CN107988158B - Three-dimensional tumor model acellular porous scaffold, construction method and application thereof - Google Patents
Three-dimensional tumor model acellular porous scaffold, construction method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of cell biology and tumor tissue engineering materials, and provides a three-dimensional tumor model acellular porous scaffold, a construction method and application thereof. Pre-freezing the whole pig lung stent, selecting a uniform part without an obvious bronchus, cutting into blocks, adding purified water and PBS (phosphate buffer solution) to clean until no blood filaments exist, removing cells by using sodium dodecyl sulfate and TritonX-100, and obtaining the cross-linked acellular pig lung three-dimensional tumor model stent by adopting a freeze drying and chemical cross-linking method. According to the invention, the acellular matrix derived from the pig lung tissue is used as a support material for constructing a tumor model, on the basis of effectively removing cell components, the pulmonary alveolus-bronchial network of the structure is kept as much as possible, the microenvironment of the natural extracellular matrix can be simulated, and the adhesion, growth and proliferation of cells are facilitated. The tissue structure of the three-dimensional tumor model constructed in vitro after cell inoculation is closer to in vivo tissue, and the three-dimensional tumor model is suitable for the screening research of anti-cancer drugs.
Description
Technical Field
The invention belongs to the field of cell biology and tumor tissue engineering materials, and provides a three-dimensional tumor model acellular porous scaffold, a construction method and application thereof.
Background
Malignant tumor is one of the major public health problems in the world, has become a common disease and frequently encountered disease seriously harming human health, and the situation faced by China is more severe. With the continuous discovery of tumor targets and anticancer drugs, the selection of a model that truly simulates in vivo tumor tissues is an important means for screening effective anticancer drugs. Clinical trials are the most effective way to study tumors and drug screening, but safety factors and ethical issues make them not widely used. The animal transplantation tumor model is one of the current main drug screening models, can provide a microenvironment of local tissues, forms tumors closer to in-vivo tumor tissues, and has more reliable drug screening results, but has the defects of longer experimental period, higher price and animal ethics. However, due to the lack of interaction between cells and extracellular matrix in a three-dimensional environment, tumor cells are changed in form and function to some extent, and the microenvironment of in vivo tumors cannot be truly reflected, so that the interaction between cells influencing gene and protein expression and cell proliferation and differentiation is changed, and the physiological relevance is lacked. In addition, the two-dimensional culture system cannot reflect the high complexity and biological characteristics of tumor tissues, and cannot accurately predict the anticancer effect of the drug.
Solid tumors grow in vivo in a three-dimensional pattern, the process of development of which is the result of constant interactions between tumor cells and their microenvironment and body. The tumor microenvironment is an important factor affecting the biological behaviors of cell secretion, adhesion, proliferation and differentiation, invasion and metastasis. The in vitro 3D tumor model can construct a cell growth system similar to that in vivo in vitro, better provide a three-dimensional space structure for tumor cell growth and a microenvironment for simulating cell growth, and embody the interaction between tumor cells and extracellular matrix (ECM). Therefore, the three-dimensional tumor model constructed in vitro is favored by more and more researchers, and is used for the research of tumor cell biological behavior and anticancer drug screening.
At present, tissue engineering technology is gradually used for constructing an in vitro three-dimensional tumor model, and research results show that a three-dimensional structure formed by inoculating tumor cells on a support material can better simulate in vivo tumor tissues than a two-dimensional plane, and particularly simulate the interaction between cells and extracellular matrix. Most of the used stent materials are natural materials and polymer synthetic materials, the polymer synthetic materials can basically meet the requirements of the stent materials in the aspects of degradation performance and plasticity, but the biocompatibility of the polymer synthetic materials is far inferior to that of the natural materials, and the natural materials have the defects of poor mechanical properties and need to be compounded with other materials. Acellular matrix biomaterials derived from organs/tissues are gradually applied in the biomedical field because they can directly retain the natural extracellular matrix microenvironment. The method mainly utilizes a decellularization technology to remove cell components and genetic materials in tissues/organs, retains original ECM components, biological activity and mechanical properties as far as possible, has a good structural network after decellularization, and provides an ideal growth environment for cells. Wherein, the porcine lung tissue is similar to human lung in physiological structure, genomics and other aspects. The acellular pig lung matrix not only contains various proteins such as collagen, laminin, fibronectin, proteoglycan and the like, but also provides a good three-dimensional space for cell growth and proliferation by the specific alveolar and bronchial structures of the acellular pig lung matrix. The decellularized pig lung scaffold disclosed in the prior art is mainly used for constructing a tissue engineering lung and providing a donor for lung transplantation treatment, and the decellularization process of the decellularized pig lung scaffold mostly adopts a perfusion method and removes all cell components by means of venation circulation of lung tissues. This method mostly needs a peristaltic pump or the like to provide pressure to introduce the decellularization reagent, and the decellularization reagent needs a large amount. In addition, at present, the decellularized lung scaffold is mainly used for replacing damaged lung organs, and the decellularization aims at the whole lung tissue, the decellularization process and the cell reinjection process and needs to ensure complete sterility, so that the operation is lack of flexibility.
The invention takes porcine lung tissue as a research object, selects uniform area without large bronchus to be cut into blocks, directly adopts sodium dodecyl sulfate and TritonX-100 to remove cell components, and obtains a porous three-dimensional tumor model scaffold after freeze drying. On the basis, the stability and the mechanical property of the stent are improved by a chemical crosslinking method. The acellular pig lung stent obtained by the invention is used for constructing an in-vitro 3D tumor model, shows good biocompatibility, has a growth mode presented by cells on the stent closer to that of a solid tumor in a human body, and is suitable for screening and researching anticancer drugs.
Disclosure of Invention
The invention aims to provide a three-dimensional tumor model scaffold, a construction method and application thereof, wherein the method effectively retains the components and the biological activity of extracellular matrix, is simple to operate and has high scaffold acquisition rate.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for constructing a three-dimensional tumor model acellular porous scaffold takes porcine lung tissue as a main material, and adopts a freeze drying technology to prepare the porous scaffold after cells are removed, and specifically comprises the following steps:
(1) freezing the whole pig lung tissue in a refrigerator at the temperature of-20 to-30 ℃ for 3 to 12 hours, and after the pig lung tissue is fixed and formed, selecting an area without obvious bronchus observed by naked eyes to be cut into blocks, wherein the size of each pig lung block is 5 to 8cm3。
(2) Washing the cut blocks obtained in the step (1) with purified water at room temperature for 3-5 times, washing with Phosphate (PBS) buffer solution with the pH value of 7.4 for 2-4 times, and removing blood filaments in the cut blocks. Magnetic stirring is adopted for each cleaning, and the cleaning time is 0.5-1 h.
(3) And (3) adding a Sodium Dodecyl Sulfate (SDS) aqueous solution with the mass fraction of 0.1-1% into the cut blocks obtained in the step (2) at room temperature, and primarily removing cells after magnetic stirring for 12-18 h to obtain the cut blocks with partial removed cells.
(4) And (3) adding a TritonX-100 solution with the volume fraction of 0.1-0.5% into the cut blocks obtained in the step (3) at room temperature, magnetically stirring for 8-15 h, and continuously removing cells.
(5) And (3) washing the cut blocks obtained in the step (4) for 3-5 times by adopting PBS (phosphate buffer solution) with the pH value of 7.4 at room temperature, wherein the washing duration time is 0.5-1 h each time, so as to obtain the acellular cut blocks.
(6) And (4) pre-freezing the acellular blocks obtained in the step (5) at the temperature of-20 to-30 ℃ for 5 to 12 hours.
(7) And (4) cutting the pre-frozen blocks in the step (6) and freeze-drying to obtain the porous acellular pig lung scaffold. The temperature of the freeze-drying cold trap is-45 to-55 ℃, and the freeze-drying time is 12 to 24 hours.
(8) And (3) slicing and cutting the compact layer on the surface of the support obtained in the step (7), immersing the support in a cross-linking agent for chemical cross-linking for 12-24 h at room temperature, and washing for 2-4 times by adopting PBS (phosphate buffer solution) with the pH of 7.4, wherein the washing time is 1h each time. After cleaning, carrying out freeze drying for 12-24 h at-45 to-55 ℃ to obtain the cross-linked acellular pig lung matrix with stable structure.
The cross-linking agent is a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 60-70% ethanol solution of ethanesulfonic acid. Wherein the concentration of EDC and NHS in the cross-linking agent is respectively 10-50 mmol/L and the concentration of ethanesulfonic acid is 20-50 mmol/L.
The acellular porous scaffold of the three-dimensional tumor model obtained by the construction method can retain the specific alveolus and a small amount of small bronchial porous structures of pig lungs, the porosity is 88.14-94.09%, the water absorption is 181.6-280.55%, the pore diameter is 115-222 μm, and the scaffold stress is 242.24-634.23 kPa (strain is 0.7).
The three-dimensional tumor model acellular porous scaffold obtained by the construction method is used as an in-vitro 3D tumor model and is used for screening research of anti-tumor drugs. The cell-free porous scaffold of the three-dimensional tumor model maintains the original good biocompatibility, and meanwhile, the specific alveolar and bronchial porous scaffold provides a three-dimensional space for cell growth and proliferation, so that a microenvironment simulating cell growth is provided for further researching the adhesion, growth, migration and invasion of tumor cells.
The invention selects the porcine lung tissue as a bracket for constructing a tumor model in vitro, and the porcine lung tissue has special alveolus and bronchus structures, and the diameter of the alveolus is 200 mu m, so the porcine lung tissue is suitable for providing a good growth and proliferation space for cells. The porcine tissue is easy to obtain and is similar to human lung in the aspects of physiology, histology and the like, and the porcine tissue can truly simulate the extracellular matrix and the tumor growth environment of a human body. Because collagen is one of the main components of the acellular pig lung scaffold, the acellular pig lung scaffold can obviously improve the mechanical property of the scaffold after being subjected to EDC/NHS chemical crosslinking treatment so as to meet the support requirement of forming a larger cell colony by long-term culture of tumor cells.
The whole pig lung tissue is frozen and then cut into pieces, and the frozen pig lung tissue is easy to cut into tissues with similar volumes and is convenient for identifying the parts without obvious bronchus. The pig lung tissue is subjected to decellularization treatment by adopting the cut blocks instead of the whole pig lung tissue, so that the decellularization treatment in a smaller area after the cut blocks is convenient, the time is saved, and the dosage of a decellularization reagent is reduced. Meanwhile, one pig lung tissue can simultaneously obtain a plurality of stent materials, so that the identity of all detection sources is ensured. In addition, the three-dimensional tumor model constructed by inoculating the scaffold with the cells can be cultured in a normal culture dish and is easy to carry out a series of tests.
The invention has the advantages that
(1) According to the invention, the acellular matrix derived from the pig lung tissue is used as a support material for constructing a tumor model, on the basis of effectively removing cell components, the pulmonary alveolus-bronchial network of the structure is kept as much as possible, the microenvironment of the natural extracellular matrix can be simulated, and the adhesion, growth and proliferation of cells are facilitated.
(2) The acellular scaffold from the tissue source has low immunogenicity and good biocompatibility. Meanwhile, the porous decellularized pig lung scaffold is beneficial to transportation of nutrient substances and oxygen. In addition, a large amount of collagen components are reserved in the scaffold, so that mechanical support is provided, and the mechanical strength required by cell growth and proliferation is ensured.
(3) The pig lung is cut into blocks instead of the whole pig lung tissue, and the cell removing process is simple and easy to implement. Meanwhile, enough supports can be obtained by the same organization, and the number of samples required by experimental detection and the accuracy of experimental results are ensured.
(4) The tissue structure of the three-dimensional tumor model constructed in vitro after cell inoculation is closer to in vivo tissue, so the three-dimensional tumor model can be used as an important research system for screening antitumor drugs and can provide more reliable and accurate experimental results compared with two-dimensional planar culture.
Drawings
FIG. 1 is an SEM image of a decellularized porcine lung matrix of the invention (example 1) prior to crosslinking;
FIG. 2 is an SEM image of a decellularized porcine lung matrix of the invention (example 3) after cross-linking;
FIG. 3 is an inverted microscope and SEM image of the decellularized porcine lung matrix (example 3) of the invention at various times after seeding with tumor cells;
FIG. 4 is a confocal laser micrograph of cell infiltration grown on acellular porcine lung matrix (example 3) according to the present invention;
FIG. 5 is a graph of HE staining and Masson staining of cell profiles grown on acellular porcine lung matrix (example 3) of the present invention.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to the drawings and examples. The present invention is not limited to the following embodiments, and all modifications and variations based on the basic idea of the present invention are within the technical scope of the claims of the present invention without departing from the spirit of the present invention.
Example 1 preparation of acellular porcine lung three-dimensional tumor model scaffold
Freezing whole pig lung tissue in a refrigerator at-20 deg.C for 6 hr, fixing, cutting into uniform parts with size of 8cm3. The pig lung was cut into pieces and placed in a beaker, 2000mL of redistilled water was added, and after 30min of magnetic stirring, fresh distilled water was replaced, and after repeating the above operation 3 times, the pieces were washed with 2000mL of PBS having a pH of 7.4 for 2 times with magnetic stirring for 30min each. After no obvious blood color exists in the tissue, 2000mL of 1% (wt%, the same below) SDS solution is added, after magnetic stirring is carried out for 6h, the 1% SDS solution is replaced and stirring is continued for 6h, so as to remove the cells. Then 2000mL of 0.5% (v%, the same applies below) TritonX-100 solution was added and stirred for 10 hours to completely remove the cells. Subsequently, 2000ml pbs wash was added 3 times, each for 1h, to completely wash the decellularized reagent. Placing the decellularized pig lung blocks in a refrigerator at the temperature of-20 ℃ for pre-freezing for 6h, and then placing the pig lung blocks in a freeze drier for drying at the temperature of-50 ℃ for 18h to obtain the porous decellularized pig lung scaffold.
Example 2 preparation of three-dimensional tumor model scaffold of decellularized pig Lung
The whole pig lungFreezing the tissue in a refrigerator at-30 deg.C for 3 hr, fixing, and cutting into 6cm pieces3. The pig lung was cut into pieces and placed in a beaker, 1000mL of redistilled water was added, the distilled water was replaced after magnetic stirring for 40min, and after 5 times of the above operation, the pieces were washed 3 times with 1500mL of PBS having a pH of 7.4 for 30min each. After the tissue has no obvious blood color, 1500mL of 0.5% SDS solution is added, magnetic stirring is carried out for 6 hours, and then the new 0.5% SDS solution is replaced and stirring is continued for 12 hours to remove cells. Then 2000mL of 0.3% TritonX-100 solution is added and stirred for 12h, and cells are thoroughly removed. Subsequently, 1500ml pbs was added for washing 3 times, each for 1h, to completely wash the decellularized reagent. Placing the decellularized pig lung blocks in a refrigerator at the temperature of-20 ℃ for pre-freezing for 12h, and then placing the pig lung blocks in a freeze dryer for drying for 24h at the temperature of-48 ℃ to obtain the porous decellularized pig lung scaffold.
Example 3 Cross-linking and Performance testing of three-dimensional tumor model scaffolds from decellularized porcine Lung
Freezing whole pig lung tissue in a refrigerator at-20 deg.C for 6 hr, fixing, cutting into uniform parts with size of 8cm3. The pig lung was cut into pieces and placed in a beaker, 2000mL of redistilled water was added, and after 30min of magnetic stirring, fresh distilled water was replaced, and after repeating the above operation 3 times, the pieces were washed with 2000mL of PBS having a pH of 7.4 for 2 times with magnetic stirring for 30min each. After the tissue has no obvious blood color, 2000mL of 1% SDS solution is added, after magnetic stirring is carried out for 6h, the 1% SDS solution is replaced by a new one, and stirring is continued for 6h, so as to remove cells. Then 2000mL of 0.5% TritonX-100 solution is added and stirred for 10h, and cells are thoroughly removed. Subsequently, 2000ml pbs wash was added 3 times, each for 1h, to completely wash the decellularized reagent. Placing the decellularized pig lung blocks in a refrigerator at the temperature of-20 ℃ for pre-freezing for 6h, and then placing the pig lung blocks in a freeze drier for drying at the temperature of-50 ℃ for 18h to obtain the porous decellularized pig lung scaffold.
Slicing and cutting off a compact layer on the surface of the pig lung scaffold material, soaking the pig lung scaffold material in 500mL of a cross-linking agent (the cross-linking agent is a mixed solution of 50 mmol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 50mmol/L N-hydroxysuccinimide and 50mmol/L ethanesulfonic acid in 60% ethanol solution, wherein the ethanesulfonic acid is used as a buffer solution), crosslinking at normal temperature for 24h, washing for 2 times at 2000mLPBS (magnetic stirring) each time for 1h, and finishing the crosslinking of the decellularized pig lung scaffold. Then, pre-freezing for 6h at the same temperature of-20 ℃, and then drying and freeze-drying for 18h at the temperature of-50 ℃ in a freeze dryer to obtain the cross-linked porous porcine lung three-dimensional tumor model scaffold.
Fig. 1 and 2 are SEM images of the prepared acellular porcine lung stent before and after cross-linking, from which it can be seen that the porous structure of the prepared three-dimensional tumor model stent can clearly see the structures of alveoli and a small number of small bronchi, the stent before cross-linking (example 1) is loose and irregular, and the stent after cross-linking (example 3) has a more compact pore structure, and the SEM result shows that the pore diameter of the stent after cross-linking is 166.98 ± 26.03 μm. The porous structure of the bracket can promote cell adhesion and better meet the growth requirement of tumor cells. In the strain range of 0-0.7, crosslinking significantly improved the elastic modulus of the scaffold, especially when the strain at maximum was 0.7 (70%), the uncrosslinked porcine lung scaffold stress was 242.24 + -22.63 kPa, and the post-crosslinking stress was 634.23 + -12.05 kPa. The number of hydrophilic groups on molecular chains is reduced by crosslinking, the molecular chains are tighter, and the water absorption of the scaffold is remarkably reduced from 280.55 +/-15.65% to 181.6 +/-17.46%. At the same time, crosslinking reduced the porosity of the scaffold (88.14 ± 3.21%), but there was no significant difference between the two compared to that before crosslinking (94.09 ± 4.62%).
Example 4 Cross-linking of decellularized porcine lung three-dimensional tumor model scaffolds
Freezing whole pig lung tissue in-30 deg.C refrigerator for 3 hr, fixing, cutting to obtain uniform part with size of 6cm3. The pig lung was cut into pieces and placed in a beaker, 1000mL of redistilled water was added, the distilled water was replaced after magnetic stirring for 40min, and after 5 times of the above operation, the pieces were washed 3 times with 1500mL of PBS having a pH of 7.4 for 30min each. After the tissue has no obvious blood color, 1500mL of 0.5% SDS solution is added, magnetic stirring is carried out for 6 hours, and then the new 0.5% SDS solution is replaced and stirring is continued for 12 hours to remove cells. Then 2000mL of 0.3% TritonX-100 solution is added and stirred for 12h, and cells are thoroughly removed. Subsequently, 150 is added0ml PBS wash 3 times, each time for 1h, to completely wash the decellularized reagent. Placing the decellularized pig lung blocks in a refrigerator at the temperature of-20 ℃ for pre-freezing for 12h, and then placing the pig lung blocks in a freeze dryer for drying for 24h at the temperature of-48 ℃ to obtain the porous decellularized pig lung scaffold.
Slicing and cutting off a compact layer on the surface of the pig lung scaffold material, soaking the pig lung scaffold material in 800mL of a cross-linking agent (the cross-linking agent is a mixed solution of 50 mmol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 50mmol/L N-hydroxysuccinimide and 20mmol/L ethanesulfonic acid in 65% ethanol solution, wherein the ethanesulfonic acid is used as a buffer solution), crosslinking at normal temperature for 12h, washing for 3 times at 2000mLPBS (magnetic stirring) for 1h each time, and finishing the crosslinking of the decellularized pig lung scaffold. Then, pre-freezing for 12h at the same temperature of-20 ℃, and placing in a freeze dryer for drying for 24h at the temperature of-48 ℃ to obtain the cross-linked porous porcine lung three-dimensional tumor model scaffold.
Example 5 decellularized porcine lung scaffolds (example 3) as tumor models
Soaking the cross-linked acellular pig lung scaffold in 75% alcohol for 3 times, soaking in PBS and DMEM medium, air drying in a super clean bench, performing ultraviolet sterilization, inoculating MCF-7 cells, and continuously culturing for 21 days. Scaffolds were removed at different time points for 1 day, 3 days, 7 days, 14 days and 21 days of culture. A part of the scaffold is directly placed under an inverted microscope to observe the growth of the cells. The other fraction was fixed in 2.5% glutaraldehyde for 3h, then washed twice with phosphate buffer pH7.4, and dehydrated with gradient alcohol (50%, 70%, 90%, 100%, 100%) for 30min per stage. After drying at normal temperature, spraying gold on the surface, placing under a scanning electron microscope, and observing the growth and proliferation conditions of cells on the acellular pig lung stent by selecting proper magnification. In addition, the tumor model cultured for 3 days was subjected to laser confocal multilayer scanning to examine the penetration of cells in the scaffold. After the tumor model section cultured for 14 days is processed, HE and Masson staining is carried out, and the distribution condition of cells in the bracket is examined.
FIG. 3 is an inverted microscope and SEM image of the acellular porcine lung matrix at different time points after inoculation of tumor cells. As can be seen from the figure, after 1 day of culture, the cells grew as a monolayer and the cells adhered tightly to the scaffold. After 3 days of culture, small colonies of cells appeared or layered on the surface of the scaffold. Along with the prolonging of the culture time, the cell proliferation is obvious, the cell colony is gradually increased, the pores of the bracket are completely filled, and the light transmittance of the bracket is reduced. The SEM can more obviously see the cell proliferation process from monolayer to multilayer, the formation of large cell colonies and the morphology and structure of the scaffold per se can not be clearly seen on the surface of the scaffold due to the cell confluency. The cell morphology also changed from the initial extended state to globular growth, and in particular after 3 days of culture, cells began to grow in a string-like aggregate, whereas cells grew in a monolayer adherent manner under two-dimensional culture conditions. Along with the prolonging of the culture time, the number of the cell balls formed by the cell aggregation on the three-dimensional tumor model support is increased, and the cell balls can further generate the aggregation phenomenon to form a larger cell colony, which shows that the cell growth on the acellular pig lung tumor model support is closer to the growth of the solid tumor in a human body.
FIG. 4 is a confocal laser micrograph of cell infiltration grown on acellular porcine lung matrix. After the tumor model is constructed for 3 days, the distribution of cells on the surface of the composite scaffold and below the surface of the composite scaffold and at a depth of 200 mu m in the scaffold is investigated by adopting laser confocal microscope multilayer scanning. The cells can be seen to permeate in different degrees in each layer, are distributed in the pore walls and pores of the scaffold and grow well, and the form of the acellular pig lung scaffold can be clearly seen. In addition, 3D images of the stacks after multi-layer scanning are presented, and the three-dimensional distribution of the cells in the scaffold can be clearly seen.
FIG. 5 is a graph of HE and Masson staining of cell profiles grown on acellular porcine lung matrix. As can be seen from the figure, the distribution of the cells in the decellularized pig lung scaffold is not uniform, the cells are distributed and concentrated in the alveolar-bronchial dense area, and larger cell colonies are formed at the edge of the scaffold due to easy contact with nutrients. Meanwhile, the Masson staining result also indicates that the main component of the acellular pig lung matrix is collagen and is intensively distributed in the bronchial region.
Claims (7)
1. A construction method of a three-dimensional tumor model acellular porous scaffold is characterized by comprising the following steps:
(1) freezing the whole pig lung tissue in a refrigerator at the temperature of-20 to-30 ℃ for 3 to 12 hours, and selecting an area without obvious bronchus observed by naked eyes to be cut into blocks after the pig lung tissue is fixed and formed;
(2) cleaning the cut blocks obtained in the step (1) by using purified water at room temperature, and cleaning by using a phosphate buffer solution to remove blood filaments in the cut blocks;
(3) placing the cut blocks obtained in the step (2) in a sodium dodecyl sulfate aqueous solution with the mass fraction of 0.1-1% at room temperature, and performing magnetic stirring for 12-18 h to primarily remove cells to obtain partially cell-removed cut blocks;
(4) placing the cut blocks obtained in the step (3) in TritonX-100 solution with the volume fraction of 0.1-0.5% at room temperature, magnetically stirring for 8-15 h, and continuously removing cells;
(5) washing the cut blocks obtained in the step (4) by adopting a phosphate buffer solution at room temperature to obtain acellular cut blocks;
(6) pre-freezing the acellular blocks obtained in the step (5) at-20 to-30 ℃ for 5 to 12 hours;
(7) cutting the pre-frozen blocks obtained in the step (6) and freeze-drying to obtain a porous acellular pig lung scaffold; the temperature of the freeze-drying cold trap is-45 to-55 ℃, and the freeze-drying time is 12 to 24 hours;
(8) slicing and cutting the compact layer on the surface of the support obtained in the step (7), immersing the support in a cross-linking agent for chemical cross-linking for 12-24 h at room temperature, washing the support by adopting a phosphate buffer solution, and freeze-drying the support for 12-24 h at-45 to-55 ℃ to obtain a cross-linked acellular pig lung matrix with a stable structure; the cross-linking agent is a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 60-70% ethanol solution of ethanesulfonic acid, wherein the ethanesulfonic acid is used as a buffer solution;
the concentrations of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the cross-linking agent are both 10-50 mmol/L; the concentration of the ethanesulfonic acid is 20-50 mmol/L.
2. The method of claim 1, wherein the phosphate buffer has a pH of 7.4.
3. The method for constructing the acellular porous scaffold for the three-dimensional tumor model according to claim 1 or 2, wherein in the step (5), the phosphate buffer solution is used for washing the cut pieces 3-5 times, and the washing duration is 0.5-1 h.
4. The method for constructing the acellular porous scaffold for the three-dimensional tumor model according to claim 1 or 2, wherein the number of times of washing with the phosphate buffer solution after crosslinking in the step (8) is 2-4, and the washing time is 1 hour each time.
5. The method for constructing the acellular porous scaffold for the three-dimensional tumor model according to claim 3, wherein the number of times of washing with the phosphate buffer solution after crosslinking in the step (8) is 2-4, and the washing time is 1 hour each time.
6. The three-dimensional tumor model acellular porous scaffold obtained by the construction method of any one of claims 1-5 is characterized in that the three-dimensional tumor model acellular porous scaffold can retain the specific alveolar and small bronchial porous structure of a pig lung, the porosity is 88.14-94.09%, the water absorption is 181.6-280.55%, the pore diameter is 115-222 μm, and the scaffold stress is 242.24-634.23 kPa.
7. The use of the three-dimensional tumor model acellular porous scaffold of claim 6 in an in vitro 3D tumor model for screening research of antitumor drugs.
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| CN109671346B (en) * | 2019-01-17 | 2021-11-19 | 南方科技大学 | Liver model and preparation method and application thereof |
| CN110894492A (en) * | 2019-12-17 | 2020-03-20 | 南通大学附属医院 | Pancreatic cancer in-vitro 3D model construction method based on pancreatic acellular scaffold |
| CN111349598B (en) * | 2020-03-30 | 2023-05-23 | 福州大学 | Decellularized scaffold template for simulating liver cancer environment |
| CN111996168B (en) * | 2020-05-18 | 2023-03-24 | 东华大学 | Construction method and application of in-vitro three-dimensional tumor cell drug-resistant model |
| CN111849864A (en) * | 2020-07-02 | 2020-10-30 | 大连理工大学 | Construction method and application of three-dimensional tumor model acellular derivative matrix scaffold |
| CN113278587B (en) * | 2021-04-29 | 2022-08-30 | 潍坊医学院 | Three-dimensional engineered breast cancer lung metastasis model, construction method and application |
| CN113293134A (en) * | 2021-04-29 | 2021-08-24 | 潍坊医学院 | Three-dimensional lung cancer model support, preparation method and application |
| CN114457018A (en) * | 2022-02-17 | 2022-05-10 | 安徽骆华生物科技有限公司 | Three-dimensional breast cancer organoid model and culture method and application thereof |
| CN116656596A (en) * | 2023-06-08 | 2023-08-29 | 北京科昕恒业生物科技有限公司 | Method for establishing in-vitro lung three-dimensional model based on acellular matrix |
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