WO2019100454A1 - 一种三维肿瘤模型脱细胞多孔支架、构建方法及其应用 - Google Patents

一种三维肿瘤模型脱细胞多孔支架、构建方法及其应用 Download PDF

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WO2019100454A1
WO2019100454A1 PCT/CN2017/115478 CN2017115478W WO2019100454A1 WO 2019100454 A1 WO2019100454 A1 WO 2019100454A1 CN 2017115478 W CN2017115478 W CN 2017115478W WO 2019100454 A1 WO2019100454 A1 WO 2019100454A1
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decellularized
tumor model
scaffold
dimensional tumor
cells
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PCT/CN2017/115478
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French (fr)
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宋克东
李文芳
李丽颖
胡雪岩
刘天庆
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大连理工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

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  • the invention belongs to the field of cell biology and tumor tissue engineering materials, and provides a three-dimensional tumor model decellularized porous stent, a construction method and application thereof.
  • the tumor microenvironment is an important factor affecting the biological behaviors such as 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, providing a three-dimensional structure of tumor cell growth and a microenvironment simulating cell growth, reflecting the interaction between tumor cells and extracellular matrix (ECM). . Therefore, the three-dimensional tumor model constructed in vitro has been favored by more and more researchers for the study of biological behavior of tumor cells and screening of anticancer drugs.
  • tissue engineering technology has been gradually used to construct in vitro three-dimensional tumor models.
  • the results show that the three-dimensional structure formed by inoculation of tumor cells into scaffolds can simulate tumor tissues in vivo better than two-dimensional planes, especially between cells and cells. Interaction between outer matrices.
  • the scaffold materials used are mostly natural materials and polymer synthetic materials.
  • the polymer synthetic materials can basically meet the requirements of scaffold materials in terms of degradation performance and plasticity, but their biocompatibility is far less than natural materials, and the drawbacks of natural materials are mainly Poor mechanical properties, and more need to be combined with other materials.
  • Organ/tissue-derived acellular matrix biomaterials are gradually being used in the biomedical field due to their ability to directly retain the natural extracellular matrix microenvironment.
  • porcine lung tissue is similar to human lung in terms of physiological structure and genomics.
  • the acellular porcine lung matrix contains not only a variety of proteins such as collagen, laminin, fibronectin and proteoglycan, but also its unique alveolar and bronchial structures provide a good three-dimensional space for cell growth and proliferation.
  • the acellular porcine lung scaffolds disclosed in the prior art are mostly used for constructing tissue engineered lungs, providing donors for lung transplantation treatment, and most of the decellularization processes are performed by perfusion method, and all cellular components are cleared by the venous circulation of lung tissues.
  • This method requires a peristaltic pump or the like to provide pressure to introduce a decellularizing reagent, and a large amount of decellularizing reagent is required.
  • the current decellularized lung scaffold is mainly used to replace the damaged lung organ. Decellularization is required to ensure complete sterility and lack of flexibility in the whole lung tissue, decellularization process and reinjection of the cell.
  • the invention adopts porcine lung tissue as the research object, selects a region with uniform and no bronchus, and directly removes cell components by using sodium dodecyl sulfate and TritonX-100, and freezes and obtains a porous three-dimensional tumor model stent. .
  • the chemical cross-linking method is used to improve the stability and mechanical properties of the stent.
  • the acellular porcine lung scaffold obtained by the invention is used for the construction of a 3D tumor model in vitro, and exhibits good biocompatibility. The growth pattern exhibited by the cells on the scaffold is closer to the solid tumor in the human body, and is suitable for screening of anticancer drugs. .
  • the object of the present invention is to provide a three-dimensional tumor model scaffold, a construction method and an application thereof, which effectively retain the composition and biological activity of the extracellular matrix, and the operation is simple and the scaffold acquisition rate is high.
  • a three-dimensional tumor model destructuring porous scaffold construction method the porcine-derived lung tissue as the main material, after the cells are removed, the porous scaffold is prepared by freeze-drying technology, specifically including the following steps:
  • the whole pig lung tissue was frozen in the refrigerator at -20 ⁇ -30 °C for 3 ⁇ 12h. After it was fixed and formed, the area with no obvious bronchus was observed by naked eyes. The size of the pig lung cut was 5 ⁇ 8cm 3 .
  • PBS phosphate
  • Magnetic cleaning is used for each cleaning, and the cleaning time is 0.5 ⁇ 1h.
  • step (3) Adding a sodium dodecyl sulfate (SDS) solution with a mass fraction of 0.1 to 1% in the dicing obtained in the step (2) at room temperature, after the magnetic stirring time is 12 to 18 hours, preliminary removal The cells are partially diced to remove the cells.
  • SDS sodium dodecyl sulfate
  • the dicing obtained in the step (4) is washed 3 to 5 times with PBS buffer having a pH of 7.4 at room temperature, and the duration of each washing is 0.5 to 1 h to obtain a decellularized dicing.
  • the acellular dicing obtained in the step (5) is pre-frozen at -20 to -30 ° C for 5 to 12 hours.
  • the step (6) is pre-frozen and then diced and freeze-dried to obtain a porous acellular porcine lung scaffold.
  • the freeze-dried cold trap temperature is -45 to -55 ° C, and the freeze-drying time is 12 to 24 h.
  • the stent After slicing the dense surface of the stent surface obtained in step (7), the stent is immersed in the cross-linking agent for chemical cross-linking for 12-24 hours at room temperature, and then washed 2 to 4 times with PBS buffer pH 7.4. , each cleaning time 1h. After washing, it was freeze-dried at -45 ⁇ -55 °C for 12-24 hours to obtain a cross-linked, structurally stable acellular porcine lung matrix.
  • the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), sulfonic acid 60 A mixed solution of ⁇ 70% ethanol solution. Among them, ethanesulfonic acid was used as a buffer. The concentrations of EDC and NHS in the cross-linking agent were 10-50 mmol/L, respectively, and the concentration of ethanesulfonic acid was 20-50 mmol/L.
  • the three-dimensional tumor model decellularized porous scaffold obtained by the above construction method can retain the pulmonary alveolar and a small amount of small bronchial porous structure, the porosity is between 88.14% and 94.09%, and the water absorption rate is between 181.6% and 280.55%.
  • the three-dimensional tumor model decellularized porous scaffold obtained by the above construction method is used as an in vitro 3D tumor model for screening research of antitumor drugs.
  • the three-dimensional tumor model decellularized porous scaffold maintains the original good biocompatibility, while its unique alveolar and bronchial porous scaffold provides three-dimensional space for cell growth and proliferation, providing simulation cells for further study of tumor cell adhesion, growth, migration and invasion.
  • the invention selects porcine lung tissue as a scaffold for constructing a tumor model in vitro, and the lung lung tissue has a special alveolar and bronchial structure, and the alveolar diameter is ⁇ 200 ⁇ m, which is suitable for providing a good growth and proliferation space for the cells.
  • Porcine-derived tissues are easily accessible and are similar to human lungs in terms of physiology and histology, and can truly simulate the extracellular matrix and tumor growth environment of the human body. Since collagen is one of the main components of the acellular porcine lung scaffold, the decellularized porcine lung scaffold of the present invention can significantly improve the mechanical properties of the scaffold after being chemically cross-linked by EDC/NHS to meet the long-term culture of tumor cells to form large cell colonies. Support needs.
  • the whole lung tissue of the pig is first frozen and then cut into pieces, and the tissue of similar volume is easily cut out after freezing, and the part without obvious bronchus is easily recognized.
  • Pig lung tissue is treated with dicing instead of whole pig lung tissue decellularization, which facilitates small area decellularization after dicing and saves time and reduces the amount of decellularization reagent.
  • a pig lung tissue can acquire multiple scaffold materials at the same time to ensure the identity of all detection sources.
  • the three-dimensional tumor model constructed by the scaffold inoculated cells can be cultured in a normal culture dish and easily subjected to a series of tests.
  • the invention adopts the acellular matrix derived from pig lung tissue as a scaffold material for constructing a tumor model, and preserves the alveolar-bronchial structure choroid as much as possible on the basis of effectively removing the cellular components, and can simulate the natural extracellular matrix microenvironment. For cell adhesion, growth and proliferation.
  • Tissue-derived decellularized scaffolds have low immunogenicity and good biocompatibility.
  • porous acellular porcine lung scaffolds facilitate the transport of nutrients and oxygen.
  • a large amount of collagen is retained in the stent to provide mechanical support to ensure the mechanical strength required for cell growth and proliferation.
  • the pig cell lung block is used instead of the whole pig lung tissue, and the decellularization process is simple and easy. At the same time, the same tissue can acquire enough scaffolds to ensure the number of samples required for experimental testing and the accuracy of the experimental results.
  • the three-dimensional tumor model constructed in vitro after inoculation of cells is closer to the body tissue, so it can be used as an important research system for anti-tumor drug screening, which will provide more reliable and accurate experimental results than two-dimensional planar culture.
  • Figure 1 is a SEM image of the acellular porcine lung matrix (Example 1) prior to cross-linking of the present invention
  • Figure 2 is a SEM image of the acellular porcine lung matrix (Example 3) after cross-linking;
  • Figure 3 is an inverted microscope and SEM image of the acellular porcine lung matrix (Example 3) of the present invention at different times after inoculation of tumor cells;
  • Figure 4 is a laser confocal micrograph of cell permeation grown on the acellular porcine lung matrix (Example 3) of the present invention.
  • Figure 5 is a HE staining and Masson staining of the distribution of cells grown on the acellular porcine lung matrix (Example 3) of the present invention.
  • the whole pig lung tissue was frozen in a refrigerator at -20 °C for 6 h. After it was fixed and formed, the naked eye was observed without obvious bronchial uniformity, and the size of the cut piece was 8 cm 3 .
  • the whole lung tissue of the pig was frozen in a refrigerator at -30 ° C for 3 h. After it was fixed and formed, the naked eye was observed without obvious bronchial uniformity, and the size of the cut piece was 6 cm 3 .
  • the decellularized pig lung diced block was pre-frozen in a refrigerator at -20 ° C for 12 h, and then dried in a freeze dryer at -48 ° C for 24 h to obtain a porous acellular porcine lung scaffold.
  • the whole pig lung tissue was frozen in a refrigerator at -20 °C for 6 h. After it was fixed and formed, the naked eye was observed without obvious bronchial uniformity, and the size of the cut piece was 8 cm 3 .
  • Place the pig lung cut into a beaker, then add 2000 mL of double distilled water, magnetically stir for 30 min, then replace with new distilled water. After repeating the above three times, wash with 2000 mL, pH 7.4 PBS for 2 times, each time magnetic stirring. 30min. After no obvious blood color in the tissue, 2000 mL of 1% SDS solution was added, and after magnetic stirring for 6 hours, the new 1% SDS solution was replaced and stirring was continued for 6 hours to remove the cells.
  • the decellularized pig lung diced block was pre-frozen for 6 h in a refrigerator at -20 ° C, and then dried in a freeze dryer at -50 ° C for 18 h to obtain a porous acellular porcine lung scaffold.
  • cross-linking agent was 50 mmol/L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt a mixed solution of acid salt, 50 mmol/L N-hydroxysuccinimide, 50 mmol/L ethanesulfonic acid in 60% ethanol solution, in which ethanesulfonic acid is used as a buffer
  • cross-linking at room temperature for 24 hours and washing twice with 2000 mL of PBS.
  • Cross-linking of the acellular porcine lung scaffold can be completed by magnetic stirring for 1 h each time. Subsequently, after pre-freezing for 6 h at -20 ° C, the mixture was freeze-dried at -50 ° C for 18 h in a freeze dryer to obtain a cross-linked porous pig lung three-dimensional tumor model scaffold.
  • FIGS. 1 and Figure 2 are SEM images of the prepared acellular porcine lung scaffolds before and after cross-linking. It can be seen from the figure that the porous structure of the prepared three-dimensional tumor model scaffold can clearly see alveolar and a small amount of small bronchial structures, cross-linking.
  • the former (Example 1) stent was loose and irregular, and the cross-linking structure of the stent after cross-linking (Example 3) was more compact and compact.
  • the pore size of the stent after cross-linking was 166.98 ⁇ 26.03 ⁇ m.
  • the porous structure of the scaffold promotes cell adhesion and better meets the needs of tumor cell growth.
  • cross-linking significantly increased the elastic modulus of the stent, especially when the maximum strain was 0.7 (70%), the uncrosslinked pig lung stent stress was 242.24 ⁇ 22.63 kPa, and cross-linking The post stress becomes 634.23 ⁇ 12.05 kPa.
  • Cross-linking reduced the number of hydrophilic groups on the molecular chain, and the molecular chains were more tight.
  • the water absorption of the scaffold decreased significantly from 280.55 ⁇ 15.65% to 181.6 ⁇ 17.46%.
  • cross-linking reduced the porosity of the stent (88.14 ⁇ 3.21%), but there was no significant difference between the two before the cross-linking (94.09 ⁇ 4.62%).
  • the whole lung tissue of the pig was frozen in a refrigerator at -30 ° C for 3 h. After it was fixed and formed, the naked eye was observed without obvious bronchial uniformity, and the size of the cut piece was 6 cm 3 .
  • the decellularized pig lung diced block was pre-frozen in a refrigerator at -20 ° C for 12 h, and then dried in a freeze dryer at -48 ° C for 24 h to obtain a porous acellular porcine lung scaffold.
  • cross-linking agent was 50 mmol/L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt a mixed solution of acid salt, 50 mmol/L N-hydroxysuccinimide, 20 mmol/L ethanesulfonic acid in 65% ethanol solution, in which ethanesulfonic acid was used as a buffer
  • cross-linking of the acellular porcine lung scaffold can be completed by magnetic stirring for 1 h each time. Subsequently, it was pre-frozen at -20 ° C for 12 h, and dried in a freeze dryer at -48 ° C for 24 h to obtain a cross-linked porous porcine lung three-dimensional tumor model scaffold.
  • Example 5 Acellular porcine lung scaffold (Example 3 As a tumor model
  • the acellular porcine lung scaffold was soaked in 75% alcohol for 3 times, then immersed in PBS and infiltrated in DMEM medium. After ultra-cleaning in the platform, the cells were inoculated with MCF-7 cells and cultured for 21 days. The stents were taken at different time points of culture for 1 day, 3 days, 7 days, 14 days, and 21 days. A portion of the scaffold was placed directly under an inverted microscope to observe cell growth. The other part of the scaffold was fixed in 2.5% glutaraldehyde for 3 h, then washed twice with phosphate buffer of pH 7.4, dehydrated with gradient alcohol (50%, 70%, 90%, 100%, 100%), dehydrated at each stage. 30min.
  • the surface was sprayed with gold and placed under a scanning electron microscope. The appropriate magnification was selected to observe the growth and proliferation of cells on the acellular porcine lung scaffold.
  • the tumor model cultured for 3 days was examined by laser confocal multilayer scanning to investigate the penetration of cells in the scaffold. After 14 days of tumor model slicing, HE and Masson staining were performed to investigate the distribution of cells in the scaffold.
  • Figure 3 is an inverted microscope and SEM image at different time points after inoculation of tumor cells from acellular lung matrix.
  • the cells grew in a single layer, and the cells adhered tightly to the scaffold.
  • small cell colonies appeared or layered on the surface of the scaffold.
  • the cell proliferation was obvious, the cell colonies gradually increased, the scaffold pores were completely filled, and the light transmittance of the scaffold decreased.
  • SEM can more clearly see the cell proliferation process, from single layer to multi-layer, and the formation of large cell colonies, the surface of the stent due to the confluence of cells, can not clearly see the shape and structure of the stent itself.
  • the cell morphology also grew from the initial stretched state to the globular growth. Especially after 3 days of culture, the cells began to show cluster growth, while the cells grew in a single layer under two-dimensional culture conditions. As the culture time prolonged, the number of cells aggregated on the three-dimensional tumor model scaffold increased, and the cell ball further agglomerated to form larger cell colonies, indicating cell growth and human body in the acellular porcine lung tumor model scaffold. Solid tumors are closer.
  • Figure 4 is a laser confocal micrograph of cell permeation grown on acellular porcine lung matrix. Three days after the tumor model was constructed, the distribution of the cells on the surface of the composite scaffold and the depth of 200 ⁇ m below the scaffold was examined by laser confocal microscopy. It can be seen that the cells have different degrees of penetration in all layers, the cells are distributed in the pore walls and pores of the scaffold, and the growth is good, and the morphology of the acellular porcine lung scaffold can also be clearly seen. In addition, a 3D map of stacked layers after multi-layer scanning is presented, and the stereoscopic distribution of cells in the stent can be clearly seen.
  • Figure 5 is a HE and Masson staining of the distribution of cells grown on the acellular porcine lung matrix. It can be seen from the figure that the distribution of cells in the acellular porcine lung scaffold is not uniform, the cells are concentrated in the alveolar-bronchial dense area, and large cell colonies are formed at the edge of the scaffold due to easy access to nutrients. At the same time, the Masson staining results also indicated that the main component of the acellular lung matrix was collagen, which was concentrated in the bronchial site.

Abstract

一种三维肿瘤模型脱细胞多孔支架、构建方法及其应用。将整个猪肺支架预冻，选取无明显支气管且均匀部位切块，加入纯净水及PBS清洗至无血丝，用十二烷基磺酸钠及TritonX-100去除细胞，采用冷冻干燥、化学交联法获得交联的脱细胞猪肺三维肿瘤模型支架。采用猪肺组织来源的脱细胞基质作为构建肿瘤模型的支架材料，在有效去除细胞成分的基础上，尽可能地保留肺泡-支气管结构脉络网，可模拟天然胞外基质微环境，利于细胞黏附、生长和增殖。经接种细胞后体外构建的三维肿瘤模型，其组织结构更接近在体组织，适用于抗癌药物的筛选研究。

Description

一种三维肿瘤模型脱细胞多孔支架、构建方法及其应用 技术领域
本发明属于细胞生物学及肿瘤组织工程材料领域，提供一种三维肿瘤模型脱细胞多孔支架、构建方法及其应用。
 背景技术
恶性肿瘤作为全球较大的公共卫生问题之一，已成为一种严重危害人类健康的常见病、多发病，我国面临的形势也越发严峻。随着肿瘤靶点及抗癌药物的不断发现，选择真实模拟体内肿瘤组织的模型是筛选有效抗癌药物的重要手段。临床试验是研究肿瘤及药物筛选最有效的途径，但安全因素及伦理问题使其得不到广泛应用。动物移植性肿瘤模型是当前主要的药筛模型之一，其能提供局部组织的微环境，形成的肿瘤更接近在体肿瘤组织，药物筛选结果更可靠，但其弊端是实验周期较长、价格较高，且存在动物伦理问题。传统细胞二维培养模型仍是目前大多数肿瘤研究的主要方法，然而由于缺乏三维环境下细胞及胞外基质之间的相互作用，肿瘤细胞在形态及功能方面都发生了一定改变，不能真实地反映体内肿瘤的微环境，使得影响基因和蛋白表达及细胞增殖分化的细胞间相互作用发生改变，缺乏生理相关性。另外，二维培养体系无法反映肿瘤组织的高度复杂性和生物学特性，不能准确预测药物的抗癌作用。
实体肿瘤在体内以三维模式生长，其发生发展过程是肿瘤细胞与其微环境及机体之间不断相互作用的结果。肿瘤微环境是影响细胞分泌、粘附、增殖分化、侵袭和转移等生物学行为的重要因素。体外3D肿瘤模型能够在体外构建与体内相近的细胞生长系统，较好地提供肿瘤细胞生长的三维空间结构及模拟细胞生长的微环境，体现肿瘤细胞与细胞外基质（ECM）之间的相互作用。因此，体外构建的三维肿瘤模型受到了越来越多研究者的青睐，用于肿瘤细胞生物学行为及抗癌药物筛选的研究。
目前，组织工程技术已逐步用于构建体外三维肿瘤模型，研究结果表明肿瘤细胞接种于支架材料后形成的三维结构能够比二维平面更好模拟体内肿瘤组织，特别是模拟细胞间及细胞-胞外基质间相互作用。其中使用的支架材料多为天然材料和高分子合成材料，高分子合成材料在降解性能及可塑性方面基本能满足支架材料的要求，但是其生物相容性远不如天然材料，而天然材料弊端主要在于机械性能差，多需要与其他材料复合。器官/组织来源的脱细胞基质生物材料因能直接保留天然胞外基质微环境被逐渐应用在生物医学领域。其主要是利用脱细胞技术去除组织/器官中的细胞成分和遗传物质，尽可能的保留原ECM成分、生物活性及机械性能，去细胞后拥有良好的结构脉络网，为细胞提供了理想的生长环境。其中，猪源性肺组织在生理结构及基因组学等方面与人体肺相似。脱细胞猪肺基质不仅包含多种蛋白如胶原、层粘连蛋白、纤维连接蛋白及蛋白多糖等，且其特有的肺泡及支气管结构为细胞生长及增殖提供了良好的三维空间。现有技术公布的脱细胞猪肺支架多用于构建组织工程肺，为肺移植治疗提供供体，且其去细胞化过程大都采用灌注法，借助肺组织的脉络循环清除所有的细胞成分。这种方法多需要蠕动泵等提供压力导入去细胞试剂，并且脱细胞试剂需求量大。另外，目前脱细胞肺支架主要用于替代损伤肺器官，脱细胞针对整个肺组织、脱细胞过程及再次注入细胞过程都需要保证完全无菌，操作缺乏灵活性。
本发明以猪源性肺脏组织为研究对象，选取均匀且无大支气管的区域切块，直接采用十二烷基磺酸钠及TritonX-100 去除细胞成分，冷冻干燥后得到多孔的三维肿瘤模型支架。在此基础上，通过化学交联法以改善支架的稳定性和机械性能。本发明获得的脱细胞猪肺支架用于体外3D肿瘤模型的构建，表现出良好的生物相容性，支架上细胞呈现的生长模式与人体内实体瘤更加接近，适用于抗癌药物的筛选研究。
 技术问题
本发明的目的是提供一种三维肿瘤模型支架、构建方法及其应用，该方法有效地保留细胞外基质的成分及生物活性，操作简单且支架获取率高。
 技术解决方案
为了达到上述目的，本发明的技术方案为：
一种三维肿瘤模型脱细胞多孔支架的构建方法，以猪源性肺脏组织为主要材料，待脱除细胞后，采用冷冻干燥技术制备多孔支架，具体包括以下步骤：
（1）将整个猪肺组织置于-20~-30℃冰箱冷冻3~12h，待其固定成型后，选取肉眼观察无明显支气管的区域切块，猪肺切块大小为5~8cm 3。
（2）室温下，将步骤（1）中得到的切块采用纯净水清洗3~5次后，采用pH=7.4的磷酸盐（PBS）缓冲液清洗2~4次，去除切块中的血丝。每次清洗均采用磁力搅拌，每次清洗时间为0.5~1h。
（3）室温下，在步骤（2）中得到的切块中加入质量分数为0.1~1%的十二烷基磺酸钠（SDS）水溶液，磁力搅拌时间为12~18h后，初步脱除细胞，得到部分脱除细胞的切块。
（4）室温下，在步骤（3）得到切块中加入体积分数为0.1~0.5%的TritonX-100溶液，磁力搅拌时间为8~15h，继续脱除细胞。
（5）室温下，将步骤（4）得到的切块采用pH为7.4的PBS缓冲液清洗3~5次，每次清洗持续时间0.5~1h，得到脱细胞切块。
（6）将步骤（5）得到的脱细胞切块置于-20~-30℃预冻5~12h。
（7）将步骤（6）预冻后切块冷冻干燥，得到多孔脱细胞猪肺支架。所述的冷冻干燥的冷阱温度为-45~-55℃，冷冻干燥时间为12~24h。
（8）切片切除步骤（7）得到的支架表面致密层后，室温条件下，将支架浸于交联剂中化学交联12~24h后，采用pH为7.4的PBS缓冲液清洗2~4次，每次清洗时间1h。清洗后，在-45~-55℃条件下对其进行冷冻干燥12~24h，获得交联的、结构稳定的脱细胞猪肺基质。
所述的交联剂为1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）、乙磺酸的60~70%乙醇溶液的混合溶液。其中乙磺酸作为缓冲液，交联剂中EDC和NHS的浓度分别为10 ~50 mmol/L，乙磺酸的浓度为20~50 mmol/L。
上述构建方法得到的三维肿瘤模型脱细胞多孔支架能够保留猪肺特有的肺泡及少量小支气管多孔结构，孔隙率在88.14%~94.09%范围之间，吸水率在181.6% ~280.55%之间，孔径在115μm~222 μm之间，支架应力在242.24~634.23 kPa (应变=0.7) 之间。
上述构建方法得到的三维肿瘤模型脱细胞多孔支架作为体外3D肿瘤模型，用于抗肿瘤药物的筛选研究。三维肿瘤模型脱细胞多孔支架保持原有良好生物相容性的同时，其特有的肺泡及支气管多孔支架为细胞生长增殖提供三维空间，为进一步研究肿瘤细胞粘附、生长、迁移和侵袭提供模拟细胞生长的微环境。
本发明选取猪源性肺脏组织作为体外构建肿瘤模型的支架，因猪肺组织具有特殊的肺泡及支气管结构，肺泡直径~200μm，适于为细胞提供良好的生长增殖空间。猪源性组织易于获取且其在生理及组织学等方面与人肺相仿，可以真实模拟人体的胞外基质及肿瘤生长环境。因胶原是脱细胞猪肺支架主要组分之一，本发明脱细胞猪肺支架经过EDC/NHS化学交联处理后，可以显著提高支架的力学性能，以满足肿瘤细胞长期培养形成较大细胞集落的支撑需求。
本发明将整个猪肺组织先冷冻后切块，因冷冻后易切取体积相仿的组织，且便于识别无明显支气管的部位。猪肺组织采用切块而非整个猪肺组织脱细胞处理，方便于切块后较小的面积脱细胞处理且节省时间及减少脱细胞试剂用量。同时一个猪肺组织可以同时获取多个支架材料，保证所有检测来源的同一性。另外，支架接种细胞构建的三维肿瘤模型可以在正常培养皿中培养并容易做一系列检测。
 有益效果
本发明的有益效果为
（1）本发明采用猪肺组织来源的脱细胞基质作为构建肿瘤模型的支架材料，在有效去除细胞成分的基础上，尽可能地保留肺泡-支气管结构脉络网，可模拟天然胞外基质微环境，利于细胞黏附、生长和增殖。
（2）采用组织来源的脱细胞支架免疫原性低且生物相容性好。同时，多孔脱细胞猪肺支架有助于营养物质和氧气的运输。另外，支架中保留了大量的胶原成分，提供力学支撑，保证细胞生长增殖所需的机械强度。
（3）采用猪肺切块而非整个猪肺组织，脱细胞过程简单易行。同时，同一组织可获取足够多支架，保证实验检测所需的样本个数及实验结果的精确性。
（4）经接种细胞后体外构建的三维肿瘤模型，其组织结构更接近在体组织，所以可作为抗肿瘤药物筛选的重要研究体系，将提供较二维平面培养更可靠、准确的实验结果。
 附图说明
图1为本发明脱细胞猪肺基质（实施例1）交联前的SEM图；
图2为本发明脱细胞猪肺基质（实施例3）交联后的SEM图；
图3为本发明脱细胞猪肺基质（实施例3）接种肿瘤细胞后不同时间的倒置显微镜及SEM图；
图4是本发明脱细胞猪肺基质（实施例3）上生长的细胞渗透的激光共聚焦显微镜照片；
图5是本发明脱细胞猪肺基质（实施例3）上生长的细胞分布的HE染色图及Masson染色图。
 本发明的实施方式
下面结合附图及实施例对本发明的具体实施方式进一步详细描述。本发明并不限于下述实施例，在不脱离前后所述宗旨的范围内，所有基于本发明基本思想的修改和变动，都属于本发明请求保护的技术范围内。
实施例 1 脱细胞猪肺三维肿瘤模型支架的制备
将整个猪肺组织置于-20℃冰箱冷冻6h，待其固定成型后，切取肉眼观察无明显大支气管均匀部位，切块大小为8cm 3。将猪肺切块置于烧杯中，随后加入2000mL二次蒸馏水，磁力搅拌30min后更换新的蒸馏水，以上操作重复3次后，用2000mL、pH=7.4的PBS同样清洗2次，每次磁力搅拌30min。待组织中无明显血色后，加入1%（wt%，下同）SDS溶液2000mL，磁力搅拌6h后，更换新的1%SDS溶液继续搅拌6h，以脱除细胞。再加入0.5% （v%，下同）TritonX-100溶液2000mL搅拌10h，彻底脱除细胞。随后，加入2000mLPBS清洗3次，每次持续时间1h，以完全洗净脱细胞试剂。将脱细胞后的猪肺切块置于-20℃的冰箱中预冻6h后，置于冷冻干燥机中-50℃干燥18h，获得多孔脱细胞猪肺支架。
实施例 2 脱细胞猪肺三维肿瘤模型支架的制备
将整个猪肺组织置于-30℃冰箱冷冻3h，待其固定成型后，切取肉眼观察无明显大支气管均匀部位，切块大小为6cm 3。将猪肺切块置于烧杯中，随后加入1000mL二次蒸馏水，磁力搅拌40min后更换新的蒸馏水，以上操作5次后，用1500mL、pH=7.4的PBS同样清洗3次，每次30min。待组织中无明显血色后，加入0.5%SDS溶液1500mL，磁力搅拌6h后，更换新的0.5%SDS溶液继续搅拌12h，以脱除细胞。再加入0.3%TritonX-100溶液2000mL搅拌12h，彻底脱除细胞。随后，加入1500mLPBS清洗3次，每次持续时间1h，以完全洗净脱细胞试剂。将脱细胞后的猪肺切块置于-20℃的冰箱中预冻12h后，然后置于冷冻干燥机中-48℃干燥24h，获得多孔脱细胞猪肺支架。
实施案例 3 脱细胞猪肺三维肿瘤模型支架的交联与性能测试
将整个猪肺组织置于-20℃冰箱冷冻6h，待其固定成型后，切取肉眼观察无明显大支气管均匀部位，切块大小为8cm 3。将猪肺切块置于烧杯中，随后加入2000mL二次蒸馏水，磁力搅拌30min后更换新的蒸馏水，以上操作重复3次后，用2000mL、pH=7.4的PBS同样清洗2次，每次磁力搅拌30min。待组织中无明显血色后，加入1%SDS溶液2000mL，磁力搅拌6h后，更换新的1%SDS溶液继续搅拌6h，以脱除细胞。再加入0.5% TritonX-100溶液2000mL搅拌10h，彻底脱除细胞。随后，加入2000mLPBS清洗3次，每次持续时间1h，以完全洗净脱细胞试剂。将脱细胞后的猪肺切块置于-20℃的冰箱中预冻6h后，置于冷冻干燥机中-50℃干燥18h，获得多孔脱细胞猪肺支架。
切片切除猪肺支架材料表面致密层后，将其浸泡于500mL交联剂中（交联剂为50 mmol/L 1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐、50 mmol/L N-羟基琥珀酰亚胺、50 mmol/L乙磺酸的60%乙醇溶液的混合溶液。其中乙磺酸作为缓冲液），常温交联24h后，2000mLPBS清洗2次，每次磁力搅拌1h，即可完成脱细胞猪肺支架的交联。随后，同样-20℃下预冻6h后，冷冻干燥机中-50℃干燥冷冻干燥18h，获得交联的多孔猪肺三维肿瘤模型支架。
图1、图2为所制备的交联前后脱细胞猪肺支架的SEM图，从图中可以看出所制备的三维肿瘤模型支架的多孔结构，可以清楚看到肺泡及少量小支气管结构，交联前（实施例1）支架较为松散且不规则，而交联后（实施例3）的支架孔结构更加致密紧凑，同时通过SEM结果可以得到交联后支架孔径为166.98±26.03μm。支架的多孔结构能够促进细胞粘附，更好的满足肿瘤细胞生长需要。在应变为0-0.7范围内，交联明显提高了支架的弹性模量，尤其是当最大应变为0.7（70%）时，未交联的猪肺支架应力是242.24±22.63kPa，而交联后应力变为634.23±12.05 kPa。交联使得分子链上的亲水基团数量减少，分子链间更加紧密，支架的吸水率显著降低，由280.55±15.65%降低到 181.6±17.46%。同时，交联降低了支架的孔隙率 (88.14±3.21%)，但是相比交联前(94.09 ±4.62%)，两者并没有显著性差异。
实施案例 4 脱细胞猪肺三维肿瘤模型支架的交联
将整个猪肺组织置于-30℃冰箱冷冻3h，待其固定成型后，切取肉眼观察无明显大支气管均匀部位，切块大小为6cm 3。将猪肺切块置于烧杯中，随后加入1000mL二次蒸馏水，磁力搅拌40min后更换新的蒸馏水，以上操作5次后，用1500mL、pH=7.4的PBS同样清洗3次，每次30min。待组织中无明显血色后，加入0.5%SDS溶液1500mL，磁力搅拌6h后，更换新的0.5%SDS溶液继续搅拌12h，以脱除细胞。再加入0.3%TritonX-100溶液2000mL搅拌12h，彻底脱除细胞。随后，加入1500mLPBS清洗3次，每次持续时间1h，以完全洗净脱细胞试剂。将脱细胞后的猪肺切块置于-20℃的冰箱中预冻12h后，然后置于冷冻干燥机中-48℃干燥24h，获得多孔脱细胞猪肺支架。
切片切除猪肺支架材料表面致密层后，将其浸泡于800mL交联剂中（交联剂为50 mmol/L 1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐、50 mmol/L N-羟基琥珀酰亚胺、20 mmol/L乙磺酸的65%乙醇溶液的混合溶液。其中乙磺酸作为缓冲液），常温交联12h后，2000mLPBS清洗3次，每次磁力搅拌1h，即可完成脱细胞猪肺支架的交联。随后，同样-20℃下预冻12h，置于冷冻干燥机中-48℃干燥24h，获得交联的多孔猪肺三维肿瘤模型支架。
实施例 5. 脱细胞猪肺支架（实施例 3 ）作为肿瘤模型
将交联后脱细胞猪肺支架经75%酒精浸泡3次，换用PBS浸泡及DMEM培养基中浸润，超净台内风干、紫外灭菌后，接种MCF-7细胞，持续培养21天。在培养1天、3天、7天、14天和21天不同时间点取出支架。一部分支架直接置于倒置显微镜下观察细胞生长情况。另一部分支架于2.5%戊二醛中固定3h，然后用pH7.4的磷酸盐缓冲液冲洗两次，梯度酒精脱水(50%，70%，90%，100%，100%)，每级脱水30min。常温干燥后，表面喷金，置于扫描电镜下，选取合适的放大倍数观察脱细胞猪肺支架上细胞生长增殖情况。另外，培养3天的肿瘤模型经过激光共聚焦多层扫描后考察了细胞在支架中的渗透情况。培养14天的肿瘤模型切片处理后进行HE及Masson染色，考察细胞在支架分布情况。
图3为脱细胞猪肺基质接种肿瘤细胞后不同时间点的倒置显微镜及SEM图。从图中可以看出，培养1天后，细胞呈单层生长，细胞与支架紧密粘附。培养3天后，出现小的细胞集落或成层生长于支架表面。随着培养时间延长，细胞增殖明显，细胞集落逐渐增大，支架孔隙完全填充，支架透光率随之下降。SEM可以更明显看到细胞增殖过程，由单层到多层，且大的细胞集落的形成，支架表面因细胞的铺满，已经无法清晰地看出支架本身的形态及结构。细胞形态也由最初的伸展状态到球状生长，尤其是培养3天后，细胞开始呈现葡串形聚集生长，而二维培养条件下细胞呈单层贴壁生长。随着培养时间延长，三维肿瘤模型支架上细胞聚集形成细胞球数量增多，且细胞球会进一步发生团聚现象，形成更大的细胞集落，表明了脱细胞猪肺肿瘤模型支架上细胞生长与人体内实体瘤更加接近。
图4是脱细胞猪肺基质上生长的细胞渗透的激光共聚焦显微镜照片。待肿瘤模型构建3天后，采用激光共聚焦显微镜多层扫描考察了复合物支架表面及其以下200µm深度细胞在支架的分布。可以看出细胞在各层均有不同程度的渗透，细胞分布在支架孔壁及孔隙中，且生长良好，同时也可以清晰地看出脱细胞猪肺支架的形态。另外，呈现了多层扫描后堆叠的3D图，可以清楚的看出细胞在支架中立体分布情况。
图5是脱细胞猪肺基质上生长的细胞分布的HE及Masson染色图。从图中可以看出，细胞在脱细胞猪肺支架分布并不是均匀的，细胞在肺泡-支气管密集区分布集中，并且在支架边缘因易接触营养物质，多形成较大细胞集落。同时，Masson染色结果也说明了脱细胞猪肺基质主要成分为胶原，且集中分布在支气管部位。

Claims (10)

  1. 一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于以下步骤：
    （1）将整个猪肺组织置于-20~-30℃冰箱冷冻3~12h，待其固定成型后，选取肉眼观察无明显支气管的区域切块；
    （2）室温下，将步骤（1）中得到的切块采用纯净水后，采用磷酸盐缓冲液清洗，去除切块中的血丝；
    （3）室温下，在步骤（2）中得到的切块放置于质量分数为0.1~1%的十二烷基磺酸钠水溶液中，磁力搅拌12~18h后，初步脱除细胞，得到部分脱除细胞的切块；
    （4）室温下，在步骤（3）得到切块放置于体积分数为0.1~0.5%的TritonX-100溶液中，磁力搅拌8~15h，继续脱除细胞；
    （5）室温下，将步骤（4）得到的切块采用磷酸盐缓冲液清洗，得到脱细胞切块；
    （6）将步骤（5）得到的脱细胞切块置于-20~-30℃预冻5~12h；
    （7）将步骤（6）预冻后切块冷冻干燥，得到多孔脱细胞猪肺支架；所述的冷冻干燥的冷阱温度为-45~-55℃，冷冻干燥时间为12~24h；
    （8）切片切除步骤（7）得到的支架表面致密层后，室温条件下，将支架浸于交联剂中化学交联12~24h后，采用磷酸盐缓冲液清洗后，在-45~-55℃条件下对其进行冷冻干燥12~24h，获得交联的、结构稳定的脱细胞猪肺基质；所述的交联剂为1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺、乙磺酸的60~70%乙醇溶液的混合溶液，其中，乙磺酸作为缓冲液。
  2. 根据权利要求1所述的一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于，所述的交联剂中1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的浓度均为10 ~50 mmol/L；乙磺酸的浓度为20~50 mmol/L。
  3. 根据权利要求1或2所述的一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于，所述的磷酸盐缓冲液的pH为7.4。
  4. 根据权利要求1或2所述的一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于，步骤（5）中磷酸盐缓冲液清洗切块3~5次，每次清洗持续时间0.5~1h。
  5. 根据权利要求3所述的一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于，步骤（5）中磷酸盐缓冲液清洗切块3~5次，每次清洗持续时间0.5~1h。
  6. 根据权利要求1或2或5所述的一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于，步骤（8）中交联后磷酸盐缓冲液清洗次数为2~4次，每次清洗时间1h。
  7. 根据权利要求3所述的一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于，步骤（8）中交联后磷酸盐缓冲液清洗次数为2~4次，每次清洗时间1h。
  8. 根据权利要求4所述的一种三维肿瘤模型脱细胞多孔支架的构建方法，其特征在于，步骤（8）中交联后磷酸盐缓冲液清洗次数为2~4次，每次清洗时间1h。
  9. 采用权利要求1-8任一所述的构建方法得到的三维肿瘤模型脱细胞多孔支架，其特征在于，所述的三维肿瘤模型脱细胞多孔支架能够保留猪肺特有的肺泡及少量小支气管多孔结构，孔隙率在88.14%~94.09%范围之间，吸水率在181.6% ~280.55%之间，孔径在115μm~222 μm之间，支架应力在242.24~634.23 kPa之间。
  10. 权利要求9所述的三维肿瘤模型脱细胞多孔支架，作为体外3D肿瘤模型，用于抗肿瘤药物的筛选研究。
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