per minute, volatilizing dichloromethane in the polylactic acid W/O emulsion in a low-temperature water bath at 45 ℃ to solidify polylactic acid to obtain a microsphere material, centrifugally collecting microspheres at 5000 revolutions per minute, cleaning the microspheres with deionized water, and freeze-drying the microspheres at-55 ℃ for 24 hours to obtain porous polylactic acid microspheres coated with Y-27632;
(4) culturing a lung cancer organoid model:
dissociating the lung cancer solid tumor tissue by using the sample dissociation liquid to obtain primary lung cancer solid tumor cells; culturing the separated tumor cells by using an RPMI-1640 culture medium containing 10% fetal bovine serum to obtain a lung cancer tumor cell suspension;
and (4) adding the porous polylactic acid microspheres coated with Y-27632 obtained in the step (3), and performing three-dimensional culture to obtain a lung cancer organoid model.
Example 2
A culture method for constructing a lung cancer organoid model based on porous microspheres specifically comprises the following steps:
(1) preparing the BMP-2 loaded sodium alginate microspheres:
weighing 30mg of sodium alginate, adding the sodium alginate into 1mL of water for full dissolution, adding 0.5mL of 0.1% (W/V) BMP-2 solution, uniformly mixing, dripping the mixture into 10mL of 4% (W/V) calcium chloride solution, stirring the mixture for 1h at 1000 rpm, finally centrifuging the mixture at 3000 rpm to collect microspheres, fully cleaning the microspheres with deionized water, and freeze-drying the microspheres;
(2) preparing polylactic acid W/O emulsion:
dissolving 11mg of polylactic acid and 3mg of levorotatory polylactic acid in dichloromethane, adjusting the concentration to be 20% and fully dissolving the polylactic acid and the levorotatory polylactic acid to be used as an oil phase; preparing 5mL of 2.0% ammonium bicarbonate aqueous solution, adding the BMP-2 loaded sodium alginate microspheres obtained in the step (1), and uniformly mixing to obtain a water phase;
adding the water phase into the oil phase, wherein the volume ratio of the water phase to the oil phase is 1: 20; mixing the two materials, and performing ultrasonic emulsification for 6min by a cell disruptor with 200W power at an interval of 1s for 1s to form polylactic acid W/O emulsion;
(3) preparing porous polylactic acid microspheres coated with BMP-2:
stirring the polylactic acid W/O emulsion prepared in the step (2) at 8000 revolutions per minute, volatilizing dichloromethane in the polylactic acid W/O emulsion in a low-temperature water bath at 45 ℃ to solidify polylactic acid to obtain a microsphere material, centrifugally collecting microspheres at 5000 revolutions per minute, cleaning the microspheres with deionized water, and freeze-drying the microspheres at-55 ℃ for 24 hours to obtain porous polylactic acid microspheres coated with BMP-2;
(4) culturing a lung cancer organoid model:
dissociating the lung cancer solid tumor tissue by using the sample dissociation liquid to obtain primary lung cancer solid tumor cells; culturing the separated tumor cells by using an RPMI-1640 culture medium containing 10% fetal calf serum to obtain a lung cancer tumor cell suspension;
and (4) adding the porous polylactic acid microspheres coated with BMP-2 obtained in the step (3), and performing three-dimensional culture to obtain the lung cancer organoid model.
Example 3
A culture method for constructing a lung cancer organoid model based on porous microspheres specifically comprises the following steps:
(1) preparing BSA loaded chitosan microspheres:
weighing 40mg of chitosan, adding the chitosan into 1mL of 2% (V/V) acetic acid aqueous solution, dissolving the chitosan, adding 0.5mL of 0.5% (W/V) bovine serum albumin aqueous solution, uniformly mixing, dripping the mixture into 30mL of n-octanol containing 4% span 80, stirring at 1200 rpm for 30 minutes, dripping 4mL of 5% (W/V) sodium tripolyphosphate to perform crosslinking reaction, continuously stirring for 1 hour, centrifugally collecting at 3000 rpm, cleaning, and freeze-drying to obtain the BSA-loaded chitosan microspheres.
(2) Preparing polylactic acid W/O emulsion:
dissolving 11mg of polylactic acid and 3mg of levorotatory polylactic acid in dichloromethane, adjusting the concentration to be 20% and fully dissolving the polylactic acid and the levorotatory polylactic acid to be used as an oil phase; preparing 5mL of ammonium bicarbonate aqueous solution with the concentration of 2.0%, adding the BSA-loaded chitosan microspheres obtained in the step (1), and uniformly mixing to obtain a water phase;
adding the water phase into the oil phase, wherein the volume ratio of the water phase to the oil phase is 1: 20; mixing the two materials, and performing ultrasonic emulsification for 6min by a cell disruptor with 200W power at an interval of 1s for 1s to form polylactic acid W/O emulsion;
(3) preparing porous BSA-coated polylactic acid microspheres:
stirring the polylactic acid W/O emulsion prepared in the step (2) at 8000 revolutions per minute, volatilizing dichloromethane in the polylactic acid W/O emulsion in a low-temperature water bath at 45 ℃ to solidify polylactic acid to obtain a microsphere material, centrifugally collecting microspheres at 5000 revolutions per minute, cleaning with deionized water, and freeze-drying at-55 ℃ for 24 hours to obtain BSA (bovine serum albumin) -coated porous polylactic acid microspheres;
(4) culturing a lung cancer organoid model:
dissociating the lung cancer solid tumor tissue by using the sample dissociation liquid to obtain primary lung cancer solid tumor cells; culturing the separated tumor cells by using an RPMI-1640 culture medium containing 10% fetal calf serum to obtain a lung cancer tumor cell suspension;
and (4) adding the BSA coated porous polylactic acid microspheres obtained in the step (3) to perform three-dimensional culture to obtain a lung cancer organoid model.
According to the invention, hydrophilic gel is taken as a loading body to load a growth promoting factor, and the growth promoting factor is taken as a water phase to be mixed and emulsified with polylactic acid oil phase to form water-in-oil emulsion so as to obtain the polylactic acid microspheres coated with gel particles, so that the growth promoting factor is coated inside the porous polylactic acid microspheres, and the proliferation and differentiation of internal tumor cells are facilitated; meanwhile, ammonium bicarbonate is used as a pore-forming agent, the emulsification process of the water phase and the oil phase is controlled, and the porous polylactic acid microspheres with uniform pores inside and on the surface are prepared, so that the tumor cells can be favorably adhered and grown on the inner pores of the porous polylactic acid microsphere support, and the requirements on the cell density and the number of the lung cancer organoid tissues in vitro are met.
The invention takes the hydrophilic gel as a loading body for promoting the growth factor, improves the biocompatibility of the hydrophilic gel and the polylactic acid microsphere scaffold, can purposefully add the factor for promoting the growth of the lung cancer tumor cells by regulating and controlling the growth factor promoting component in the hydrophilic gel, is beneficial to the culture of in vitro lung cancer organoids, and has the advantages of simple preparation process, mature reaction condition and strong operability.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A culture method for constructing a lung cancer organoid model based on porous microspheres is characterized by comprising the following steps:
(1) preparing gel particles loaded with growth promoting factors:
adding the growth promoting factor into the hydrophilic gel material, centrifuging, cleaning, and freeze-drying to obtain gel particles loaded with the growth promoting factor;
(2) preparing polylactic acid W/O emulsion:
a) dissolving polylactic acid and levorotatory polylactic acid in an organic solvent to be fully dissolved to be used as an oil phase; preparing an ammonium bicarbonate aqueous solution with the concentration of 2.0-6.0%, adding the gel particles loaded with the growth promoting factors obtained in the step (1), and uniformly mixing to obtain a water phase;
b) adding the water phase into the oil phase, mixing the two phases, and performing ultrasonic emulsification for 5-30 min by using a cell disruption instrument with the power of 200W in a mode of ultrasonic for 1s at intervals of 1s to form a polylactic acid W/O emulsion;
(3) preparing porous polylactic acid microspheres coated with growth promoting factors:
stirring the polylactic acid W/O emulsion prepared in the step (2) at 600-1000 rpm, volatilizing an organic solvent in the polylactic acid W/O emulsion in a low-temperature water bath to solidify polylactic acid to separate out a microsphere material, centrifugally collecting microspheres at 3000-6000 rpm, cleaning the microspheres with deionized water, and freeze-drying the microspheres at-45 to-55 ℃ for 12-24 hours to obtain porous polylactic acid microspheres coated with growth promoting factors;
(4) culturing a lung cancer organoid model:
dissociating the lung cancer solid tumor tissue by using the sample dissociation liquid to obtain primary lung cancer solid tumor cells; culturing the separated tumor cells by using a special culture medium to obtain a lung cancer tumor cell suspension; and (4) adding the porous polylactic acid microspheres coated with the growth promoting factors obtained in the step (3) to perform three-dimensional culture to obtain the lung cancer organoid model.
2. The culture method for constructing the lung cancer organoid model based on porous microspheres according to claim 1, wherein the growth promoting factor in the step (1) is selected from BSA; BMP 4; BMP 2; b27; ITS-X; y-27632; EGF; one of Human FGF 4.
3. The culture method for constructing the lung cancer organoid model based on the porous microspheres according to claim 1, wherein the hydrophilic gel material is selected from one of gelatin, chitosan and sodium alginate.
4. The culture method for constructing the lung cancer organoid model based on the porous microspheres according to claim 1, wherein the concentration of the polylactic acid and the L-polylactic acid in the oil phase in the step (1) is 10-30% in an organic solvent, and the organic solvent is dichloromethane, ethyl acetate or chloroform.
5. The culture method for constructing the lung cancer organoid model based on the porous microspheres as claimed in claim 1, wherein the volume ratio of the water phase to the oil phase in step (1) is 1 (10-40).
6. The culture method for constructing the lung cancer organoid model based on porous microspheres according to claim 1, wherein the average diameter of the porous polylactic acid microspheres coated with the growth promoting factors is 2 ± 1 μm.
7. The culture method for constructing the lung cancer organoid model based on the porous microspheres as claimed in claim 1, wherein the special culture medium comprises 70-90% of basal medium and 10-30% of fetal bovine serum.
8. The culture method for constructing the lung cancer organoid model based on the porous microspheres according to claim 7, wherein the basic culture medium is one selected from RPMI-1640 culture medium, DMEM culture medium, F-12 culture medium or DMEM/F-12 culture medium.
9. The lung cancer organoid model constructed by the culture method for constructing the lung cancer organoid model based on the porous microspheres according to any one of claims 1 to 8.
10. Use of the lung cancer organoid model of claim 9 in the development of lung cancer tumor vaccines, medicaments.
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