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CN105695392B - A kind of cultural method that liver cell vitro differentiation phenotype and function can be improved - Google Patents

  • ️Tue Nov 13 2018
A kind of cultural method that liver cell vitro differentiation phenotype and function can be improved Download PDF

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Publication number
CN105695392B
CN105695392B CN201510937399.0A CN201510937399A CN105695392B CN 105695392 B CN105695392 B CN 105695392B CN 201510937399 A CN201510937399 A CN 201510937399A CN 105695392 B CN105695392 B CN 105695392B Authority
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China
Prior art keywords
hepatocytes
culture
scaffold
cells
silk protein
Prior art date
2015-12-15
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Expired - Fee Related
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CN201510937399.0A
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Chinese (zh)
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CN105695392A (en
Inventor
王秀丽
魏国峰
徐红
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Dalian Medical University
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Dalian Medical University
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2015-12-15
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2015-12-15
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2018-11-13
2015-12-15 Application filed by Dalian Medical University filed Critical Dalian Medical University
2015-12-15 Priority to CN201510937399.0A priority Critical patent/CN105695392B/en
2016-06-22 Publication of CN105695392A publication Critical patent/CN105695392A/en
2018-11-13 Application granted granted Critical
2018-11-13 Publication of CN105695392B publication Critical patent/CN105695392B/en
Status Expired - Fee Related legal-status Critical Current
2035-12-15 Anticipated expiration legal-status Critical

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    • 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
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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    • 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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Abstract

本发明公开一种可提高肝细胞体外分化表型及功能的培养方法，其特征在于：所述的方法按照以下步骤进行：采用肝细胞为种子细胞，以多孔蚕丝蛋白支架作为肝细胞体外培养载体，将肝细胞、肝源性间质细胞和细胞外基质按一定比例接种到多孔蚕丝蛋白支架上，并将多孔蚕丝蛋白支架置于模拟肝细胞的体外培养微环境中进行培养；该方法所建立的三维肝细胞共培养体系，不但可以用于组织工程化肝细胞功能单元，而且还可用于体外评价药物对肝代谢酶的影响及肝毒性检测，为新药研发提供新的筛选评价工具。

The invention discloses a culture method capable of improving the in vitro differentiation phenotype and function of hepatocytes, which is characterized in that: the method is carried out according to the following steps: using hepatocytes as seed cells, and using porous silk protein scaffolds as hepatocyte in vitro culture carriers , hepatocytes, liver-derived mesenchymal cells and extracellular matrix were seeded on the porous silk protein scaffold according to a certain ratio, and the porous silk protein scaffold was placed in an in vitro culture microenvironment simulating liver cells for cultivation; the method established The three-dimensional hepatocyte co-culture system can not only be used for tissue engineering hepatocyte functional units, but also can be used for in vitro evaluation of drug effects on liver metabolic enzymes and hepatotoxicity detection, providing a new screening and evaluation tool for new drug development.

Description

一种可提高肝细胞体外分化表型及功能的培养方法A culture method capable of improving the differentiation phenotype and function of hepatocytes in vitro

技术领域technical field

本发明涉及一种肝细胞的体外培养方法，特别是一种可提高肝细胞体外分化表型及功能的培养方法。The invention relates to an in vitro culture method of hepatocytes, in particular to a culture method capable of improving the differentiation phenotype and function of hepatocytes in vitro.

背景技术Background technique

原代肝细胞不但是构建组织工程化肝脏的理想种子细胞来源，而且也是对药物体外ADME/T性质进行准确评价的重要模型。但肝细胞在传统的体外平面培养过程中极易出现“去分化”，即出现活性的降低，表型的改变以及功能活性（包括合成和代谢活性）的降低甚至完全丧失。研究证实：新鲜分离的人肝细胞在悬浮培养条件下，其P450酶活性仅维持4h；在胶原包被的培养板内，贴壁细胞的P450酶表达量及酶活性在24－48h后便会显著降低。而由于酶功能活性的降低，肝细胞单层培养法在肝毒性药物筛选中的灵敏度仅有50％。因此如何在体外培养中克服或延缓肝细胞的“去分化”现象，从而有效维持肝细胞的理想分化状态，即维持其表型及相关功能一直是肝细胞培养及应用研究中所亟待解决的“瓶颈”问题。Primary hepatocytes are not only an ideal seed cell source for constructing tissue engineered liver, but also an important model for accurate evaluation of ADME/T properties of drugs in vitro. However, hepatocytes are prone to "dedifferentiation" in the traditional in vitro planar culture process, that is, there is a decrease in activity, a change in phenotype, and a decrease in or even complete loss of functional activity (including synthetic and metabolic activities). Studies have confirmed that the P450 enzyme activity of freshly isolated human hepatocytes can only be maintained for 4 hours under suspension culture conditions; in the collagen-coated culture plate, the P450 enzyme expression and enzyme activity of adherent cells will decrease after 24-48 hours. Significantly lower. However, due to the reduction of enzyme functional activity, the sensitivity of hepatocyte monolayer culture method in the screening of hepatotoxic drugs is only 50%. Therefore, how to overcome or delay the "dedifferentiation" phenomenon of hepatocytes in vitro culture, so as to effectively maintain the ideal differentiation state of hepatocytes, that is, maintain their phenotype and related functions, has always been an urgent problem to be solved in the culture and application research of hepatocytes. Bottleneck problem.

发明内容Contents of the invention

本发明是为了解决现有技术所存在的上述不足，提出一种能够理想维持肝细胞生长活性，并显著提高体外培养肝细胞表型及功能活性的新培养方法。The present invention aims to solve the above-mentioned shortcomings in the prior art, and proposes a new culture method that can ideally maintain the growth activity of hepatocytes and significantly improve the phenotype and functional activity of hepatocytes cultured in vitro.

本发明的技术解决方案是：一种可提高肝细胞体外分化表型及功能的培养方法，其特征在于：所述的方法按照以下步骤进行：采用肝细胞为种子细胞，以多孔蚕丝蛋白支架作为肝细胞体外培养载体，将肝细胞、肝源性间质细胞和细胞外基质按一定比例接种到多孔蚕丝蛋白支架上，并将多孔蚕丝蛋白支架置于模拟肝细胞的体外培养微环境中进行培养，The technical solution of the present invention is: a culture method that can improve the differentiation phenotype and function of liver cells in vitro, characterized in that: the method is carried out according to the following steps: use liver cells as seed cells, and use porous silk protein scaffolds as Liver cell in vitro culture carrier, hepatocytes, liver-derived mesenchymal cells and extracellular matrix are seeded on a porous silk protein scaffold in a certain proportion, and the porous silk protein scaffold is placed in an in vitro culture microenvironment simulating liver cells for cultivation ,

所述的将肝细胞、肝源性间质细胞和细胞外基质按一定比例接种到多孔蚕丝蛋白支架上的具体操作步骤如下：将新鲜分离的肝细胞和肝星形间质细胞按照细胞数3:1的比例混合，并将获得的混合细胞与细胞外基质混合，获得细胞-基质混合物，所述的细胞外基质由胶原和基质胶按照体积比为1:1的比例混合而成，然后将细胞-基质混合物多点接种至多个多孔蚕丝蛋白支架，并保证肝细胞数量为600,000cells/支架，基质胶的体积为30μL/支架，The specific operation steps of seeding hepatocytes, hepatic stromal cells and extracellular matrix on the porous silk protein scaffold according to a certain ratio are as follows: Freshly separated hepatocytes and hepatic stellate stromal cells are divided into 3 :1 ratio, and the obtained mixed cells are mixed with the extracellular matrix to obtain the cell-matrix mixture, and the extracellular matrix is formed by mixing collagen and Matrigel at a volume ratio of 1:1, and then The cell-matrix mixture was inoculated onto multiple porous silk protein scaffolds at multiple points, and the number of hepatocytes was guaranteed to be 600,000 cells/scaffold, and the volume of matrigel was 30 μL/scaffold.

所述的将多孔蚕丝蛋白支架置于模拟肝细胞的体外培养微环境中进行培养的具体操作步骤如下：将接种完成的多孔蚕丝蛋白支架按照1个支架/孔的密度放置到多孔细胞培养板中，并将多孔细胞培养板置于细胞培养箱中，在37℃、5%CO2的条件下凝胶化2h，然后向多孔细胞培养板的每个孔中加入共培养基，所述的共培养基为肝细胞培养基和星形间质细胞培养基按照体积比为1:1的比例混合而成。The specific operation steps of placing the porous silk protein scaffold in an in vitro culture microenvironment simulating liver cells are as follows: place the inoculated porous silk protein scaffold in a porous cell culture plate at a density of 1 scaffold/well , and put the multi-well cell culture plate in the cell culture incubator, gelatinize at 37°C and 5% CO 2 for 2 hours, then add the co-culture medium to each well of the multi-well cell culture plate, the co-culture medium The medium is prepared by mixing hepatocyte medium and astrocytic medium at a volume ratio of 1:1.

本发明同现有技术相比，具有如下优点：Compared with the prior art, the present invention has the following advantages:

本发明所公开的肝细胞的体外培养方法，该培养方法利用细胞与细胞之间以及细胞与细胞外基质之间复杂的相互作用，形成与体内肝细胞类似的微环境，使肝细胞的生长形态更接近体内三维生长模式；它采用多孔丝素蛋白作为培养支架，同时添加胶原和基质胶作为培养基质，构建出一个复合培养体系，该复合培养体系不但能够为肝细胞提供三维生长空间，而且其具有生物相容性、材料可塑性和支持肝细胞生长及组织化的特点，此外，其多孔特征也可大大提高培养体系内的物质传递，从而有利于肝细胞在体外培养过程中维持其活性；多孔蚕丝蛋白支架所特有的生物相容性、机械强度特征及生物降解特性，使得该培养体系易于实现肝细胞体内移植及回收，这不但可推进肝细胞替代治疗，而且还可能为探讨有关肝细胞移植的相关研究提供理想的研究模型体系；该培养方法所建立的模型内，细胞间彼此建立细胞通讯，且可分化形成毛细胆管结构，说明所构建的肝细胞培养体系更具备组织化特征，从而有利于肝细胞在体外培养过程中维持其表型及功能；该方法所建立的培养体系中，肝细胞的白蛋白分泌和尿素合成功能均显著高于平面培养组及“三明治”培养组，更接近在体肝细胞的功能活性，同时该培养体系内肝细胞的代谢酶活性显著高于传统培养方法（平面培养组和“三明治”培养组），更利于对药物代谢活性及肝毒性进行准确的体外评价，从而可能为新药研发提供新的评价工具。The in vitro culture method of liver cells disclosed in the present invention utilizes complex interactions between cells and between cells and extracellular matrix to form a microenvironment similar to that of liver cells in vivo, so that the growth morphology of liver cells It is closer to the three-dimensional growth mode in vivo; it uses porous silk fibroin as a culture scaffold, and adds collagen and matrigel as a culture substrate to construct a composite culture system, which can not only provide a three-dimensional growth space for liver cells, but also It has the characteristics of biocompatibility, material plasticity and support for the growth and organization of hepatocytes. In addition, its porous feature can also greatly improve the material transfer in the culture system, which is conducive to the maintenance of the activity of hepatocytes during in vitro culture; porous The unique biocompatibility, mechanical strength characteristics and biodegradation characteristics of silk protein scaffolds make the culture system easy to realize transplantation and recovery of hepatocytes in vivo, which can not only promote the replacement therapy of hepatocytes, but also may provide a basis for exploring related hepatocyte transplantation. The related research provides an ideal research model system; in the model established by this culture method, cells establish cell communication with each other, and can differentiate to form capillary bile duct structure, which shows that the constructed hepatocyte culture system has more organizational characteristics, thus has It is beneficial for hepatocytes to maintain their phenotype and function during in vitro culture; in the culture system established by this method, the albumin secretion and urea synthesis functions of hepatocytes are significantly higher than those of the plane culture group and the "sandwich" culture group, closer to The functional activity of hepatocytes in vivo, and the metabolic enzyme activity of hepatocytes in this culture system are significantly higher than those of traditional culture methods (plane culture group and "sandwich" culture group), which is more conducive to accurate in vitro drug metabolism activity and liver toxicity. Evaluation, which may provide a new evaluation tool for new drug development.

附图说明Description of drawings

图1为多孔蚕丝蛋白支架的扫描电镜图（A：盐析法制备；B: 有机溶剂法制备）。Figure 1 is a scanning electron microscope image of a porous silk protein scaffold (A: prepared by salting out method; B: prepared by organic solvent method).

图2为利用活细胞荧光标记方法显示大鼠原代肝细胞（红色）和星形间质细胞（蓝色荧光）在多孔蚕丝蛋白支架上的生长形态（A：3D共培养组；B：对照组）和细胞活性（C）。Figure 2 shows the growth morphology of rat primary hepatocytes (red) and astrocytes (blue fluorescence) on porous silk protein scaffolds using fluorescent labeling of living cells (A: 3D co-culture group; B: control group) and cell viability (C).

图3为HE染色显示利用本发明所述方法培养的大鼠肝细胞的形态结构（A1-2：本发明培养组；B：对照组）。Fig. 3 is HE staining showing the morphological structure of rat hepatocytes cultured by the method of the present invention (A1-2: the culture group of the present invention; B: the control group).

图4为扫描电镜（SEM）检测显示大鼠原代肝细胞在多孔蚕丝蛋白支架上的生长形貌及特征（A1-2）；透射电镜（TEM）检测显示大鼠原代肝细胞形成细胞连接（B）。Figure 4 shows the growth morphology and characteristics of rat primary hepatocytes on porous silk protein scaffolds detected by scanning electron microscope (SEM) (A1-2); transmission electron microscope (TEM) detection shows that primary rat hepatocytes form cell junctions (B).

图5为本发明所述方法培养的大鼠肝细胞的白蛋白分泌(A)和尿素合成功能(B)。Fig. 5 shows albumin secretion (A) and urea synthesis function (B) of rat hepatocytes cultured by the method of the present invention.

图6为利用qRT-PCR方法检测本发明所述方法对大鼠原代肝细胞代谢酶基因表达水平的影响图例之一。Fig. 6 is one of the illustrations of detecting the effect of the method of the present invention on the gene expression level of metabolic enzymes in primary rat hepatocytes by using qRT-PCR method.

图7为利用qRT-PCR方法检测本发明所述方法对大鼠原代肝细胞代谢酶基因表达水平的影响图例之二。Fig. 7 is the second illustration of the influence of the method of the present invention on the expression level of metabolic enzyme genes in primary rat hepatocytes detected by qRT-PCR method.

图8为利用qRT-PCR方法检测本发明所述方法对大鼠原代肝细胞代谢酶基因表达水平的影响图例之三。Fig. 8 is the third example of detecting the effect of the method of the present invention on the gene expression level of metabolic enzymes in primary rat hepatocytes by using qRT-PCR method.

图9为本发明所述方法对大鼠原代肝细胞代谢酶CYP3A1活性的影响。Fig. 9 shows the effect of the method of the present invention on the activity of metabolic enzyme CYP3A1 in primary rat hepatocytes.

具体实施方式Detailed ways

下面将结合附图说明本发明的具体实施方式。如图1至图7所示：The specific implementation manner of the present invention will be described below with reference to the accompanying drawings. As shown in Figure 1 to Figure 7:

多孔蚕丝蛋白支架的制备：Preparation of porous silk protein scaffold:

首先制备浓度为6-7%的丝素蛋白溶液，采用盐析-干燥法制备多孔丝素蛋白支架，通过盐颗粒大小控制支架孔径为500-600 μm, 干燥后充分浸润于去离子水内，然后采用标准打孔器制备直径5mm，厚度3mm的三维支架（即多孔蚕丝蛋白支架）。多孔蚕丝蛋白支架在接种细胞前需经高温高压灭菌处理，且浸泡在培养基内进行预平衡12 h。First prepare a silk fibroin solution with a concentration of 6-7%, and prepare a porous silk fibroin scaffold by salting out-drying method. The pore size of the scaffold is controlled by the size of the salt particles to be 500-600 μm, and fully soaked in deionized water after drying. Then a three-dimensional scaffold with a diameter of 5 mm and a thickness of 3 mm (that is, a porous silk protein scaffold) was prepared using a standard puncher. The porous silk protein scaffold needs to be sterilized by high temperature and high pressure before inoculating cells, and soaked in the medium for pre-equilibration for 12 h.

大鼠原代肝细胞的分离培养：Isolation and culture of primary rat hepatocytes:

大鼠经麻醉后采用胶原酶两步灌流法（0.5mg/ml,型胶原酶）分离获取大鼠（成年，雄性Sprague Dawley大鼠）原代肝细胞，经过梯度percoll液分离后进行纯化、富集。通常可获得2×108细胞总量，且活性细胞>90%. 新鲜分离的肝细胞培养于William’s E培养基添加0.5 U/mL 胰岛素(Invitrogen), 7 ng/mL 胰高血糖素 (Sigma), 7.5 μg/mL 氢化考地松(Sigma) 和 1% 抗生素( Invitrogen)，After rats were anesthetized, two-step perfusion with collagenase (0.5mg/ml, Type collagenase) to obtain rat (adult, male Sprague Dawley rat) primary hepatocytes, purified and enriched after gradient percoll liquid separation. Usually 2×10 8 total cells can be obtained, and the viable cells are >90%. Freshly isolated hepatocytes were cultured in William's E medium supplemented with 0.5 U/mL insulin (Invitrogen), 7 ng/mL glucagon (Sigma) , 7.5 μg/mL hydrocortisone (Sigma) and 1% antibiotic (Invitrogen),

大鼠原代肝星形间质细胞购置美国ScienCell，培养于Stellate 培养基，细胞生长至近90%融合度时进行1：3常规传代培养，Rat primary hepatic stellate stromal cells were purchased from ScienCell in the United States and cultured in Stellate medium. When the cells grew to nearly 90% confluence, they were routinely subcultured at 1:3.

所有细胞均培养于37 ℃，5%CO2 细胞培养箱内。All cells were cultured at 37°C in a 5% CO2 incubator.

进行大鼠肝细胞的体外培养：In vitro culture of rat hepatocytes:

以上述步骤中获得的大鼠原代肝细胞为种子细胞，以多孔蚕丝蛋白支架作为肝细胞体外培养载体，将肝细胞、肝源性间质细胞和细胞外基质按一定比例接种到多孔蚕丝蛋白支架上，并将多孔蚕丝蛋白支架置于模拟肝细胞的体外培养微环境中进行培养，The primary rat hepatocytes obtained in the above steps were used as seed cells, and the porous silk protein scaffold was used as the carrier for in vitro culture of liver cells. on the scaffold, and the porous silk protein scaffold was cultured in an in vitro culture microenvironment simulating hepatocytes,

将新鲜分离的肝细胞和肝星形间质细胞按照细胞数3:1的比例混合，并将获得的混合细胞与细胞外基质混合，获得细胞-基质混合物，所述的细胞外基质由胶原和基质胶按照体积比为1:1的比例混合而成（所述的胶原和基质胶均选用BD Biosciences的产品），然后将细胞-基质混合物多点接种至多个多孔蚕丝蛋白支架，并保证肝细胞数量为600,000cells/支架，基质胶的体积为30μL/支架，Freshly isolated hepatocytes and hepatic stellate cells were mixed according to the ratio of cell number 3:1, and the obtained mixed cells were mixed with extracellular matrix to obtain a cell-matrix mixture, and the extracellular matrix was composed of collagen and Matrigel is mixed according to the volume ratio of 1:1 (both the collagen and Matrigel are products of BD Biosciences), and then the cell-matrix mixture is multi-point seeded on multiple porous silk protein scaffolds to ensure that the hepatic cells The quantity is 600,000cells/stent, the volume of Matrigel is 30μL/stent,

将接种完成的多孔蚕丝蛋白支架按照1个支架/孔的密度放置到多孔细胞培养板中，并将多孔细胞培养板置于细胞培养箱中，在37℃、5%CO2的条件下充分凝胶化2h，然后向多孔细胞培养板的每个孔中缓慢加入共培养基，所述的共培养基为肝细胞培养基和星形间质细胞培养基按照体积比为1:1的比例混合而成，The inoculated porous silk protein scaffolds were placed in a porous cell culture plate at a density of 1 scaffold/well, and the porous cell culture plate was placed in a cell culture incubator, and fully condensed under the conditions of 37°C and 5% CO 2 . After gelling for 2 hours, slowly add co-culture medium to each well of the multi-well cell culture plate. The co-culture medium is a mixture of hepatocyte culture medium and astrocyte culture medium at a ratio of 1:1 by volume made,

通过上述步骤，构建出肝细胞的三维共培养模型。Through the above steps, a three-dimensional co-culture model of hepatocytes was constructed.

同批次新鲜分离的大鼠原代肝细胞以相同密度分别直接接种于经胶原包被的24孔板作为对照组1（平面培养组）；同时设定传统三明治培养组为对照组2。Rat primary hepatocytes freshly isolated from the same batch were directly inoculated on collagen-coated 24-well plates at the same density as control group 1 (plane culture group); at the same time, the traditional sandwich culture group was set as control group 2.

建立评价肝细胞三维共培养模型的形态学检测平台：Establish a morphological detection platform for evaluating the three-dimensional co-culture model of hepatocytes:

肝细胞生长形态及活性检测：为了更好的观察三维共培养体系内不同细胞，首先采用活细胞标记染料DiI 和DiD（Invitrogen）分别标记原代肝细胞和星形间质细胞，培养5天后于激光共聚焦显微镜下直接观察。细胞活性染色采用calcein-AM/EthD-1 染色试剂盒(Invitrogen)，37℃孵育2h，其余操作按说明书进行。染色后于激光共聚焦显微镜下观察、拍照。Hepatocyte growth morphology and activity detection: In order to better observe the different cells in the three-dimensional co-culture system, the living cell labeling dyes DiI and DiD (Invitrogen) were used to label the primary hepatocytes and astrocytes respectively, and cultured for 5 days. Direct observation under confocal laser microscopy. Cell viability was stained with calcein-AM/EthD-1 staining kit (Invitrogen), incubated at 37°C for 2 hours, and other operations were performed according to the instructions. After staining, the cells were observed and photographed under a confocal laser microscope.

H&E染色：样品收集后经PBS冲洗、4%多聚甲醛室温下固定24 h，由形态学中心制备石蜡切片。再依次经过脱蜡、H&E染色以及封片，制备H&E染色切片。正视光学显微镜下观察，拍照。H&E staining: After the samples were collected, they were rinsed with PBS, fixed with 4% paraformaldehyde at room temperature for 24 h, and paraffin sections were prepared from the Morphological Center. After dewaxing, H&E staining and mounting, the H&E stained sections were prepared. Observe under an optical microscope and take pictures.

扫描电镜和透射电镜观察：所收集样品经PBS充分清洗后，2.5%戊二醛溶液前固定4-6 h，PBS冲洗后，2%锇酸后固定2 h，再次PBS充分清洗后，部分样品置于真空干燥箱内干燥过夜。干燥后样品经喷金15min后，于扫描电镜下观察拍照。其余样品送至透射电镜检测中心，经树脂包埋后制备超薄切片，于透射电镜下观察、拍照。Scanning electron microscope and transmission electron microscope observation: After the collected samples were fully washed with PBS, pre-fixed with 2.5% glutaraldehyde solution for 4-6 h, after washing with PBS, post-fixed with 2% osmic acid for 2 h, and after fully washed with PBS again, some samples Dry overnight in a vacuum oven. After drying, the sample was sprayed with gold for 15 minutes, observed and photographed under a scanning electron microscope. The rest of the samples were sent to the transmission electron microscope testing center, and were embedded in resin to prepare ultra-thin sections, observed and photographed under the transmission electron microscope.

建立评价肝细胞三维共培养模型的功能活性检测平台：Establish a functional activity detection platform for evaluating the three-dimensional co-culture model of hepatocytes:

白蛋白含量测定及尿素合成测定：在培养过程中定期收集各培养组内培养基，-80℃保存后批量测量白蛋白及尿素含量。其中，白蛋白测定采用大鼠白蛋白ELISA试剂盒，尿素合成的测定采用QuantiChrom尿素检测试剂盒。具体操作均依照说明书完成。Determination of albumin content and urea synthesis: during the cultivation process, the medium in each culture group was collected regularly, and the albumin and urea content were measured in batches after storage at -80°C. Among them, rat albumin ELISA kit was used for the determination of albumin, and QuantiChrom urea detection kit was used for the determination of urea synthesis. The specific operations are completed in accordance with the manual.

定量RT-PCR测定代谢酶基因表达：定期收集样品，直接采用TRIZOL裂解细胞，RNAeasy 试剂盒(Qiagen).提取总RNA后，采用PrimeScriptTM RT试剂盒和 SYBR Premix ExTaqTM II试剂盒 （TaKaRa）进行定量RT-PCR扩增。依据各目的基因的Ct值及内参基因的Ct值进行定量分析（ABI smart I），确定各目的基因的表达水平。所扩增基因的引物序列如下：Quantitative RT-PCR determination of gene expression of metabolic enzymes: Collect samples regularly, lyse cells directly with TRIZOL, RNAeasy kit (Qiagen). After extracting total RNA, use PrimeScript TM RT kit and SYBR Premix ExTaq TM II kit (TaKaRa) for Quantitative RT-PCR amplification. Quantitative analysis (ABI smart I) was performed based on the Ct value of each target gene and the Ct value of the internal reference gene to determine the expression level of each target gene. The primer sequences of the amplified genes are as follows:

定量RT-PCR检测的引物序列Primer sequences for quantitative RT-PCR detection

GeneGene ForwardForward ReverseReverse rGAPDHrGAPDH CAAGGTCATCCATGACAACTTTGCAAGGTCATCCATGACAACTTTG GGGCCATCCACAGTCTTCTGGGGCCATCCACAGTCTTCTG rCYP1A2rCYP1A2 CGGTGATTGGCAGAGATCGGCGGTGATTGGCAGAGATCGG GTCCCTCGTTGTGCTGTGGGTCCCTCGTTGTGCTGTGG rCYP2B1rCYP2B1 CTCCAAAAACCTCCAGGAAATCCTCCTCCAAAAACCTCCAGGAAATCCTC GTGGATAACTGCATCAGTGTATGGCGTGGATAACTGCATCAGTGTATGGC rCYP3A1rCYP3A1 GAGGAGTAATTTGCTGACAGAACCTGCGAGGAGTAATTTGCTGACAGAACCTGC CCAGGAAT CCCCTGTTTCTTGAACCAGGAAT CCCCTGTTTCTTGAA

r: Rat-specific primerr: Rat-specific primer

肝细胞代谢酶活性测定：于培养第6天，更换无血清培养基，并加入50μM睾酮，37℃孵育4h后收集培养基，4℃离心（12000rpm，10min）后收集上清液保存至-80℃冰箱。带样品收集结束后，采用HPLC方法进行检测CYP3A代谢产物6β羟基睾丸酮的水平（检测条件：A(methanol): B (water) = 52:48, 0-15 min, B 48-30, 15-20 min30-20, UV 254 nm）。Hepatocyte metabolic enzyme activity assay: On the 6th day of culture, replace the serum-free medium, add 50 μM testosterone, incubate at 37°C for 4 hours, collect the medium, centrifuge at 4°C (12000rpm, 10min), collect the supernatant and store it at -80 ℃ refrigerator. After the samples were collected, the HPLC method was used to detect the level of CYP3A metabolite 6β-hydroxytestosterone (detection conditions: A (methanol): B (water) = 52:48, 0-15 min, B 48-30, 15-20 min30-20, UV 254nm).

结果：result:

1.肝细胞生长形态及活性：如图2所示，在多孔蚕丝蛋白支架上原代肝细胞生长状态良好，细胞活性染色显示大部分细胞均为绿色荧光，说明其活性理想。仅有少量细胞死亡，显示红色荧光。细胞标记图片提示，肝细胞在三维共培养体系呈现聚团生长模式，且易于与星形间质细胞彼此聚合成团；而对照组细胞较为分散，未见细胞彼此聚合成团现象。分析肝细胞聚合成团有理由肝细胞在体外培养过程更好的维持其表型及功能。1. Growth morphology and activity of hepatocytes: As shown in Figure 2, the primary hepatocytes grew well on the porous silk protein scaffold, and cell viability staining showed that most of the cells were green fluorescent, indicating that their activity was ideal. Only a few cells died, showing red fluorescence. The pictures of the cell markers indicated that the hepatocytes in the three-dimensional co-culture system exhibited an aggregated growth pattern, and were easy to aggregate with the astrocytes into clusters; while the cells in the control group were more dispersed, and no cells aggregated into clusters with each other. The analysis of hepatocyte aggregates has reasons that hepatocytes can better maintain their phenotype and function during in vitro culture.

2. H&E染色（图3）结果进一步证明肝细胞团的形成及形态特征。可见：大部分肝细胞与星形间质细胞在多孔蚕丝蛋白支架上聚团生长。其中，肝细胞呈现多角形，可见单核或双核，而星形间质细胞分布于肝细胞团内，二者紧密聚合在一起，支持两种细胞间可能存在细胞-细胞间相互作用。2. The results of H&E staining (Figure 3) further proved the formation and morphological characteristics of hepatocyte clusters. It can be seen that most of the hepatocytes and astrocytes aggregate and grow on the porous silk protein scaffold. Among them, the hepatocytes were polygonal, with single or double nuclei, and the stellate interstitial cells were distributed in the hepatocyte mass, and the two closely aggregated together, supporting the possible existence of cell-cell interaction between the two types of cells.

3. 如图4所示，扫描电镜结果显示肝细胞紧密生长于多孔蚕丝蛋白支架表面及三维孔隙，细胞形态规则，且分泌大量的细胞外基质。彼此间相互连接，形成类组织化的表观形貌特征。透色电镜分析进一步显示肝细胞与肝细胞之间能够建立细胞紧密连接,彼此间存在交流，呈现“类组织化”特征。这些都支持肝细胞在体外培养过程中的良好活性、组织化特征以及理想表型。3. As shown in Figure 4, the results of scanning electron microscopy showed that hepatocytes grew closely on the surface of the porous silk protein scaffold and the three-dimensional pores, the cells were regular in shape, and secreted a large amount of extracellular matrix. They are connected with each other to form the appearance characteristics of quasi-organization. Transmission electron microscope analysis further showed that hepatocytes could establish tight junctions between hepatocytes, and communicate with each other, presenting a "like tissue" feature. These all support the good viability, organizational characteristics and ideal phenotype of hepatocytes during in vitro culture.

如图5所示，肝细胞白蛋白分泌及尿素合成检测结果表明：与对照组比较，基于丝蛋白支架的三维共培养组内肝细胞能够分泌较高水平的白蛋白；此外其尿素合成水平也比对照组有显著提高（P<0.05）。该结果说明，原代肝细胞在该培养体系下能够维持理想的合成功能活性。As shown in Figure 5, the detection results of albumin secretion and urea synthesis in hepatocytes showed that: compared with the control group, the hepatocytes in the three-dimensional co-culture group based on silk protein scaffolds could secrete a higher level of albumin; in addition, the level of urea synthesis was lower. Compared with the control group, it was significantly improved (P<0.05). The results indicated that primary hepatocytes could maintain ideal synthetic functional activity in this culture system.

肝细胞代谢酶表达水平的评价及酶活性测定：肝细胞代谢酶的改变是对肝细胞代谢功能进行评价的重要指标。如图6、7、8、9所示，经三维共培养后大鼠原代肝细胞的多种细胞色素代谢酶基因表水平显著高于对照组，且通过特异性底物代谢分析发现：与对照培养组比较，三维培养体系内肝细胞对睾酮的代谢提高，证明其CYP3A的代谢活性在该培养体系内也能够理想维持，从而为肝细胞的理想功能提供了有一实验论据。Evaluation of the expression level of hepatocyte metabolic enzymes and determination of enzyme activity: the change of hepatic metabolic enzymes is an important index for evaluating the metabolic function of hepatic cells. As shown in Figures 6, 7, 8, and 9, the gene expression levels of various cytochrome metabolic enzymes in rat primary hepatocytes after three-dimensional co-culture were significantly higher than those in the control group, and it was found through the analysis of specific substrate metabolism: Compared with the control culture group, the metabolism of testosterone by hepatocytes in the three-dimensional culture system was improved, which proved that the metabolic activity of CYP3A could also be maintained ideally in this culture system, thus providing an experimental argument for the ideal function of hepatocytes.

Claims (1)

1.一种可提高肝细胞体外分化表型及功能的培养方法，其特征在于：所述的方法按照以下步骤进行：采用肝细胞为种子细胞，以多孔蚕丝蛋白支架作为肝细胞体外培养载体，将肝细胞、肝源性间质细胞和细胞外基质按一定比例接种到多孔蚕丝蛋白支架上，并将多孔蚕丝蛋白支架置于模拟肝细胞的体外培养微环境中进行培养，1. A culture method that can improve hepatocyte differentiation phenotype and function in vitro, characterized in that: the method is carried out according to the following steps: adopting hepatocytes as seed cells, using porous silk protein scaffold as hepatocyte in vitro culture carrier, Hepatocytes, liver-derived mesenchymal cells and extracellular matrix were seeded on the porous silk protein scaffold in a certain proportion, and the porous silk protein scaffold was placed in an in vitro culture microenvironment simulating hepatocytes for cultivation. 所述的将肝细胞、肝源性间质细胞和细胞外基质按一定比例接种到多孔蚕丝蛋白支架上的具体操作步骤如下：将新鲜分离的肝细胞和肝星形间质细胞按照细胞数3:1的比例混合，并将获得的混合细胞与细胞外基质混合，获得细胞-基质混合物，所述的细胞外基质由胶原和基质胶按照体积比为1:1的比例混合而成，然后将细胞-基质混合物多点接种至多个多孔蚕丝蛋白支架，并保证肝细胞数量为600,000cells/支架，基质胶的体积为30μL/支架，The specific operation steps of seeding hepatocytes, hepatic stromal cells and extracellular matrix on the porous silk protein scaffold according to a certain ratio are as follows: Freshly separated hepatocytes and hepatic stellate stromal cells are divided into 3 :1 ratio, and the obtained mixed cells are mixed with the extracellular matrix to obtain the cell-matrix mixture, and the extracellular matrix is formed by mixing collagen and Matrigel at a volume ratio of 1:1, and then The cell-matrix mixture was inoculated onto multiple porous silk protein scaffolds at multiple points, and the number of hepatocytes was guaranteed to be 600,000 cells/scaffold, and the volume of matrigel was 30 μL/scaffold. 所述的将多孔蚕丝蛋白支架置于模拟肝细胞的体外培养微环境中进行培养的具体操作步骤如下：将接种完成的多孔蚕丝蛋白支架按照1个支架/孔的密度放置到多孔细胞培养板中，并将多孔细胞培养板置于细胞培养箱中，在37℃、5%CO2的条件下凝胶化2h，然后向多孔细胞培养板的每个孔中加入共培养基，所述的共培养基为肝细胞培养基和星形间质细胞培养基按照体积比为1:1的比例混合而成，The specific operation steps of placing the porous silk protein scaffold in an in vitro culture microenvironment simulating liver cells are as follows: place the inoculated porous silk protein scaffold in a porous cell culture plate at a density of 1 scaffold/well , and put the multi-well cell culture plate in the cell culture incubator, gelatinize at 37°C and 5% CO 2 for 2 hours, then add the co-culture medium to each well of the multi-well cell culture plate, the co-culture medium The culture medium is prepared by mixing hepatocyte medium and astrocytic medium at a volume ratio of 1:1. 所述的多孔蚕丝蛋白支架按照如下步骤制备：首先制备浓度为6-7%的丝素蛋白溶液，采用盐析-干燥法制备多孔丝素蛋白支架，通过盐颗粒大小控制支架孔径为500-600 μm,干燥后充分浸润于去离子水内，然后采用标准打孔器制备直径5mm，厚度3mm的三维支架，即多孔蚕丝蛋白支架，并且多孔蚕丝蛋白支架在接种细胞前需经高温高压灭菌处理，且浸泡在培养基内进行预平衡12 h。The porous silk fibroin scaffold is prepared according to the following steps: first, a silk fibroin solution with a concentration of 6-7% is prepared, and a porous silk fibroin scaffold is prepared by a salting-out-drying method, and the pore diameter of the scaffold is controlled to be 500-600 by the size of the salt particles. μm, fully immersed in deionized water after drying, and then use a standard puncher to prepare a three-dimensional scaffold with a diameter of 5mm and a thickness of 3mm, that is, a porous silk protein scaffold, and the porous silk protein scaffold must be sterilized by high temperature and high pressure before inoculating cells , and pre-equilibrated by immersing in the culture medium for 12 h.

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