CN211445769U - 3D support for constructing in-vitro tumor model - Google Patents
3D support for constructing in-vitro tumor model Download PDFInfo
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- CN211445769U CN211445769U CN201922013014.8U CN201922013014U CN211445769U CN 211445769 U CN211445769 U CN 211445769U CN 201922013014 U CN201922013014 U CN 201922013014U CN 211445769 U CN211445769 U CN 211445769U
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Abstract
The utility model relates to a 3D support for constructing external tumour model, the 3D support includes support body, aperture, support first floor, support second floor, support third layer, the aperture has been seted up on the support body, the support body is laminated structure, is respectively: the bracket comprises a first bracket layer, a second bracket layer and a third bracket layer, wherein the first bracket layer, the second bracket layer and the third bracket layer are sequentially arranged from outside to inside, and small holes penetrate through the first bracket layer, the second bracket layer and the third bracket layer. When in use, the tumor cells are inoculated in the stent body, and can be used for constructing an in vitro tumor model. Its advantage does: the three-dimensional structure is more beneficial to cell growth than a two-dimensional structure, and can truly reflect the biological behavior of cancer; the support has moderate fluffiness rate, large pore diameter, large porosity, high water absorption and moderate degradation rate.
Description
Technical Field
The utility model belongs to the technical field of the medical instrument technique and specifically relates to a 3D support for making up external tumour model.
Background
Cell culture work is widely applied to various fields such as biology, medicine, toxicology, new drug research and development, bioengineering and the like, and a technical means for researching and observing morphological structures and life activities of cells becomes a necessary screening and pre-experimental means for life research.
Currently, conventional cell culture modes include two-dimensional cell culture, performed in cell culture plates such as 2, 4, 6, 12, 24, 48, 96-well cell culture plates or flasks, bioreactors, etc., where the cells grow adherently in a monolayer in culture medium in a two-dimensional manner. However, several studies have found that gene expression, signal transduction, and morphology of cells cultured in two dimensions in vitro may differ from those of cells grown in three dimensions in vivo.
The prior art has the following defects:
1. by using the existing 2D cell culture plate, the cell morphology is changed, the polarity is changed, the skeleton is reconstructed, and the biological behavior of the cell is changed;
2. the defect that the microenvironment of cells cultured by using a cell culture dish or a cell culture plate is too simple is known, the physical, chemical and biological signals and other complex signal stimulation exist in the microenvironment of colorectal cancer cells in vivo, and the physical, chemical and biological signals are difficult to be completely simulated by using the cell culture plate or the cell culture plate;
3. the 2D cell culture plate or culture dish has a limited culture area, the colon cancer cell culture needs 2-3 days for cell passage, time and labor are wasted, the cell culture efficiency is not high, and the passage is complicated as an exogenous operation. Therefore, the biological behavior of the cultured cells by using the cell culture plate or the cell culture dish is greatly different from that of the in vivo tumor cells.
4. The construction of the in vitro colon cancer model by using nude mice requires a large amount of scientific research investment, requires SPF-level animal houses (which are difficult to meet by basic scientific research institutions), requires complex ethical auditing procedures, and has low success rate and long period for constructing the in vitro colon cancer model.
To the above-mentioned defect, the inventor has designed the utility model discloses: the prepared 3D scaffold has the advantages of moderate degradation rate, moderate expansion rate, high water absorption, high porosity and large pore diameter, and can be used for constructing an in vitro tumor model.
The 3D stent for constructing the in vitro tumor model of the utility model has not been reported at present.
Disclosure of Invention
The utility model aims at the not enough of prior art, provide a 3D support for constructing external tumour model.
In order to achieve the purpose, the utility model adopts the technical proposal that:
A3D support for constructing an external tumor model comprises a support body (1), a 3D support local amplification module (3), a support third layer (4), small holes (5), a support first layer (6) and a support second layer (7), wherein the 3D support local amplification module (3) is arranged in the support body (1), the small holes (5) are uniformly distributed on the support body (1), the support body (1) is of a layered structure and is respectively the support third layer (4), the support first layer (6) and the support second layer (7), the support first layer (6), the support second layer (7) and the support third layer (4) are sequentially arranged from outside to inside, and the small holes (5) penetrate through the support first layer (6), the support second layer (7) and the support third layer (4);
the thickness of the 3D support is 6.26 mm-6.30 mm, and the aperture of the small hole (5) of the 3D support is 242 mu m-558 mu m; the porosity of the 3D scaffold is 92.61% -94.85%;
the bracket body (1) is cylindrical.
As a preferred embodiment of the utility model, the inside of the 3D bracket also comprises tumor cells (2), and the tumor cells (2) are positioned in the pores (5).
As a preferred embodiment of the present invention, the water absorption of the 3D scaffold is 3819.03% -3925.81%.
As a preferred embodiment of the present invention, the first layer (6) of the scaffold is a silk fibroin solution, the second layer (7) of the scaffold is a chitosan solution, and the third layer (4) of the scaffold is an alginate solution.
As a preferred embodiment of the utility model, the thickness of the first layer (6), the second layer (7) and the third layer (4) of the bracket are the same.
Preferably, the preparation method of the 3D scaffold for constructing the in vitro tumor model is as follows:
the method comprises the following steps: preparing a silk fibroin solution:
(1) cutting clean silkworm cocoon shell into 0.5-1cm2Weighing the pieces, storing in a vacuum bag for later use, preparing 1000ml of 0.5% sodium carbonate solution, placing in a water bath kettle, heating to boil, adding 5g of silkworm cocoon shells into the 0.5% sodium carbonate solution, immersing, stirring with a stirring rod, boiling for 3 times, and each time for 1 hour;
(2) washing the silkworm cocoons with natural water for 3 times, then washing with deionized water for 3 times, and then drying in an oven at 60 ℃ for 10 hours;
(3) dissolving the dried silkworm cocoons in the step (2) in a lithium bromide solution, preparing a 9M lithium bromide solution, stirring and heating the solution by using a magnetic stirrer at 60 ℃, adding the dried fibroin, and fully dissolving the fibroin; cooling to room temperature, and filtering with a Buchner funnel;
(4) and (3) dialysis: putting the filtrate into a dialysis bag, dialyzing with deionized water in a refrigerator at 4 ℃ for 5 days, and changing water once every 3 hours to remove small molecular substances in the silk fibroin solution to prepare the silk fibroin solution;
(5) concentration and purification: placing the obtained silk fibroin liquid in dialysis bag, adding into polyethylene glycol 6000 powder, drying, concentrating, collecting liquid, centrifuging at 3500r/min for 15min, removing insoluble substances, collecting supernatant, and storing in refrigerator at 4 deg.C for one week;
(6) and (3) concentration determination: taking 3 weighing bottles, numbering, cleaning, putting into a constant-temperature oven at 60 ℃, drying, fully cooling, weighing the three, and recording as M1; putting 10ml of silk fibroin solution into a weighing bottle, and weighing M2; putting the silk fibroin solution into a 60 ℃ oven for 12h, taking out the silk fibroin solution, cooling, weighing the cooled silk fibroin solution as M3, and taking the average value of the three according to the formula that the concentration of the silk fibroin solution is (M3-Ml)/(M2-M1) 100% to obtain the concentration of the silk fibroin which is 1.5%;
step two: preparing a chitosan solution:
weighing 1.5g of CS powder, adding deionized water to a constant volume of 100ml, and then adding 2ml of glacial acetic acid solution to obtain 1.5% yellowish CS solution with high viscosity;
step three: preparing an alginate solution:
weighing 1.5g of sodium alginate, preparing 100ml of solution by using distilled water, stirring for 1.5h in a constant-temperature water bath at 50 ℃, preparing a sodium alginate solution with the concentration of 1.5%, and standing and defoaming for 24h at 10 ℃ for later use;
step four: constructing a silk fibroin chitosan three-dimensional scaffold:
(1) cross-linked scaffold
1) Mixing the silk fibroin solution prepared in the step one, the chitosan solution prepared in the step two and the alginate solution prepared in the step three according to the weight ratio of 1:1:1, and uniformly stirring;
2) continuously immersing into 95% ethanol water solution containing 50mmol/L EDC and 18mmol/L NHS;
3) adding 1ml of mixed solution into each hole of a 24-hole culture plate, adding 200 mul of mixed solution into each hole of a 96-hole culture plate, removing bubbles, sequentially placing the culture plate in a refrigerator at 4 ℃ for crosslinking for 24 hours, freezing in the refrigerator at-20 ℃ for 12 hours, freezing in the refrigerator at-80 ℃ for 12 hours, and freezing and drying in a freeze dryer for 72 hours to obtain the SF/CS three-dimensional porous scaffold, and performing Co60 irradiation sterilization for later use.
The utility model has the advantages that:
1. the 3D in-vitro culture system is used for replacing 2D cell culture for colon cancer in-vitro cell culture, so that the biological behavior of colon cancer cells in vivo is simulated more truly, the cell growth is facilitated, the biological behavior of cancer can be reflected truly, and a more reliable basic research basis is provided for diagnosis and treatment of cancer.
2. The support has moderate fluffiness rate, large pore diameter, large porosity, high water absorption and moderate degradation rate.
3. The thickness of the first layer of the bracket, the thickness of the second layer of the bracket and the thickness of the third layer of the bracket are equal, and the used raw materials are as follows: the silk fibroin solution, the chitosan solution and the alginate solution are equal in parts by weight, and the pores are uniformly arranged, so that a better environment is provided for cell growth, and the success rate is high when the silk fibroin solution, the chitosan solution and the alginate solution are used for constructing a tumor in-vitro model.
4. The utility model discloses but composite support independent use or with the cooperation of conventional cell culture device use, and through rational design bearing structure's structure and size, make that it can be better compare with two-dimensional cultivation.
Drawings
Fig. 1 is a schematic diagram of cells seeded on the 3D scaffold of the present invention.
Fig. 2 is the internal part of the 3D support of the present invention.
Fig. 3 is an enlarged view of a portion of the inside of fig. 2.
FIG. 4 is a schematic diagram of the morphological structure of cells seeded on a 3D scaffold.
Fig. 5 is a schematic diagram of the layered structure of the 3D scaffold of the present invention.
Detailed Description
The present invention will be further described with reference to the following detailed description. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents also fall within the scope of the appended claims.
1. A stent body;
2. a tumor cell;
3. a 3D stent local magnification module;
4. a third layer of the scaffold;
5. a small hole;
6. a scaffold first layer;
7. a scaffold second layer.
Example 1
Referring to fig. 1, fig. 1 is a schematic view of cells seeded on the 3D scaffold of the present invention.
A3D support for constructing an in vitro tumor model comprises a support body 1, wherein the support body 1 is cylindrical, tumor cells are further arranged inside the 3D support, and the tumor cells are inoculated inside the 3D support.
Referring to fig. 2-3, fig. 2 is a schematic view of an internal part of a 3D bracket according to the present invention, and fig. 3 is a schematic view of an enlarged internal part of the bracket shown in fig. 2.
On the basis of fig. 1, the 3D stent for constructing an in vitro tumor model further comprises a 3D stent local amplification module 3 and small holes 5, wherein the stent body 1 comprises the 3D stent local amplification module 3 inside, the small holes 5 are uniformly distributed on the stent body 1, and the aperture of the small hole 5 of the 3D stent is 242 μm to 558 μm; the porosity of the 3D scaffold is 92.61% -94.85%.
Referring to fig. 4, fig. 4 is a schematic diagram of the morphological structure of cells seeded on a 3D scaffold.
In use, tumor cells 2 are seeded into the pores 5.
Referring to fig. 5, fig. 5 is a schematic diagram of a layered structure of the 3D scaffold of the present invention.
On the basis of fig. 1-4, the 3D support for constructing the in vitro tumor model further comprises a support third layer 4, a support first layer 6 and a support second layer 7, wherein the support first layer 6, the support second layer 7 and the support third layer 4 are sequentially arranged from outside to inside, and the small holes 5 penetrate through the support first layer 6, the support second layer 7 and the support third layer 4. The thickness of the 3D support is 6.26 mm-6.30 mm.
Example 2
A preparation method and a use method of a 3D scaffold for constructing an in vitro tumor model are as follows:
the preparation method comprises the following steps:
the method comprises the following steps: preparing a silk fibroin solution:
(1) cutting clean silkworm cocoon shell into 0.5-1cm2Weighing the pieces, storing in vacuum bag, preparing 0.5% sodium carbonate solution 1000ml, heating in water bath to boil, adding 5g cocoon shell into 0.5% sodium carbonate solution, soaking, stirring with stirring rod, boiling for 3 times1 hour;
(2) washing the silkworm cocoons with natural water for 3 times, then washing with deionized water for 3 times, and then drying in an oven at 60 ℃ for 10 hours;
(3) dissolving the dried silkworm cocoons in the step (2) in a lithium bromide solution, preparing a 9M lithium bromide solution, stirring and heating the solution by using a magnetic stirrer at 60 ℃, adding the dried fibroin, and fully dissolving the fibroin; cooling to room temperature, and filtering with a Buchner funnel;
(4) and (3) dialysis: putting the filtrate into a dialysis bag, dialyzing with deionized water in a refrigerator at 4 ℃ for 5 days, and changing water once every 3 hours to remove small molecular substances in the silk fibroin solution to prepare the silk fibroin solution;
(5) concentration and purification: placing the obtained silk fibroin liquid in dialysis bag, adding into polyethylene glycol 6000 powder, drying, concentrating, collecting liquid, centrifuging at 3500r/min for 15min, removing insoluble substances, collecting supernatant, and storing in refrigerator at 4 deg.C for one week;
(6) and (3) concentration determination: taking 3 weighing bottles, numbering, cleaning, putting into a constant-temperature oven at 60 ℃, drying, fully cooling, weighing the three, and recording as M1; putting 10ml of silk fibroin solution into a weighing bottle, and weighing M2; putting the silk fibroin solution into a 60 ℃ oven for 12h, taking out the silk fibroin solution, cooling, weighing the cooled silk fibroin solution as M3, and taking the average value of the three according to the formula that the concentration of the silk fibroin solution is (M3-Ml)/(M2-M1) 100% to obtain the concentration of the silk fibroin which is 1.5%;
step two: preparing a chitosan solution:
weighing 1.5g of CS powder, adding deionized water to a constant volume of 100ml, and then adding 2ml of glacial acetic acid solution to obtain 1.5% yellowish CS solution with high viscosity;
step three: preparing an alginate solution:
weighing 1.5g of sodium alginate, preparing 100ml of solution by using distilled water, stirring for 1.5h in a constant-temperature water bath at 50 ℃, preparing a sodium alginate solution with the concentration of 1.5%, and standing and defoaming for 24h at 10 ℃ for later use;
step four: constructing a silk fibroin chitosan three-dimensional scaffold:
(1) cross-linked scaffold
1) Mixing the silk fibroin solution prepared in the step one, the chitosan solution prepared in the step two and the alginate solution prepared in the step three according to the weight ratio of 1:1:1, and uniformly stirring;
2) continuously immersing into 95% ethanol water solution containing 50mmol/L EDC and 18mmol/L NHS;
3) adding 1ml of mixed solution into each hole of a 24-hole culture plate, adding 200 mul of mixed solution into each hole of a 96-hole culture plate, removing bubbles, sequentially placing the culture plate in a refrigerator at 4 ℃ for crosslinking for 24 hours, freezing in the refrigerator at-20 ℃ for 12 hours, freezing in the refrigerator at-80 ℃ for 12 hours, and freezing and drying in a freeze dryer for 72 hours to obtain the SF/CS three-dimensional porous scaffold, and performing Co60 irradiation sterilization for later use.
The using method comprises the following steps:
cell inoculation on three-dimensional scaffold to construct in-vitro tumor model
The scaffold material was soaked in culture medium for 24h, pre-wetted and placed in 24-well plates. 100ul of cell suspension with adjusted concentration is dripped on the material, and then the culture plate is placed on a vibrator to vibrate for 5min, so that the cells are evenly inoculated in the three-dimensional scaffold material. After all three groups are placed in the same incubator for 1h, 200ul of culture medium is added to the edge, the stent material is just infiltrated, and the three groups are continuously placed in the incubator for observation.
The experimental results are as follows:
(1) from the comparison of the real images of the scaffolds, the scaffolds of the experimental group and the control group have small differences in appearance, are cylindrical (24-well cell culture plate is a mold), and are off-white. The control bracket is taken out of the mold, the edge of the control bracket is rough, and the experimental bracket is taken out of the mold, and the edge of the experimental bracket is smooth. Under the condition of applying the same pressure (adjusting to the same scale by using a vernier caliper, clamping the bracket at the same position for 5min, taking down the bracket, standing for 5min under the same environment, observing and taking a picture), the contrast group shows obvious dents, the experimental group has better elasticity, and no obvious dents appear. The experimental group and the control group are both formed by freeze-drying 200 microliter of raw material solution, and the thicknesses of the two groups of brackets are respectively measured by vernier calipers: the thickness of the experimental scaffold (6.28 +/-0.02 mm) is larger than that of the control scaffold (5.70 +/-0.01 mm) (P is less than 0.05), so that the experimental scaffold is supposed to be fluffy, namely the pore diameter is larger and the porosity is higher than that of the control scaffold.
(2) The scaffolds were cut into 3 μm slices with a paraffin slicer and observed under an optical microscope: the aperture of the cross section of the experimental group bracket is obviously larger than that of the control group bracket. After counting 1000 wells in each group by ImageJ software, the control group scaffold pore size (255 +/-85 μm) is smaller than the experimental group scaffold pore size (400 +/-158 μm) (P < 0.05).
(3) In order to compare two sets of scaffolds more finely microscopically and to overcome the disadvantage of optical microscopy that only two-dimensional structures can be observed, we observed the three-dimensional internal pore structure of the scaffolds with a scanning electron microscope. When two groups of supports are subjected to metal spraying and observed under a scanning electron microscope, the internal pore diameter of an experimental group is larger than that of a control group, and the arrangement is more regular. The pore channel wall structure is smoother, the smooth pore channel wall structure is more favorable for liquid flow, transportation of cell nutrients and oxygen and discharge of metabolic waste from the viewpoint of hydrodynamics, and all experimental group scaffolds are more suitable for cell growth and serve as in-vitro cell culture models.
(4) The porosity of the experimental group is higher than that of the control group, and the porosity of the stent of the SF/Cs/Alg (1:1:1) group in the experimental group is the highest (93.73 +/-1.12%) (the optimal ratio is 1:1: 1). Water absorption: the SF/Cs/Alg (1:1:2) group scaffolds had the highest water absorption (5261.70 + -69.95%), followed by SF/Cs/Alg (1:1:1) group scaffolds (3872.42 + -53.39%). The control SF/Cs (1:1) group scaffolds had the lowest water absorption (2473.38 + -60.00%). the SF/Cs (1:1) and SF/Cs/Alg (1:1:1) group scaffolds were statistically different in water absorption (P < 0.001). The expansion rates of the SF/Cs/Alg (1:1:2) group stents are the highest, and are followed by SF/Cs/Alg (1:1:1), andSF/Cs/Alg (1:1:0.5), SF/Cs (1:1), scaffold of the high order of the scaffold, collapsed by SF/Cs/Alg (1:1:1), and SF/Cs/Alg (1:1:0.5), SF/Cs (1:1: 1). Experimental results show that no significant statistical difference is seen in the expansion rates of the SF/Cs (1:1) group stents and SF/Cs/Alg (1:1:1) (P ═ 0.071> 0.05). Degradation rate of the SF/Cs/Alg (1:1:2) group stent is the highest, and the SF/Cs/Alg (1:1:1) group stent is the next. SF/Cs and SF/Cs/Alg (1:1:0.5) have similar degradation rates.
(5) Results of CCK8 value-added experiments: the colon cancer cell HCT-116 cells on the 3D bracket (SF/Cs/Alg1:1:1) prepared by the utility model are obviously faster than the proliferation of 2D cells.
(6) When the cells are inoculated for 3 days, no obvious difference is seen between the experimental group and the control group. On day 7 of cell inoculation, the experimental cells proliferated faster than the control, and the experimental cells were more likely to form spherical cell masses.
(7) When the cells are inoculated for 1 day, no obvious difference is seen between the experimental group and the control group. On day 3 of cell inoculation, the cells of the experimental group proliferated faster than those of the control group, the cells of the control group grew annularly, and the cells of the experimental group grew in a cake shape. On day 7 of cell inoculation, the cells of the experimental group proliferated faster than those of the control group, the cells of the control group grew in a cake shape, and the cells of the experimental group grew in a spherical shape.
(8) HCT-116 cells were inoculated into the control group scaffolds (SF/Cs (1:1)), the experimental group scaffolds (SF/Cs/Alg (1:1:1)) and the left axilla of nude mice, respectively, 14 days later, the cells on the scaffolds were fixed with 4% paraformaldehyde, the nude mice were sacrificed, subcutaneous tumor blocks were fixed with formalin, and three groups of tissues were subjected to HE staining, and the results showed that: compared with the tumor model constructed by the control group of scaffolds, the in-vitro tumor model constructed by the experimental group of scaffolds is closer to the tissue morphology of subcutaneous tumor formation of nude mice.
It should be noted that the utility model discloses composite support can independent use or use with the cooperation of conventional cell culture device, and through rational design bearing structure's structure and size, makes that it can be better compare with two-dimensional cultivation.
The 3D bracket of the utility model has moderate fluffiness, large aperture, large porosity, high water absorption and moderate degradation rate, more truly simulates the biological behavior of colon cancer cells in vivo, is more conducive to cell growth, can truly reflect the biological behavior of cancer, has a layered structure, and overcomes the defects of difficult operation, poor rigidity and easy damage of the three-dimensional bracket in the prior art; the stent body can bear normal mechanical operation in transfer and cell culture, and the porous structure of the stent is ensured not to deform and damage.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of improvements and additions can be made without departing from the principles of the present invention, and these improvements and additions should also be regarded as the protection scope of the present invention.
Claims (5)
1. A3D support for constructing an external tumor model is characterized by comprising a support body (1), a 3D support local amplification module (3), a support third layer (4), small holes (5), a support first layer (6) and a support second layer (7), wherein the 3D support local amplification module (3) is arranged in the support body (1), the small holes (5) are uniformly distributed on the support body (1), the support body (1) is of a layered structure and is respectively the support third layer (4), the support first layer (6) and the support second layer (7), the support first layer (6), the support second layer (7) and the support third layer (4) are sequentially arranged from outside to inside, and the small holes (5) penetrate through the support first layer (6), the support second layer (7) and the support third layer (4);
the thickness of the 3D support is 6.26 mm-6.30 mm, and the aperture of the small hole (5) of the 3D support is 242 mu m-558 mu m; the porosity of the 3D scaffold is 92.61% -94.85%;
the bracket body (1) is cylindrical.
2. The 3D scaffold for constructing an in vitro tumor model according to claim 1, further comprising tumor cells (2) within the 3D scaffold, the tumor cells (2) being located within the pores (5).
3. The 3D scaffold for constructing an in vitro tumor model according to claim 1, wherein the water uptake of the 3D scaffold is 3819.03% -3925.81%.
4. The 3D scaffold for constructing an in vitro tumor model according to claim 1, wherein the first layer (6) of the scaffold is a silk fibroin solution, the second layer (7) of the scaffold is a chitosan solution, and the third layer (4) of the scaffold is an alginate solution.
5. 3D scaffold for constructing an in vitro tumor model according to claim 1, characterized in that the thickness of the scaffold first layer (6), the scaffold second layer (7) and the scaffold third layer (4) are the same.
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| CN110724668B (en) * | 2019-11-20 | 2023-12-05 | 上海市第五人民医院 | 3D (three-dimensional) bracket for constructing in-vitro tumor model and preparation method and application thereof |
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