WO2023241094A1 - Tissue engineering liver on basis of plant decellularized scaffold and preparation method therefor - Google Patents

Tissue engineering liver on basis of plant decellularized scaffold and preparation method therefor Download PDF

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WO2023241094A1
WO2023241094A1 PCT/CN2023/078369 CN2023078369W WO2023241094A1 WO 2023241094 A1 WO2023241094 A1 WO 2023241094A1 CN 2023078369 W CN2023078369 W CN 2023078369W WO 2023241094 A1 WO2023241094 A1 WO 2023241094A1
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plant
decellularized scaffold
preparation
decellularized
celery
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任昊桢
王经琳
赵远锦
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南京鼓楼医院
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Definitions

  • the invention belongs to the field of biomedical materials, and specifically relates to a tissue engineering liver based on a plant decellularized scaffold and a preparation method.
  • liver decellularized scaffolds refer to the removal of immunogenic cellular components while retaining non-immunogenic ECM, retaining the natural 3D structure of the tissue, and providing a stable physical structure for subsequent cell-cell interactions and cell-ECM interactions.
  • Commonly used liver decellularized scaffolds are mainly derived from human and animal livers. Due to species differences, this Unavoidable immune rejection may result, thus limited availability. Therefore, in the biomedical field, there is an urgent need for a biocompatible, non-animal-derived decellularized scaffold that can replace animal tissue.
  • the present invention provides a tissue engineering liver based on a plant decellularized scaffold and a preparation method thereof, which helps the proliferation and adhesion of liver cells with the help of the natural porous microstructure of the decellularized celery scaffold.
  • a method for preparing tissue-engineered liver based on plant decellularized scaffolds including the following steps:
  • the celery is celery with a hollow hole structure, which is similar to the structure of liver lobules.
  • step S2 the concentration of the sodium dodecyl sulfate solution is 10 wt%.
  • step S2 the celery stems were immersed in sodium dodecyl sulfate solution for 5 days, and the solvent was changed every day.
  • step S2 in the trition x-100 solution containing hypochlorous acid, the content of hypochlorous acid is 0.1v/v%, and the content of trition x-100 is 1v/v%.
  • step S2 the celery stems are rinsed with a trition x-100 solution containing hypochlorous acid for two days.
  • the decellularized scaffold obtained in step S2 is washed with PBS solution to provide an environment suitable for cell survival.
  • step S3 mix the hepatocyte mixture and Matrigel with a volume ratio of 1:4 and plant it on the decellularized scaffold. After incubating it in the incubator for 30 minutes, add culture medium to help the hepatocytes grow on the decellularized scaffold. adhesion and proliferation.
  • a tissue-engineered liver based on plant decellularized scaffold prepared by the above method A tissue-engineered liver based on plant decellularized scaffold prepared by the above method.
  • the beneficial effects of the invention are: plants are natural renewable materials with abundant sources and are easy to obtain, providing a low-cost substitute for animal tissues.
  • Celery stem tissue has a natural vascular bundle-like porous structure, is cheap, easy to obtain, non-toxic, safe, and can be eaten in daily life.
  • the present invention obtains a decellularized scaffold by soaking celery stems in sodium dodecyl sulfate (SDS) solution, and then rinsing the celery stems with trition x-100 solution containing hypochlorous acid.
  • SDS sodium dodecyl sulfate
  • the method is simple and the operation is Convenient, low-cost, safe, non-toxic, cheap and easily available compared to other sources of decellularized scaffolds.
  • the prepared decellularized scaffold has high biocompatibility, suitable porosity and hydrophilicity, and excellent mechanical properties, providing a suitable biological microenvironment for the growth and proliferation of cells, and its natural 3D microenvironment cannot easily replicated by current 3D printing and microfluidic technologies.
  • a method for preparing tissue-engineered livers based on plant decellularized scaffolds was developed.
  • the tissue-engineered liver prepared by this method has high biocompatibility and can maintain the morphology and function of liver cells for a period of time. In addition, it can also maintain certain liver functions in vitro.
  • the tissue-engineered liver based on the plant decellularized scaffold provided by the present invention shows broad application prospects in tissue repair and regeneration, disease treatment and other aspects.
  • Figure 1 shows the electron microscopy characterization of the celery scaffold prepared by the present invention before and after decellularization.
  • the ratio bars are 100 ⁇ m (i), 20 ⁇ m (ii), and 5 ⁇ m (iii);
  • Figure 2 is a confocal scanning fluorescence image of the bioengineered liver tissue prepared by the present invention after culture for 14 days.
  • the scale bars are all 100 ⁇ m;
  • Figure 3 shows the bioengineered liver tissue prepared by the present invention, and the cells cultured in 2D conventional plane and matrix gel.
  • Immunofluorescence image of ALB (red), nuclei stained blue, scale bar is 50 ⁇ m;
  • Figure 4 shows the expression of liver function of bioengineered liver tissue prepared by the present invention, and cells cultured in 2D conventional plane culture and matrix gel culture.
  • This embodiment provides a tissue-engineered liver based on a plant decellularized scaffold, which is prepared by the following method. The specific steps are as follows:
  • the prepared tissue-engineered livers were cultured for another 14 days, while hepatocytes suspended in Matrigel were seeded on 24-well plates as a control group.
  • cell number was determined using GFP staining.
  • the number of GFP-positive cells was higher than that on the first day.
  • the number of GFP-positive cells increased with the prolongation of culture time, indicating that the decellularized scaffold promoted cell proliferation, confirming its high biocompatibility.
  • hepatocytes Due to the lack of extracellular matrix components and cell-cell interactions, hepatocytes lose their differentiated state over time when cultured in 2D dishes.
  • the composition of Matrigel is heterogeneous, with over 1500 different proteins in its composition, including the most common proteins such as laminin and type IV collagen.
  • Metabolic activity in the scaffolds albumin secretion, glycogen synthesis and mRNA expression levels of key hepatic transcription factors and functional genes were quantified. As shown in Figure 3, the fluorescence image of albumin confirmed that the number of albumin-positive cells was greater in both the Matrigel and acellular scaffold groups compared with the dish group.
  • Hepatocyte-specific gene expression was performed during culture to determine the extent to which liver-specific functions are maintained over time under different culture conditions.
  • the genes detected in each group included ALB, cytochrome P-4503A4 (CYP3A4), cytochrome P-450 1A2 (CYP1A2), cytokeratin 18 (CK18), and asialoglycoprotein receptor (ASGPR).
  • ALB cytochrome P-4503A4
  • CYP1A2 cytochrome P-450 1A2
  • CK18 cytokeratin 18
  • ASGPR asialoglycoprotein receptor
  • the mRNA levels in the decellularized scaffold group were lower than those in the other two groups, and similar results were also shown in CK18 and ASGPR1. This culture condition improves the functional status of the cells. Therefore, the liver engineering structure constructed on the scaffold has a more stable liver phenotype.

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Abstract

Provided in the present invention is a method for preparing a tissue engineering liver on the basis of a plant decellularized scaffold. The method comprises the following steps: S1, cutting out the stem of hollow water celery; S2, immersing the stem of the celery in sodium dodecyl sulfate solution, and then rinsing the stem of the celery using trition x-100 solution containing hypochlorous acid to obtain a decellularized scaffold; and S3, implanting hepatocytes on the decellularized scaffold and culturing same.

Description

一种基于植物脱细胞支架的组织工程肝脏和制备方法A kind of tissue engineering liver based on plant decellularized scaffold and preparation method 技术领域Technical field
本发明属于生物医学材料领域，具体涉及一种基于植物脱细胞支架的组织工程肝脏和制备方法。The invention belongs to the field of biomedical materials, and specifically relates to a tissue engineering liver based on a plant decellularized scaffold and a preparation method.
背景技术Background technique
近年来，肝脏脱细胞支架是指去除免疫原性细胞成分的同时保留非免疫原性的ECM，保留组织的天然的3D结构，为后续的细胞间相互作用和细胞ECM相互作用提供具有稳定物理结构的空间和微环境的生物材料，其促进新组织形成等特点在组织工程中发挥了重要作用并得到了广泛的应用，常用的肝脏脱细胞支架主要来源于人和动物肝脏，由于物种差异，这可能导致不可避免的免疫排斥反应，因而可用性有限。因此，在生物医学领域，急需一种可替代动物组织的具有生物相容性的非动物源性脱细胞支架。In recent years, liver decellularized scaffolds refer to the removal of immunogenic cellular components while retaining non-immunogenic ECM, retaining the natural 3D structure of the tissue, and providing a stable physical structure for subsequent cell-cell interactions and cell-ECM interactions. Biomaterials with unique space and microenvironment, which promote the formation of new tissues, play an important role in tissue engineering and have been widely used. Commonly used liver decellularized scaffolds are mainly derived from human and animal livers. Due to species differences, this Unavoidable immune rejection may result, thus limited availability. Therefore, in the biomedical field, there is an urgent need for a biocompatible, non-animal-derived decellularized scaffold that can replace animal tissue.
发明内容Contents of the invention
本发明针对现有技术中的不足，提供一种基于植物脱细胞支架的组织工程肝脏及其制备方法，其借助脱细胞后的芹菜支架天然多孔的微结构，帮助肝细胞的增殖和黏附。In view of the deficiencies in the prior art, the present invention provides a tissue engineering liver based on a plant decellularized scaffold and a preparation method thereof, which helps the proliferation and adhesion of liver cells with the help of the natural porous microstructure of the decellularized celery scaffold.
为实现上述目的，本发明采用以下技术方案：In order to achieve the above objects, the present invention adopts the following technical solutions:
一种基于植物脱细胞支架的组织工程肝脏的制备方法，包括以下步骤：A method for preparing tissue-engineered liver based on plant decellularized scaffolds, including the following steps:
S1、截取空心水芹菜的茎部并清洗干净；S1. Cut off the stems of hollow water celery and clean them;
S2、将芹菜茎部浸没在十二烷基硫酸钠(SDS)溶液中，然后利用含有次氯酸的trition x-100溶液对芹菜茎部进行漂洗，获得脱细胞支架；S2. Immerse the celery stems in sodium dodecyl sulfate (SDS) solution, and then rinse the celery stems with trition x-100 solution containing hypochlorous acid to obtain a decellularized scaffold;
S3、将肝细胞种植在脱细胞支架上并进行培养。S3. Plant the hepatocytes on the decellularized scaffold and culture them.
为优化上述技术方案，采取的具体措施还包括：In order to optimize the above technical solutions, specific measures taken also include:
进一步地，步骤S1中，所述芹菜为具有空心孔状结构的芹菜，该结构与肝小叶的结构相似。Further, in step S1, the celery is celery with a hollow hole structure, which is similar to the structure of liver lobules.
进一步地，步骤S2中，所述十二烷基硫酸钠溶液的浓度为10wt％。Further, in step S2, the concentration of the sodium dodecyl sulfate solution is 10 wt%.
进一步地，步骤S2中，芹菜茎部浸没在十二烷基硫酸钠溶液中5天，每天更换溶剂。 Further, in step S2, the celery stems were immersed in sodium dodecyl sulfate solution for 5 days, and the solvent was changed every day.
进一步地，步骤S2中，所述含有次氯酸的trition x-100溶液中，次氯酸的含量为0.1v/v％，trition x-100的含量为1v/v％。Further, in step S2, in the trition x-100 solution containing hypochlorous acid, the content of hypochlorous acid is 0.1v/v%, and the content of trition x-100 is 1v/v%.
进一步地，步骤S2中，利用含有次氯酸的trition x-100溶液对芹菜茎部进行漂洗两天。Further, in step S2, the celery stems are rinsed with a trition x-100 solution containing hypochlorous acid for two days.
进一步地，脱细胞支架制备完成后，利用PBS溶液对步骤S2得到的脱细胞支架清洗，以提供适合细胞生存的环境。Further, after the preparation of the decellularized scaffold is completed, the decellularized scaffold obtained in step S2 is washed with PBS solution to provide an environment suitable for cell survival.
进一步地，步骤S3中，将体积比为1:4的肝细胞混液与基质胶混匀种植在脱细胞支架上，在培养箱孵育30分钟后加入培养基培养，帮助肝细胞在脱细胞支架上的黏附和增殖。Further, in step S3, mix the hepatocyte mixture and Matrigel with a volume ratio of 1:4 and plant it on the decellularized scaffold. After incubating it in the incubator for 30 minutes, add culture medium to help the hepatocytes grow on the decellularized scaffold. adhesion and proliferation.
一种通过上述方法制备得到的基于植物脱细胞支架的组织工程肝脏。A tissue-engineered liver based on plant decellularized scaffold prepared by the above method.
本发明的有益效果是：植物是一种天然的可再生材料，来源丰富，易于获取，为动物组织提供了一种低成本的替代品。芹菜茎部组织具有天然的维管束状的多孔结构，且其价格便宜、易得、无毒、安全，在生活中可食用。本发明通过将芹菜茎部浸泡在十二烷基硫酸钠(SDS)溶液中，然后利用含有次氯酸的trition x-100溶液对芹菜茎部进行漂洗，获得了脱细胞支架，方法简单，操作方便，价格低廉，与其他来源的脱细胞支架相比，安全无毒、价格便宜且易于获得。所制备的脱细胞支架具有较高的生物相容性，适宜的孔隙率和亲水性以及优异的力学性能，为细胞的生长和增殖提供适宜的生物微环境，且其天然的3D微环境不能被当前的3D打印和微流控技术简单的复制。通过进一步将脱细胞支架和hiPSC-Heps(肝细胞)有效地整合，开发出一种基于植物脱细胞支架的组织工程肝脏的制备方法。通过该方法制备得到的组织工程肝脏具有较高的生物相容性，并能在一段时间内维持肝细胞的形态和功能，此外，还可以在体外维持一定的肝脏功能。本发明提供的基于植物脱细胞支架的组织工程肝脏在组织修复和再生、疾病治疗等方面展示出了广泛的应用前景。The beneficial effects of the invention are: plants are natural renewable materials with abundant sources and are easy to obtain, providing a low-cost substitute for animal tissues. Celery stem tissue has a natural vascular bundle-like porous structure, is cheap, easy to obtain, non-toxic, safe, and can be eaten in daily life. The present invention obtains a decellularized scaffold by soaking celery stems in sodium dodecyl sulfate (SDS) solution, and then rinsing the celery stems with trition x-100 solution containing hypochlorous acid. The method is simple and the operation is Convenient, low-cost, safe, non-toxic, cheap and easily available compared to other sources of decellularized scaffolds. The prepared decellularized scaffold has high biocompatibility, suitable porosity and hydrophilicity, and excellent mechanical properties, providing a suitable biological microenvironment for the growth and proliferation of cells, and its natural 3D microenvironment cannot Easily replicated by current 3D printing and microfluidic technologies. By further effectively integrating decellularized scaffolds and hiPSC-Heps (liver cells), a method for preparing tissue-engineered livers based on plant decellularized scaffolds was developed. The tissue-engineered liver prepared by this method has high biocompatibility and can maintain the morphology and function of liver cells for a period of time. In addition, it can also maintain certain liver functions in vitro. The tissue-engineered liver based on the plant decellularized scaffold provided by the present invention shows broad application prospects in tissue repair and regeneration, disease treatment and other aspects.
附图说明Description of the drawings
图1为本发明制备的芹菜支架脱细胞前后的电镜表征，比标尺为100μm(i)、20μm(ii)、5μm(iii)；Figure 1 shows the electron microscopy characterization of the celery scaffold prepared by the present invention before and after decellularization. The ratio bars are 100 μm (i), 20 μm (ii), and 5 μm (iii);
图2为本发明制备的生物工程肝组织培养14天后的共聚焦扫描荧光图像，比例尺均为100μm；Figure 2 is a confocal scanning fluorescence image of the bioengineered liver tissue prepared by the present invention after culture for 14 days. The scale bars are all 100 μm;
图3为本发明制备的生物工程肝组织，与在2D常规平面培养，以及基质凝胶培养的细胞 的ALB(红色)的免疫荧光图像，核被染成蓝色，比例尺为50μm；Figure 3 shows the bioengineered liver tissue prepared by the present invention, and the cells cultured in 2D conventional plane and matrix gel. Immunofluorescence image of ALB (red), nuclei stained blue, scale bar is 50 μm;
图4为本发明制备的生物工程肝组织，与在2D常规平面培养，以及基质凝胶培养的细胞肝功能的表达。Figure 4 shows the expression of liver function of bioengineered liver tissue prepared by the present invention, and cells cultured in 2D conventional plane culture and matrix gel culture.
具体实施方式Detailed ways
以下结合具体实施例对本发明作进一步说明。The present invention will be further described below in conjunction with specific examples.
本实施例提供一种基于植物脱细胞支架的组织工程肝脏，通过以下方法制备，具体步骤如下：This embodiment provides a tissue-engineered liver based on a plant decellularized scaffold, which is prepared by the following method. The specific steps are as follows:
S1、截取具有空心孔状结构水芹菜的茎部并清洗干净；S1. Cut off the stem of water celery with a hollow hole structure and clean it;
S2、将芹菜茎部浸没在10wt％十二烷基硫酸钠(SDS)溶液中5天，每天更换溶剂；然后利用含有0.1v/v％次氯酸的1v/v％trition x-100溶液对芹菜茎部进行漂洗两天，获得脱细胞支架，可以观察新鲜的芹菜组织部由绿色变为透明(组织细胞成分去除)；利用PBS溶液对得到的脱细胞支架清洗残留的溶剂成分，以提供适合细胞生存的环境；图1展示了天然的芹菜茎部(上)与制备得到的脱细胞支架(下)的多孔结构；S2. Immerse the celery stems in 10wt% sodium dodecyl sulfate (SDS) solution for 5 days, changing the solvent every day; then use 1v/v% trition x-100 solution containing 0.1v/v% hypochlorous acid to treat Rinse the celery stems for two days to obtain a decellularized scaffold. You can observe the fresh celery tissue turning from green to transparent (tissue cell components are removed); use PBS solution to clean the remaining solvent components of the decellularized scaffold to provide a suitable The environment for cell survival; Figure 1 shows the porous structure of the natural celery stem (top) and the prepared decellularized scaffold (bottom);
S3、将体积比为1:4的肝细胞混液与基质胶混匀种植在脱细胞支架上，在培养箱孵育30分钟后加入培养基培养，帮助肝细胞在脱细胞支架上的黏附和增殖，得到组织工程肝脏。S3. Mix the hepatocyte mixture and Matrigel with a volume ratio of 1:4 and plant it on the decellularized scaffold. After incubating it in the incubator for 30 minutes, add culture medium to help the adhesion and proliferation of hepatocytes on the decellularized scaffold. Tissue engineered liver was obtained.
对本实施例得到的基于植物脱细胞支架的组织工程肝脏进行实验测试：Experimental testing was performed on the tissue engineered liver based on the plant decellularized scaffold obtained in this example:
一、基于植物脱细胞支架的组织工程肝脏相容性的测试1. Testing the liver compatibility of tissue engineering based on plant decellularized scaffolds
为了检查脱细胞支架的生物相容性，将制备的组织工程肝脏再培养14天，而将混悬在基质胶的肝细胞接种在24孔板上作为对照组。如图2所示，使用GFP染色测定细胞数量。在接种后的第三天，GFP阳性细胞的数量高于第一天。此外，GFP阳性细胞的数量随着培养时间的延长而增加，这表明去细胞化的支架促进了细胞增殖，证实了其具有较高的生物相容性。To check the biocompatibility of the decellularized scaffolds, the prepared tissue-engineered livers were cultured for another 14 days, while hepatocytes suspended in Matrigel were seeded on 24-well plates as a control group. As shown in Figure 2, cell number was determined using GFP staining. On the third day after inoculation, the number of GFP-positive cells was higher than that on the first day. Furthermore, the number of GFP-positive cells increased with the prolongation of culture time, indicating that the decellularized scaffold promoted cell proliferation, confirming its high biocompatibility.
二、基于植物脱细胞支架的组织工程肝脏的肝功能表征2. Characterization of liver function in tissue-engineered liver based on plant decellularized scaffolds
由于缺乏细胞外基质成分和细胞间相互作用，肝细胞在2D培养皿中培养时会随着时间的推移而失去分化状态。然而，基质胶的组成是异质的，其组成中有超过1500种不同的蛋白质，包括最常见的蛋白质，如层粘连蛋白和IV型胶原蛋白。为了评估移植的肝细胞在去细胞化肝 支架中的代谢活性，对关键肝转录因子和功能基因的白蛋白分泌、糖原合成和mRNA表达水平进行了定量分析。如图3所示，白蛋白的荧光图像证实，与dish组相比，基质胶和脱细胞支架组中白蛋白阳性细胞的数量都更大。在培养过程中进行肝细胞特异性基因表达，以确定在不同培养条件下长期维持肝特异性功能的程度。每组检测到的基因包括ALB，细胞色素P-4503A4(CYP3A4)，细胞色素P-450 1A2(CYP1A2)，细胞角蛋白18(CK18)和去唾液酸糖蛋白受体(ASGPR)。如图4所示，基质胶和去细胞支架组ALB的表达高于dish组，表明肝细胞功能较好。进一步研究了关键CYP酶CYP1A2和CYP3A4的基因表达水平，它们与药物代谢和解毒密切相关。脱细胞支架组的mRNA水平低于其他两组，CK18和ASGPR1中也显示出相似的结果。这种培养条件改善了细胞的功能状态。因此，在支架上构建的肝脏工程结构具有更稳定的肝脏表型。Due to the lack of extracellular matrix components and cell-cell interactions, hepatocytes lose their differentiated state over time when cultured in 2D dishes. However, the composition of Matrigel is heterogeneous, with over 1500 different proteins in its composition, including the most common proteins such as laminin and type IV collagen. To evaluate transplanted hepatocytes in decellularized livers Metabolic activity in the scaffolds, albumin secretion, glycogen synthesis and mRNA expression levels of key hepatic transcription factors and functional genes were quantified. As shown in Figure 3, the fluorescence image of albumin confirmed that the number of albumin-positive cells was greater in both the Matrigel and acellular scaffold groups compared with the dish group. Hepatocyte-specific gene expression was performed during culture to determine the extent to which liver-specific functions are maintained over time under different culture conditions. The genes detected in each group included ALB, cytochrome P-4503A4 (CYP3A4), cytochrome P-450 1A2 (CYP1A2), cytokeratin 18 (CK18), and asialoglycoprotein receptor (ASGPR). As shown in Figure 4, the expression of ALB in the Matrigel and decellularized scaffold groups was higher than that in the dish group, indicating better liver cell function. The gene expression levels of key CYP enzymes CYP1A2 and CYP3A4, which are closely related to drug metabolism and detoxification, were further studied. The mRNA levels in the decellularized scaffold group were lower than those in the other two groups, and similar results were also shown in CK18 and ASGPR1. This culture condition improves the functional status of the cells. Therefore, the liver engineering structure constructed on the scaffold has a more stable liver phenotype.
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。 The above are only preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above-mentioned embodiments. All technical solutions that fall under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims (9)

  1. 一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，包括以下步骤：A method for preparing tissue-engineered liver based on plant decellularized scaffolds, which is characterized by including the following steps:
    S1、截取空心水芹菜的茎部；S1. Cut off the stem of hollow water celery;
    S2、将芹菜茎部浸没在十二烷基硫酸钠溶液中，然后利用含有次氯酸的trition x-100溶液对芹菜茎部进行漂洗，获得脱细胞支架；S2. Immerse the celery stems in sodium dodecyl sulfate solution, and then rinse the celery stems with trition x-100 solution containing hypochlorous acid to obtain a decellularized scaffold;
    S3、将肝细胞种植在脱细胞支架上并进行培养。S3. Plant the hepatocytes on the decellularized scaffold and culture them.
  2. 根据权利要求1所述的一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，The preparation method of tissue engineering liver based on plant decellularized scaffold according to claim 1, characterized in that:
    步骤S1中，所述芹菜为具有空心孔状结构的芹菜。In step S1, the celery is celery with a hollow hole structure.
  3. 根据权利要求1所述的一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，The preparation method of tissue engineering liver based on plant decellularized scaffold according to claim 1, characterized in that:
    步骤S2中，所述十二烷基硫酸钠溶液的浓度为10wt％。In step S2, the concentration of the sodium dodecyl sulfate solution is 10 wt%.
  4. 根据权利要求1所述的一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，The preparation method of tissue engineering liver based on plant decellularized scaffold according to claim 1, characterized in that:
    步骤S2中，芹菜茎部浸没在十二烷基硫酸钠溶液中5天，每天更换溶剂。In step S2, the celery stems were immersed in sodium dodecyl sulfate solution for 5 days, and the solvent was changed every day.
  5. 根据权利要求1所述的一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，The preparation method of tissue engineering liver based on plant decellularized scaffold according to claim 1, characterized in that:
    步骤S2中，所述含有次氯酸的trition x-100溶液中，次氯酸的含量为0.1v/v％，trition x-100的含量为1v/v％。In step S2, in the trition x-100 solution containing hypochlorous acid, the content of hypochlorous acid is 0.1v/v%, and the content of trition x-100 is 1v/v%.
  6. 根据权利要求1所述的一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，The preparation method of tissue engineering liver based on plant decellularized scaffold according to claim 1, characterized in that:
    步骤S2中，利用含有次氯酸的trition x-100溶液对芹菜茎部进行漂洗两天。In step S2, the celery stems are rinsed with trition x-100 solution containing hypochlorous acid for two days.
  7. 根据权利要求1所述的一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，The preparation method of tissue engineering liver based on plant decellularized scaffold according to claim 1, characterized in that:
    脱细胞支架制备完成后，利用PBS溶液对步骤S2得到的脱细胞支架清洗。After the preparation of the decellularized scaffold is completed, use PBS solution to wash the decellularized scaffold obtained in step S2.
  8. 根据权利要求1所述的一种基于植物脱细胞支架的组织工程肝脏的制备方法，其特征在于，The preparation method of tissue engineering liver based on plant decellularized scaffold according to claim 1, characterized in that:
    步骤S3中，将体积比为1:4的肝细胞混液与基质胶混匀种植在脱细胞支架上，在培养箱孵育30分钟后加入培养基培养。In step S3, mix the hepatocyte mixture and Matrigel with a volume ratio of 1:4 and plant them on the decellularized scaffold. Incubate in the incubator for 30 minutes and then add culture medium for culture.
  9. 一种通过权利要求1-8任一项制备方法得到的基于植物脱细胞支架的组织工程肝脏。 A tissue-engineered liver based on a plant decellularized scaffold obtained by the preparation method of any one of claims 1-8.
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