CN116515157A - Novel three-dimensional porous material and preparation method and application thereof - Google Patents
Novel three-dimensional porous material and preparation method and application thereof Download PDFInfo
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- CN116515157A CN116515157A CN202310341417.3A CN202310341417A CN116515157A CN 116515157 A CN116515157 A CN 116515157A CN 202310341417 A CN202310341417 A CN 202310341417A CN 116515157 A CN116515157 A CN 116515157A
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- dimensional porous
- chip
- porous material
- foam metal
- solution
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Classifications
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Abstract
The invention discloses a novel three-dimensional porous material, a preparation method and application thereof, belonging to the fields of biology, chemistry and material science. The invention takes foam metal as a template, uses natural polymer solution to completely replicate the structure of the foam metal, and removes the foam metal to obtain the three-dimensional porous material. After the surface of the three-dimensional porous material is modified and activated by functional groups, the three-dimensional porous material can be coupled with biomolecules; and integrating the obtained biomolecule-modified three-dimensional porous material with a chip to obtain the three-dimensional porous microfluidic chip. The three-dimensional porous microfluidic chip can be used in biomedicine, such as separation, in-vitro culture, transplantation after culture and the like of circulating tumor cells. The material has the advantages of easy batch preparation, high light transmittance, good porosity and biocompatibility, degradability and the like, and can realize efficient separation of target objects and rapid in-situ amplification and transplantation of target cells. The invention can provide a new platform for the biomedical fields such as cancer research, tumor treatment and the like.
Description
Technical Field
The invention belongs to the fields of biology, chemistry and material science, and particularly relates to a novel three-dimensional porous material, a preparation method and application thereof.
Background
Circulating tumor cells are tumor cells that shed from a primary or metastatic tumor lesion into the peripheral blood. In recent years, in vitro culture of circulating tumor cells in blood has received extensive attention from researchers. The in vitro culture of the circulating tumor cells is beneficial to the downstream molecular analysis and the drug sensitivity test, and has great guiding significance for the deep knowledge of cancer metastasis and the personalized treatment of cancer patients. However, the number of circulating tumor cells in blood is extremely small, and only a very small number thereof has proliferation capacity, so that the in vitro culture is difficult. There is a need to develop a new method for in vitro culture of circulating tumor cells that is simple and efficient.
The microfluidic chip has the advantages of easy integration, controllability, less consumption of reagents, high analysis speed, high throughput and the like, and has wide application in the fields of liquid biopsy, gene and protein analysis, organ chip and drug research and the like. However, the existing preparation of the microfluidic chip still has the problems of complicated operation, high price, difficult recycling and the like, and limits the application of the microfluidic chip in biomedicine.
The natural polymer materials such as cellulose, chitin and the like have the advantages of environmental friendliness, reproducibility, degradability, good biocompatibility and the like, and are widely applied to the fields of foods, industry, medicines and the like, but are relatively rarely applied to the biomedical field related to cancers.
Disclosure of Invention
The primary aim of the invention is to provide a preparation method of a novel three-dimensional porous material and the three-dimensional porous material obtained by the method.
The invention further aims at providing a novel three-dimensional porous microfluidic chip and a preparation method thereof.
It is a further object of the present invention to provide applications of the three-dimensional porous material and the three-dimensional porous microfluidic chip.
The aim of the invention is achieved by the following technical scheme:
a preparation method of a three-dimensional porous material comprises the following steps:
(1) After the natural polymer solution and the cross-linking agent are uniformly mixed, the foam metal is immersed in the natural polymer solution, and the natural polymer solution is centrifuged to fully infiltrate the foam metal by the liquid material.
(2) Centrifuging the foam metal infiltrated with the high polymer material to remove redundant liquid material in the pores, then placing the foam metal into a coagulant for solidification, and then taking out and heating the foam metal to further solidify the liquid material on the surface of the foam metal, thereby obtaining the foam metal with a layer of high polymer material attached on the surface.
(3) Repeating the steps (1) and (2) for a plurality of times to obtain the foam metal coated with the polymer material.
(4) Immersing foam metal coated with high molecular material into acid solution to remove foam metal, immersing in water to remove adsorbed impurities, and obtaining the three-dimensional porous material.
In the step (1), the foam metal comprises foam nickel, foam copper, foam aluminum and foam alloy.
In step (1), the metal foam is preferably treated by: sequentially placing the foam metal into organic reagent, and respectively ultrasonically cleaning in water for 1-60 minutes. The organic reagent comprises acetone, toluene and ethanol.
In the step (1), the natural polymer comprises cellulose and chitin.
In the step (1), the crosslinking agent is preferably epichlorohydrin.
In step (1), the rotational speed of the centrifugation is preferably 1000-9000rpm.
In step (2), the rotational speed of the centrifugation is preferably 1000-5000rpm.
In the step (2), the coagulating agent comprises ultrapure water, ethanol and sodium sulfate solution, and the curing time in the coagulating agent is preferably 30-120 minutes.
In the step (4), the acidic solution comprises nitric acid, hydrochloric acid and sulfuric acid solution. The concentration of the acidic solution is preferably 5-14mol/L. The time for immersing the foamed metal encapsulating the polymer material in the acidic solution is preferably 1 to 12 hours. The time for soaking and washing in water is preferably 12-48 hours.
A novel three-dimensional porous material can be prepared by the method. The three-dimensional porous material has adjustable size, the pore diameter is equivalent to that of the foam metal material, and the three-dimensional porous material has self-supporting property, degradability and excellent biocompatibility.
A novel three-dimensional porous microfluidic chip comprises a chip bottom plate, a chip cover plate, a biomolecule modified three-dimensional porous material and a chip clamp. Wherein, be equipped with the passageway of placing three-dimensional porous material in the middle of the chip bottom plate, be equipped with inlet and liquid outlet on the chip apron, the position of inlet and liquid outlet corresponds with the passageway both ends on the chip bottom plate, and the chip anchor clamps are used for pressing from both sides tight chip bottom plate, chip apron, make it can not leak liquid. The biomolecules are preferably antibodies, streptavidin, lectins, growth factors, nucleic acid aptamers, etc., for specific recognition and capture of targets including cells, exosomes, proteins, free RNAs, etc.
The three-dimensional porous material modified by the biological molecule is preferably prepared by a method comprising the following steps: immersing the three-dimensional porous material after being treated by Plasma into a silanization reagent for reaction to obtain the three-dimensional porous material treated by the silanization reagent; and then activating the porous material by using a chemical cross-linking agent, and adding biomolecules for incubation to obtain the biomolecule-modified three-dimensional porous material. The time for treating the three-dimensional porous material by Plasma is preferably 1-6 minutes; the silanization reagent comprises carboxyethyl silanetriol sodium salt, gamma-aminopropyl triethoxysilane and the like, and can be used for derivatizing carboxyl, amino and other functional groups on the surface of the material; the concentration of the silylating agent is preferably 1 to 25% by mass; the three-dimensional porous material is preferably reacted in the silylating agent for a time period of 1 to 10 hours.
The preparation method of the three-dimensional porous microfluidic chip comprises the following steps: cutting the three-dimensional porous material modified by the biological molecules into a size matched with a channel on a chip bottom plate, placing the three-dimensional porous material in the channel, covering a chip cover plate, and clamping an upper layer and a lower layer of the chip by adopting a clamp to obtain the chip with the three-dimensional porous material embedded inside, namely the three-dimensional porous microfluidic chip. The preparation method of the three-dimensional porous microfluidic chip is simple, the batch preparation is easy, and the chip bottom plate, the cover plate and the clamp can be reused.
The three-dimensional porous material or the three-dimensional porous microfluidic chip is applied to biomedicine, such as separation and detection of specific cells, exosomes, proteins and free RNAs in body fluid, in-situ culture of the specific cells in the body fluid, implantation of the cultured cells into an animal body, model construction and the like.
The method for separating and culturing circulating tumor cells in blood based on the three-dimensional porous material and the three-dimensional porous microfluidic chip and implanting the cultured cells into an animal body to construct a cancer animal model comprises the following steps:
(1) The bovine serum albumin solution is introduced into a three-dimensional porous microfluidic chip modified by biomolecules (such as antibodies or nucleic acid aptamers, etc.) for specifically recognizing tumor cells, and after incubation and closure, the three-dimensional porous microfluidic chip is washed with Phosphate Buffered Saline (PBS).
(2) Taking fresh blood of a cancer patient or a tumor-bearing animal, introducing the fresh blood into the chip obtained in the step (1), and capturing circulating tumor cells in the blood in the chip by the biomolecules after flushing the fresh blood with PBS.
(3) Dispersing trophoblast cells into a collagen solution to obtain a mixed solution of collagen and cells.
(4) Introducing the mixed solution of the collagen and the cells obtained in the step (3) into the chip for capturing tumor cells obtained in the step (2), standing to gel the collagen, then adding a complete culture medium into the chip, and replacing the culture medium in the chip every day to enable the captured circulating tumor cells to proliferate in situ in the chip.
(5) And (5) detaching the clamp of the chip, opening the cover plate, and taking out the three-dimensional porous material containing the amplified tumor cells.
(6) Implanting the three-dimensional porous material containing the amplified tumor cells obtained in the step (5) into an animal body, suturing and feeding, and finding that tumors grow out at the implantation site to obtain the cancer animal model.
In the step (1), the concentration of the bovine serum albumin solution is preferably 1-10% by mass, and the incubation time of the bovine serum albumin solution is preferably 1-100 minutes.
In step (2), the fresh blood of the cancer patient or tumor-bearing animal is preferably 0.5-10mL, and the fresh blood is preferably introduced into the chip at a flow rate of 1-200 mu L/min.
In step (3), the trophoblast cells include cells, preferably fibroblasts, that secrete growth factors to promote proliferation of tumor cells. The collagen solution is preferably formulated by a method comprising the steps of: and diluting the collagen solution to 1-3mg/mL, and regulating the pH to 7.2-7.4 by using an alkali solution. The alkali comprises sodium hydroxide and potassium hydroxide.
The invention has the following advantages and effects:
the invention provides a three-dimensional porous material prepared based on a natural polymer solution and a sacrificial template method, which has the advantages of high light transmittance, porosity, excellent self-supporting property and biocompatibility, degradability in organisms and the like. The three-dimensional porous material can be integrated with a chip after being subjected to functional modification, and the obtained three-dimensional porous microfluidic chip can realize high-efficiency and high-flux separation of a target object, rapid in-situ amplification of target cells and transplantation of cells after amplification. The invention has simple and easy operation, low cost and high repeatability, can be completed in general chemistry and biochemistry laboratories, and provides a new platform for the biomedical fields such as tumor research and treatment.
Drawings
FIG. 1 is a scanning electron microscope image of nickel foam.
FIG. 2 is a physical and microscopic block diagram of a three-dimensional porous cellulosic material prepared from a cellulose solution; fig. 2 (a) is a physical view, and fig. 2 (B) is a scanning electron microscope view of a three-dimensional porous cellulose material.
FIG. 3 is a fluorescence microscopy image of Dylight 488-labeled secondary antibodies characterizing anti-epithelial cell adhesion factor antibody modified three-dimensional porous cellulose material.
FIG. 4 is a graph showing the mass change of a three-dimensional porous cellulose material during 28 days of immersion in a cellulase solution.
Fig. 5 (a) is an assembled schematic diagram of a three-dimensional porous microfluidic chip, wherein: 1-three-dimensional porous material, 2-chip cover plate, 3-chip bottom plate and 4-clamp. Fig. 5 (B) is a physical diagram of a solution introduced into a three-dimensional porous microfluidic chip, wherein the three-dimensional porous microfluidic chip after 5-assembly has 6-liquid inlet, 7-liquid outlet, 8-liquid inlet pipe and 9-liquid outlet pipe.
FIG. 6 is the results of capturing and culturing in situ mouse breast cancer cells for 7 days using an anti-epithelial cell adhesion factor antibody modified three-dimensional porous cellulose chip; wherein: a is Hoechst staining, B is FITC-CK staining, C is a superposition graph of the two staining, 1 represents the staining result of fibroblasts, and 2 represents the staining result of mouse breast cancer cells.
FIG. 7 is a fluorescence microscope photograph of circulating tumor cells captured from blood of breast cancer model mice by an anti-epithelial cell adhesion factor antibody modified three-dimensional porous cellulose chip; wherein: a is Hoechst staining, B is PE-CD45 staining, C is FITC-CK staining, and D is a superposition diagram of the three staining.
FIG. 8 is a fluorescence microscope photograph of tumor cells after 60 days of capture and in situ culture of anti-epithelial cell adhesion factor antibody modified three-dimensional porous cellulose chip from breast cancer model mouse blood; wherein: a is Hoechst staining, B is FITC-CK staining, and C is a superposition graph of the two stains.
Fig. 9 is a photograph of a tumor-bearing nude mouse obtained after a three-dimensional porous cellulose material containing amplified tumor cells is implanted in the subcutaneous position of the lower abdomen of the nude mouse for 50 days, and the inset in the black dotted line is a tumor photograph obtained by dissecting the nude mouse after the nude mouse is sacrificed.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail with reference to the accompanying drawings and preferred examples, but the embodiments of the present invention are not limited thereto.
Example 1
(1) 7g of sodium hydroxide and 12g of urea were dissolved in 81mL of ultrapure water and placed in a refrigerator for pre-cooling to-12 ℃. 4g of cotton wool was weighed and added thereto, and vigorously stirred with a mechanical stirrer for 10 minutes, to obtain a cellulose solution having a mass fraction of 4%.
(2) The foam nickel (30 mm. Times.4 mm. Times.1 mm) was sequentially ultrasonically washed with acetone, ethanol, and ultra-pure water for 30 minutes, respectively, and then oven dried. FIG. 1 is a scanning electron microscope image of nickel foam showing its three-dimensional porous structure.
(3) The 4% cellulose solution and epichlorohydrin (mass ratio of 27:1) are mixed and stirred uniformly, poured into a 2mL centrifuge tube for filling, and then the dried foam nickel is immersed into the centrifuge tube, and centrifuged at 8000rpm for 5 minutes to fully infiltrate the viscous mixed solution into the foam nickel.
(4) And (3) placing the foam nickel obtained in the step (3) into a new 2mL centrifuge tube, centrifuging at 3000rpm for 3 minutes to remove redundant mixed solution in pores, soaking the obtained foam nickel in ethanol for 1 hour, and drying to obtain the foam nickel with a layer of cellulose attached to the surface.
(5) Repeating the steps (3) and (4) for three times again to obtain the foam nickel wrapping the cellulose.
(6) Immersing the foam nickel coated with cellulose obtained in the step (5) into a nitric acid solution of 7mol/L for 3 hours, etching to remove nickel, immersing and cleaning in ultrapure water for 24 hours to remove adsorbed nitric acid and other metal ions, and drying to obtain the three-dimensional porous cellulose material capable of being self-supported. Fig. 2 (a) is a physical diagram of a three-dimensional porous cellulose material, and fig. 2 (B) is a scanning electron microscope diagram thereof, which shows that the prepared material has a certain self-supporting property and flexibility, and the three-dimensional porous structure of the foam nickel is completely duplicated.
(7) Sterilizing the three-dimensional porous cellulose material obtained in the step (6) at high temperature, treating for 5 minutes by using Plasma, immersing the three-dimensional porous cellulose material into 5% carboxyethyl silanetriol sodium salt solution, incubating for 4 hours at room temperature, and washing the three-dimensional porous cellulose material by using ultrapure water to obtain carboxylated three-dimensional porous cellulose material; then immersing the mixture into a mixed solution containing 10mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 10mg/mL of N-hydroxysuccinimide (NHS), and incubating the mixture at room temperature for 40 minutes to activate carboxyl groups; after washing with PBS, it was immersed in 50. Mu.g/mL streptavidin solution and incubated for 4 hours at room temperature; after washing with PBS, the mixture was immersed in a 10. Mu.g/mL solution of biotinylated anti-epithelial cell adhesion factor antibody (available from eBioscience under the trade designation 13-5791-82), incubated at room temperature for 1 hour, and washed with PBS to obtain the anti-epithelial cell adhesion factor antibody-modified three-dimensional porous cellulose material. FIG. 3 is a fluorescent photograph taken after incubation of a material modified with a Dylight 488-labeled secondary antibody solution with an anti-epithelial cell adhesion factor antibody, showing successful modification of the material surface antibody.
(8) Immersing the three-dimensional porous cellulose material modified by the anti-epithelial cell adhesion factor antibody obtained in the step (7) in a cellulase solution (100 unit/g), wherein fig. 4 is a mass change curve of the cellulose material in the process of 28 days, and the three-dimensional porous cellulose material is found to be gradually degraded.
(9) And (3) sterilizing the chip bottom plate, the cover plate and the clamp at high temperature, putting the three-dimensional porous cellulose material modified by the anti-epithelial cell adhesion factor antibody obtained in the step (7) into a straight-shaped channel (the channel size is 25mm multiplied by 4mm multiplied by 0.6 mm) on the chip bottom plate, effectively attaching the chip bottom plate, covering the chip cover plate, and clamping the upper layer and the lower layer of the chip by the clamp, so that no liquid leakage occurs, thereby obtaining the three-dimensional porous cellulose microfluidic chip. Fig. 5 (a) is a schematic diagram of chip assembly including a three-dimensional porous cellulose material 1, a chip cover plate 2, a chip base plate 3, and a jig 4.
(10) On a sterile console, 40 mouse breast cancer cells (4T 1) are dispersed in 1mL of PBS solution, the solution is introduced into the chip obtained in the step (9) at a flow rate of 50 mu L/min, and the chip is washed for 3 minutes at a flow rate of 50 mu L/min by PBS, so that the chip capturing the breast cancer cells is obtained.
(11) On a sterile bench, a collagen solution was taken, diluted to a concentration of 1mg/mL with DMEM high-sugar basal medium (available from GIBCO Co., ltd., cat# 11995065), and pH was adjusted to 7.2-7.4 with 0.2mol/L sodium hydroxide solution. Will 10 5 The individual fibroblasts were dispersed in the above collagen solution to obtain a mixed solution of collagen and fibroblasts.
(12) Introducing the solution obtained in step (11) into the chip obtained in step (10), and placing in a constant temperature incubator (37 ℃ C., 5% CO) 2 ) Standing for 2 hr to gel collagen, adding DMEM high sugar complete culture medium (prepared by mixing 89% DMEM high sugar basic culture medium, 10% foetus calf serum and 1% penicillin-streptomycin solution), culturing in a constant temperature incubator for 7 days, and changing culture medium every day. After 7 days, the chip was removed, 1mg/mL collagenase solution was introduced into the chip, incubated at 37℃for 2 hours to degrade collagen, and then washed with PBS at a flow rate of 50. Mu.L/min for 10 minutes to wash out collagen and most of fibroblasts in the chip. Then, 4% paraformaldehyde solution is firstly introduced into the chip for fixing for 30 minutes, triton X-100 is introduced after PBS flushing for 10 minutes, and 5% bovine serum albumin solution is introduced after PBS flushing for sealing for 2 hours; finally, a staining agent solution containing Hoechst 33258 (blue fluorescence) and FITC-CK (green fluorescence) is added into the chip to incubate for 2 hours at 4 ℃, after washing for 10 minutes by PBS, the chip is placed under an inverted fluorescence microscope to observe. The amplified tumor cells were labeled simultaneously with the blue fluorescence of Hoechst 33258 and the green fluorescence of FITC-CK (diameter greater than 10 μm), while the remaining fibroblasts were labeled individually with the blue fluorescence of Hoechst 33258, and fig. 6 is a representative cell photograph. By counting under a microscope, the number of breast cancer cells in the chip was found to proliferate from the initial 38.+ -. 5 to 499.+ -. 65 under the co-culture condition, by a factor of 13.13. The results show that the three-dimensional porous microfluidic chip provided by the invention can be used for efficient capture and in-situ culture of cancer cells.
Example 2
(1) 7g of sodium hydroxide and 12g of urea were dissolved in 81mL of ultrapure water and placed in a refrigerator for pre-cooling to-12 ℃. 4g of cotton wool was weighed and added thereto, and vigorously stirred with a mechanical stirrer for 10 minutes, to obtain a cellulose solution having a mass fraction of 4%.
(2) The nickel foam (30 mm. Times.8 mm. Times.1.5 mm) was sequentially ultrasonically washed with acetone, ethanol, and ultra-pure water for 30 minutes, respectively, and then oven dried.
(3) The cellulose solution and epichlorohydrin (mass ratio of 27:1) were mixed and stirred uniformly, poured into a 2mL centrifuge tube and filled, and then the dried foam nickel was immersed therein, and centrifuged at 9000rpm for 4 minutes to allow the viscous mixed solution to fully infiltrate the foam nickel.
(4) Transferring the foam nickel obtained in the step (3) into an empty 2mL centrifuge tube, centrifuging at 3000rpm for 3 minutes to remove redundant solution in pores, soaking the obtained foam nickel in ultrapure water for 30 minutes, and drying to obtain the foam nickel with a layer of cellulose attached to the surface.
(5) Repeating the steps (3) and (4) for three times again to obtain the foam nickel with the surface coated with cellulose.
(6) Immersing the foam nickel with the cellulose coated on the surface obtained in the step (5) into a nitric acid solution of 7mol/L for 3 hours to etch and remove metallic nickel, immersing in ultrapure water for 24 hours to remove adsorbed impurities, taking out and drying to obtain the three-dimensional porous cellulose material.
(7) And (3) sterilizing the three-dimensional porous cellulose material obtained in the step (6) by ultraviolet irradiation, treating Plasma for 5 minutes, then incubating in 5% carboxyethyl silanetriol sodium salt solution for 4 hours, incubating in a mixed solution containing 10mg/mL EDC & HCl and 10mg/mL NHS for 40 minutes, incubating in 50 mug/mL streptavidin solution for 4 hours, and finally incubating in 10 mug/mL biotinylated anti-epithelial cell adhesion factor antibody solution (purchased from eBioscience company and having the product number of 13-5791-82) for 1 hour, and washing with PBS to obtain the three-dimensional porous cellulose material modified by the anti-epithelial cell adhesion factor antibody.
(8) And (3) after the chip bottom plate, the cover plate and the clamp are sterilized at high temperature, cutting the material obtained in the step (7) to 25mm multiplied by 4mm, putting the material into a straight-shaped channel (the channel size is 25mm multiplied by 4mm multiplied by 0.6 mm) on the chip bottom plate, effectively attaching the material and the channel, covering the chip cover plate, and clamping the upper layer and the lower layer of the chip by the clamp to prevent liquid leakage, thus obtaining the three-dimensional porous cellulose chip.
(9) Introducing 5% sterile bovine serum albumin solution into the chip obtained in the step (8), incubating and sealing for 60 minutes, and then flushing with PBS.
(10) 2mL of blood from a fresh breast cancer mouse was introduced into the chip obtained in the step (9) at a flow rate of 50. Mu.L/min (FIG. 5 (B)), and the blood cells were washed out by washing with PBS for 3 minutes. Then sequentially introducing 4% paraformaldehyde into the chip for fixing for 30 minutes, allowing Triton X-100 to permeate for 10 minutes, and sealing for 2 hours by using 5% bovine serum albumin solution; finally, a staining agent solution containing Hoechst 33258 (blue fluorescence), FITC-CK (green fluorescence) and PE-CD45 (red) is added into the chip to incubate for 2 hours at 4 ℃, after the chip is washed by PBS, the chip is placed under an inverted fluorescence microscope to observe. The circulating tumor cells are identified as blue fluorescence capable of simultaneously marking Hoechst 33258 and green fluorescence of FITC-CK, and the size is larger than 10 mu m; FIG. 7 is a diagram of captured tumor cells. The results show that the chip can be used for capturing circulating tumor cells in blood of tumor-bearing mice.
(11) In a sterile operation table, taking 2mL of blood which is the same as that in the step (10), introducing the blood into the chip obtained in the step (9) at a flow rate of 50 mu L/min, and washing the blood cells by using PBS for 3 minutes to obtain the chip for capturing the circulating tumor cells.
(12) On a sterile bench, a collagen solution was taken, diluted to a concentration of 1mg/mL with DMEM high-sugar basal medium (available from GIBCO Co., ltd., cat# 11995065), and pH was adjusted to 7.2-7.4 with 0.2mol/L sodium hydroxide solution. Will 10 5 The individual fibroblasts were dispersed in the above collagen solution to obtain a mixed solution of collagen and fibroblasts.
(13) Introducing the mixed solution obtained in the step (12) into the chip obtained in the step (11), and then placing the chip in a constant temperature incubator (37 ℃ C., 5% CO) 2 ) Standing for 30 min to gel collagen, adding DMEM high sugar complete culture medium (prepared by mixing 89% DMEM high sugar basic culture medium, 10% foetus calf serum and 1% penicillin-streptomycin solution), placing into incubator, changing culture medium every day, and culturing for 60 days to obtain tumor cells containing amplified tumor cellsIs a chip of the chip (a).
(14) 1mg/mL collagenase solution is introduced into the chip in the step (13), the chip is incubated for 2 hours at 37 ℃ to degrade collagen, and then the PBS is used for washing the collagen and most of fibroblasts in the chip. Then 4% paraformaldehyde is sequentially introduced for fixing for 30 minutes, triton X-100 is penetrated for 10 minutes, and 5% bovine serum albumin solution is blocked for 2 hours; then, a staining agent solution containing Hoechst 33258 (blue fluorescence) and FITC-CK (green fluorescence) was introduced and incubated at 4℃for 2 hours, and after washing with PBS, the chip was observed under an inverted fluorescence microscope. The circulating tumor cells were identified as simultaneously labeled Hoechst 33258 blue fluorescence and FITC-CK green fluorescence, and were greater than 10 μm in size. FIG. 8 is a fluorescent image after incubation, tumor cells grown in clusters. The results show that the chip can be successfully used for capturing and in-situ culturing of circulating tumor cells in blood.
(15) Detaching the clamp of the chip obtained in the step (13), opening a cover plate of the clamp, and taking out the three-dimensional porous cellulose material containing the amplified tumor cells; the skin of the lower abdomen mammary gland position of the nude mice is cut off, and the wound is sutured after the taken out materials are put in, so that the nude mice are continuously fed. On day 41, a tumor growth was observed at the nude mice implantation site; by day 50, tumor size was as long as 700mm 3 The method comprises the steps of carrying out a first treatment on the surface of the After the nude mice were sacrificed and dissected, no trace of the three-dimensional porous material was seen at the implantation site, and fig. 9 is a picture of the tumor-bearing nude mice obtained.
The embodiment shows that the three-dimensional porous chip prepared by the method has a good separation effect on target cells, can realize in-situ rapid amplification and implantation, and can be used in biomedical fields such as cancer research, tumor treatment and the like.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a three-dimensional porous material is characterized by comprising the following steps: the method comprises the following steps:
(1) Uniformly mixing a natural polymer solution and a cross-linking agent, immersing foam metal in the mixture, and centrifuging to fully infiltrate the foam metal with a liquid material;
(2) Centrifuging the foam metal infiltrated with the high polymer material to remove redundant liquid materials in pores, then placing the foam metal into a coagulant for solidification, and then taking out the foam metal and heating the foam metal to further solidify the liquid materials on the surface of the foam metal;
(3) Repeating the steps (1) and (2) for a plurality of times to obtain foam metal coated with high polymer materials;
(4) Immersing foam metal coated with high molecular material into acid solution to remove foam metal, immersing in water to remove adsorbed impurities, and obtaining the three-dimensional porous material.
2. The method for preparing a three-dimensional porous material according to claim 1, wherein: the natural polymer comprises cellulose and chitin; the cross-linking agent comprises epichlorohydrin; the foam metal comprises foam nickel, foam copper, foam aluminum and foam alloy; the coagulant comprises ultrapure water, ethanol and sodium sulfate solution; the acidic solution comprises nitric acid, hydrochloric acid and sulfuric acid solution.
3. A three-dimensional porous material, characterized by: obtained by the production process according to claim 1 or 2.
4. A three-dimensional porous microfluidic chip, characterized in that: a three-dimensional porous material of claim 3, comprising a chip base plate, a chip cover plate, a biomolecular modification, a chip fixture; the chip clamp is used for clamping the chip bottom plate and the chip cover plate; the biological molecule is used for specifically identifying and capturing a target object.
5. The three-dimensional porous microfluidic chip of claim 4, wherein: the three-dimensional porous material of claim 3 modified by biomolecules is prepared by a method comprising the steps of: immersing the three-dimensional porous material after being treated by Plasma into a silanization reagent for reaction to obtain the three-dimensional porous material treated by the silanization reagent; then activating the porous material by using a chemical cross-linking agent, and adding biomolecules for incubation to obtain a three-dimensional porous material modified by the biomolecules; the silylation agent is used for derivatizing functional groups on the surface of the material, wherein the functional groups comprise carboxyl and amino.
6. The method for preparing the three-dimensional porous microfluidic chip according to claim 4 or 5, wherein the method comprises the following steps: the method comprises the following steps: cutting the three-dimensional porous material modified by the biological molecules into a size matched with a channel on a chip bottom plate, placing the three-dimensional porous material in the channel, covering a chip cover plate, and clamping an upper layer and a lower layer of the chip by adopting a clamp to obtain the three-dimensional porous microfluidic chip.
7. Use of the three-dimensional porous material of claim 3 or the three-dimensional porous microfluidic chip of claim 4 or 5 in biomedical science.
8. The use according to claim 7, characterized in that: the biomedical application comprises the separation and detection of specific cells, exosomes, proteins and free RNAs in body fluid, in-situ culture of specific cells in body fluid, and cell transplantation into animal body after culture to construct a model.
9. A method for separating and culturing circulating tumor cells in blood based on the three-dimensional porous material of claim 3 or the three-dimensional porous microfluidic chip of claim 4 or 5, characterized in that: the method comprises the following steps:
(1) Introducing bovine serum albumin solution into a three-dimensional porous microfluidic chip modified by biomolecules for specifically recognizing tumor cells, incubating and sealing, and then flushing with PBS;
(2) Taking fresh blood of a cancer patient or a tumor-bearing animal, introducing the fresh blood into the chip obtained in the step (1), and capturing circulating tumor cells in the blood in the chip by the biomolecules after flushing the fresh blood by using PBS;
(3) Dispersing trophoblast cells into a collagen solution to obtain a mixed solution of collagen and cells;
(4) Introducing the mixed solution of the collagen and the cells obtained in the step (3) into the chip for capturing tumor cells obtained in the step (2), standing to gel the collagen, then adding a complete culture medium into the chip, and replacing the culture medium in the chip every day to enable the captured circulating tumor cells to proliferate in situ in the chip.
10. A method of constructing an animal model of cancer based on the three-dimensional porous material of claim 3 or the three-dimensional porous microfluidic chip of claim 4 or 5, characterized in that: the method comprises the following steps: the method of claim 9, wherein the captured circulating tumor cells can be proliferated in situ in the chip, the three-dimensional porous material containing the tumor cells after proliferation is taken out from the chip, and is implanted into animals for feeding, and the tumor animal model is obtained after the tumor grows out at the implantation site.
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