WO2016127908A1 - 一种三维复合细胞聚集体模型及其制备方法与应用 - Google Patents

一种三维复合细胞聚集体模型及其制备方法与应用 Download PDF

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WO2016127908A1
WO2016127908A1 PCT/CN2016/073472 CN2016073472W WO2016127908A1 WO 2016127908 A1 WO2016127908 A1 WO 2016127908A1 CN 2016073472 W CN2016073472 W CN 2016073472W WO 2016127908 A1 WO2016127908 A1 WO 2016127908A1
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cad
fusion protein
cell
microspheres
dimensional composite
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杨军
张妍
李素华
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南开大学
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  • the invention relates to the technical field of cell culture in vitro, in particular to a three-dimensional composite cell aggregate model and a preparation method and application thereof.
  • cells are in a highly informative microenvironment that includes a variety of temporal/space-changing physicochemical signals that are regulated by various signal stimuli to achieve their specific biological functions.
  • This interaction between the cell and the microenvironment includes various complex biochemical, biomechanical and bioelectrical signal stimuli responses from surrounding cells, extracellular matrices and soluble factors.
  • cells interact with cells through direct contact or paracrine secretion of soluble factors.
  • This intercellular communication function is an important link to maintain the structure and function of cells, tissues and organs, and is also a key factor regulating the repair and reconstruction of tissues in vitro and in vivo.
  • Three-dimensional cell culture refers to the co-culture of a material carrier having a three-dimensional structure with various kinds of cells in vitro, so that cells can migrate and grow in the three-dimensional spatial structure of the carrier to form a three-dimensional structure.
  • Cell-carrier complex The design and preparation of the carrier material in the culture mode can not only simulate the in vivo environment to maximize the expression of the cell function, but also exhibit the advantages of cell culture intuitiveness and conditional controllability, and is the current in vitro and in vivo tissue structure and One of the most common means of functional reconstruction.
  • ESCs embryonic stem cells
  • MSCs Mesenchymal stem cells
  • Various tissue cells such as tendon, ligament, nerve, liver, heart muscle and endothelium can be used as ideal seed cells for tissue damage repair caused by aging and lesions.
  • the differentiation of embryonic stem cells should first be induced in a serum-free medium to form cell aggregates, ie, embryoid bodies (EB). After that, mimic the conditions of in vivo embryogenesis, sequentially change the growth environment of the embryoid bodies, and induce differentiation. Specific cells.
  • the existing methods for amplification and differentiation of MSCs are mainly two-dimensional plate culture. Since the growth mode of cells in the three-dimensional culture system is closer to the body, the complex in vivo cell microenvironment can be more realistically simulated, and the differentiation of stem cells in vitro can be promoted.
  • the mesenchymal stem cell aggregates were found to enhance the secretion of cytokines in mesenchymal stem cells and significantly increase the efficiency of cell differentiation.
  • problems such as decreased long-term culture cell activity, low induction differentiation efficiency, unstable induction effect, and inaccurate in vivo induction effects in the culture and directed differentiation of the above two, which are urgently needed to be solved. Apply conversions.
  • microspheres are mainly common natural extracellular matrix or synthetic chemical materials, and have not deviated from traditional cell culture.
  • Cadherin is an important component of intercellular adhesion and has a function of mediating cell-to-cell specific linkage. It has been shown in the literature that E-cadherin fusion proteins can promote a variety of cell proliferation and increase cell viability. For example, the T. AKAike team led the construction of the murine E-cadherin extracellular domain and the IgG protein Fc-end recombinant protein mE-cadherin-Fc. The experimental results showed that with collagen control, mouse hepatic primary cells could adhere to the surface of mE-Cadherin-Fc coated membrane better mediated by cell membrane protein E-cadherin, and the DNA synthesis activity decreased and tryptophan The expression of synthase remained unchanged.
  • the research group also applied mE-cadherin-Fc to the culture of mouse embryonic stem cells. It was found that embryonic stem cells did not form colonies on the surface of the plate, which could amplify and significantly inhibit cell self-differentiation. . Current research on human cadherin proteins is rarely reported.
  • the present inventors have previously cultured human bone marrow mesenchymal stem cells two-dimensionally on a culture plate coated with human-derived cell membrane protein E-cadherin, and transformed cell-to-cell interaction between cells and matrix, and found that in hE- The Cad-Fc fusion protein matrix significantly improved the adhesion, proliferation and liver-directed differentiation of two-dimensional cultured mesenchymal stem cells.
  • fusion proteins with E-cadherin function were prepared by genetic engineering techniques and used for surface modification of biomaterial microspheres to prepare E-cadherin-mediated composite cells of biomaterial microspheres and stem cells. Aggregates, establish a three-dimensional culture technique that promotes the expansion and directed differentiation of mesenchymal stem cells more effectively, and interacts with cell-cell, cell-extracellular matrix and cell-soluble factors, and constructs mesenchymal stem cells in vitro. Tissue engineering of various types of tissue and organ analogs or equivalents has important theoretical research significance and application development value.
  • the technical problem to be solved by the present invention is to provide a three-dimensional composite cell aggregate model, which fully considers the characteristics of strong mutual contact between cells in a three-dimensional aggregate microenvironment, and proposes to modify the surface-controlled release cells with intercellular adhesion factor for the first time.
  • the microspheres of growth factors promote the microsphere/stem cell composite microspheres by co-culture of microspheres with cells by cadherin and adhesion-linked signal molecules, which is a stem cell culture technique for regulating the biological activity of aggregate cells from aggregates. .
  • Another technical problem to be solved by the present invention is to provide a method for preparing the above three-dimensional composite cell aggregate model.
  • Another technical problem to be solved by the present invention is to provide an application of the above three-dimensional composite cell aggregate model.
  • the technical solution of the present invention is:
  • a three-dimensional composite cell aggregate model is a stem cell aggregate containing microspheres inside, and a fusion protein molecule of a genetically engineered cadherin protein family is immobilized on the surface of the microsphere, and the fusion protein molecule and stem cells are self-owned by stem cells.
  • the cadherin adhesion factor is constructed, and the microspheres are co-cultured with stem cells.
  • the microspheres are prepared by natural or chemically synthesized polymers, and have a particle size ranging from 0.5 to 40 ⁇ m.
  • the polymer is a natural polymer, including collagen, gelatin, alginic acid, chitosan, hyaluronic acid; or a chemically synthesized polymer, including polylactic acid, polyglycolic acid , polylactic acid-glycolic acid, polyethylene glycol or polystyrene.
  • the inside or the surface of the microsphere can release or fix the cell active factor.
  • the stem cells are embryonic stem cells or mesenchymal stem cells.
  • the above three-dimensional composite cell aggregate model the fusion protein molecule of the genetically engineered cadherin family, including the extracellular domain of cadherin protein and the Fc domain of immunoglobulin, and mediated by Fc or Chemical bonding, used for various forms of material bulk or / and material surface modification, improve the hydrophilicity of the material and cadherin-mediated cell-specific adhesion, improve the biocompatibility of the material.
  • the type of the fusion protein molecule is E-cadherin protein.
  • the fusion protein molecule is genetically engineered to recombine the E-cadherin extracellular domain and the immunoglobulin Fc domain gene to construct a double function comprising E-cadherin and Fc.
  • the fusion protein gene sequence is obtained by gene transfection, protein expression, and separation and purification.
  • the fusion protein molecule is immobilized on the surface of the microsphere by Fc, and after mixing with the stem cells, the microsphere/cell composite aggregate containing the microsphere is prepared by centrifugation or hanging drop method. Then maintain its three-dimensional structure for long-term culture, in order to achieve the three-dimensional cell-matrix interaction and cell-cell interaction through tissue engineering principles and techniques, effectively improve the internal structure and cell function regulation of cell aggregates. .
  • the fusion protein molecule is The eukaryotic expression gene hE-cad-Fc recombinantly constructed by the extracellular domain hE-cad of the human epithelial cell cadherin adhesion factor hE-cadherin and the Fc domain domain Fc gene of human immunoglobulin IgG 1
  • the base sequence is the sequence described in Sequence Listing ⁇ 400>1.
  • the fusion protein molecule is a hE-cad-Fc fusion prepared by transfection of a gene plasmid by eukaryotic gene transfection, protein expression and protein A affinity chromatography column.
  • the protein was subjected to SDS-PAGE and Western blot analysis, and the base sequence thereof was the sequence of the sequence ⁇ 400>1.
  • the specific step of obtaining the gene plasmid is: using a recombinant gene PCR method to amplify the target gene hE-cad and Fc sequences, and respectively connecting and embedding in eukaryotic cells by double restriction enzyme digestion.
  • the gene plasmid vector pcDNA3.1 the eukaryotic expression gene plasmid pcDNA3.1-E-cad-Fc containing the bifunctional target gene hE-cad-Fc fragment was obtained, and the base sequence thereof is described in the sequence table ⁇ 400>2. sequence.
  • the fusion protein molecule is used in a concentration range of 0.1-20 ⁇ g/ml.
  • the stem cells are mixed with the above hE-cad-Fc matrix microspheres, and the hanging cells are suspended.
  • Methods such as centrifugation and centrifugation induce stem cells to form three-dimensional aggregates, or envelop cells in a three-dimensional culture environment such as a hydrogel or a porous scaffold to spontaneously form cell aggregates.
  • the natural polymer is collagen, gelatin, alginate, chitosan, hyalonic acid. Or a polymer obtained by chemically modifying the above materials.
  • the chemically synthesized polymer is polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid (PLGA), polyethylene glycol (PEG). Or polystyrene (PS) or a polymer obtained by chemically modifying the above materials.
  • the obtained microspheres have a particle diameter ranging from 0.5 to 40 ⁇ m.
  • the hE-cad-Fc fusion protein is used at a concentration of 0.1-20 ⁇ g/ml.
  • the mixing ratio of the cells to hE-cad-Fc matrix microspheres is 1:1-10:1.
  • the method for preparing the three-dimensional composite cell aggregate model is prepared by hanging drop culture for 48h-72h or after centrifugation for 12-24h.
  • the application of the above three-dimensional composite cell aggregate model can be used to improve the adhesion, proliferation, bulk amplification of mesenchymal stem cells, and the functional expression of differentiated cells differentiated and differentiated by time-dependent and directed regulation of mesenchymal stem cells.
  • the application of the above three-dimensional composite cell aggregate model can be used to study fine Cell-cell, cell-extracellular matrix, and cell-solubility factor interactions, as well as in vitro mesenchymal stem cell expansion culture, construction of tissue engineered various organelle analogs or equivalents.
  • the present invention utilizes submicron-sized microspheres to release cytokines and mediates the interaction between biological materials and cells by human E-cadherin to form a three-dimensional composite cell aggregate model that can regulate cell biological activity from the inside out.
  • the model can be used for the study of proliferation and differentiation of stem cells in three-dimensional state, and overcomes the shortcomings of three-dimensional culture of traditional stem cells, which are caused by the influence of mass transfer resistance on cell heterogeneous growth and contact inhibition and proliferation, and construct and optimize stem cells in vitro.
  • Three-dimensional biomimetic culture techniques, as well as tissue engineering to construct various types of tissue and organ analogs or equivalents provide theoretical and technical support.
  • the present invention utilizes genetic engineering principles and techniques to fuse the extracellular domain of human epithelial cell E-cadherin with the human immunoglobulin IgG 1 Fc fragment to prepare a fusion protein having E-cadherin protein and Fc dual function.
  • the fusion protein of the present invention forms a monolayer on the surface of the material by Fc-mediated self-assembly, thereby realizing surface modification of the material, improving the hydrophilicity of the material, improving the biocompatibility of the material, and enhancing the specificity of the cells and materials. Interaction and further regulation of intracellular signaling pathways.
  • the hE-cad-Fc fusion protein of the invention promotes stem cell proliferation and differentiation function expression under the condition of three-dimensional aggregate culture by matrix fixation on the surface of the microsphere, and promotes the batch of seed cells related to tissue engineering and regenerative medicine. Amplification and induction of differentiation.
  • the fusion protein matrix immobilization method of the invention is simple and easy, and the modification efficiency is high. Good stability and reduced experimental costs.
  • the three-dimensional aggregate model constructed by the present invention can more effectively realize three-dimensional biomimetic culture of cells, and to investigate the interaction between cell-cell, cell-extracellular matrix and cell-soluble factor, and to examine specific cytokines in three-dimensional aggregates.
  • the basic and applied researches on the regulation and mechanism of biological behaviors such as growth, migration, proliferation and differentiation, and the molecular regulation mechanism of stem cell differentiation, provide stem cell culture techniques.
  • Figure 1 is a schematic diagram of the construction of a recombinant plasmid
  • Figure 2 is the identification of recombinant plasmids
  • Figure 3 is a purification fusion protein identification
  • Figure 4 is a quantitative detection of the amount of protein immobilized on the surface of PLGA microspheres by different concentrations of hE-cad-Fc fusion protein
  • Figure 5 is an immunofluorescence assay for matrix-immobilized hE-cad-Fc fusion protein on the surface of PLGA microspheres
  • Figure 6 is a graph showing the effect of hE-cad-Fc fusion protein-mediated surface-modified PLGA microspheres on the proliferation of mesenchymal stem cells
  • Figure 7 is a diagram showing the effect of hE-cad-Fc fusion protein-mediated surface-modified PLGA microspheres on the morphology of mesenchymal stem cells
  • Figure 8 is a schematic diagram of a three-dimensional composite cell aggregate model
  • Figure 9 is a photomicrograph of the morphology of simple cell aggregates and microspheres/cell complex aggregates
  • Figure 10 is a three-dimensional complex of hE-cad-Fc fusion protein-based surface-modified PLGA microspheres The effect of mesenchymal stem cell aggregate cell proliferation;
  • Figure 11 is a graph showing the effect of hE-cad-Fc fusion protein-mediated surface-modified PLGA microspheres on the cell viability of three-dimensional composite mesenchymal stem cell aggregates.
  • Figure 12 is a graph showing the effect of hE-cad-Fc fusion protein-mediated surface-modified PLGA microspheres on cell membrane signaling pathway in three-dimensional composite mesenchymal stem cell aggregates.
  • the PCR product was analyzed by 1% agarose gel electrophoresis, and the PCR product was digested with Hind III and Not I, and inserted between the Hind III and Not I sites of pcDNA3.1-Fc. .
  • the plasmid pcDNA3.1-Fc was kindly provided by Prof. Chi Chi Min Hong, Tokyo Institute of Technology.
  • the recombinant expression plasmid was identified by Hind III and Not I double digestion electrophoresis, and it was revealed that a DNA fragment identical in size to the target gene was excised, and the plasmid vector was named pcDNA3.1-hE-cad-Fc.
  • Sequencing assay showed that the full length of pcDNA3.1-hE-cad-Fc sequence was 3020 bases, and the base sequence of the gene plasmid was the sequence of ⁇ 400>2 in the sequence listing.
  • the plasmid pcDNA3.1-hE-cad-Fc containing the gene of interest constructed in Example 1 was transfected into 293F cells, cultured in suspension for 72 hours, and the cell culture supernatant was collected, and the hE-cad-Fc fusion protein was purified by rProtein AFF column. .
  • the purified fusion protein was detected by immunoblotting using an anti-E-cadherin antibody. As shown in Figure 3, there were no other non-specific exposure bands in the figure.
  • the band with a molecular weight of approximately 120 KD was a reduced E-cadherin (Fig. 3 1)
  • the strip at 240 KD is a non-reducing E-cadherin (2 in Figure 3).
  • the hE-cad-Fc fusion protein is prepared by the present invention, and the base sequence thereof is the sequence of ⁇ 400>1 in the sequence table, and the non-reduced state of the fusion protein can form a stable dimer structure.
  • microspheres with a particle size range of 15 ⁇ 5 ⁇ m were prepared by chemically synthesizing polymer PLGA, and then the above hE-cad-Fc fusion protein solution was diluted to 10 ⁇ g/ml, soaked in microspheres, and incubated at 37 °C. The supernatant was discarded by centrifugation, and the unfixed fusion protein hE-cad-Fc was removed by rinsing with PBS three times to obtain hE-cad-Fc matrix-modified surface-modified PLGA microspheres, which was named hE-cad- Fc-PLGA.
  • the present invention detects the protein concentration of hE-cad-Fc solution against PLGA micro by Elisa method.
  • the hE-cad-Fc matrixing method is the same as above. Namely: after obtaining hE-cad-Fc-PLGA, add 1% BSA for 1 h at room temperature, add anti-E-cadherin antibody for 1 h at 37 °C, wash 3 times with PBST, add biotin-labeled secondary antibody for 30 min at 37 °C, wash with PBS 3 Then, TBA coloring solution was added and incubated at 37 ° C for 10 min in the dark.
  • the stop solution was added, and 100 ⁇ l of the supernatant was centrifuged and added to a 96-well plate. The result was obtained by reading the plate at a wavelength of 450 nm using a microplate reader within 10 min.
  • the concentration of hE-cad-Fc solution increased, the amount of matrix-derived hE-cad-Fc protein on the surface of PLGA microspheres increased gradually.
  • the distribution of hE-cad-Fc fusion protein on the surface of the microspheres was detected by E-cadherin immunofluorescence staining of PLGA and hE-cad-Fc-PLGA.
  • the fusion protein was fixed, it was blocked with 1% BSA for 1 h at room temperature, and anti-E-cadherin antibody was added to incubate at 4 ° C overnight.
  • the fluorescently labeled secondary antibody was added at 37 ° C for 1 h, washed with PBS 3 times, and the microspheres were added dropwise. On the slide, the film was observed under a laser confocal microscope. The results showed that the hE-cad-Fc-PLGA surface was evenly covered with hE-cad-Fc fusion protein.
  • Example 4 Effect of hE-cad-Fc-PLGA on proliferation and morphology of mesenchymal stem cells
  • the invention adopts the CCK-8 method to detect the effects of PLGA microspheres and hE-cad-Fc-PLGA on the proliferation of umbilical cord mesenchymal stem cells.
  • hE-cad-Fc-PLGA, unfixed protein PLGA microspheres (control group) and mesenchymal stem cells were mixed in a ratio of 1:1, taking 5000 mesenchymal cells per well, 5000 per well.
  • Mesenchymal cells + 5000 PLGA microspheres, and 5000 mesenchymal cells per well + 5000 h hE-cad-Fc-PLGA microspheres were added to a 96-well plate and cultured in a 37 ° C 5% CO 2 incubator.
  • the morphology of the cells was observed by cytoskeleton immunofluorescence staining, and the experimental grouping was the same as above.
  • 2*10 4 mesenchymal cells per well, cells and microspheres were 1:1 planted in 24-well plates, cultured in a 37 ° C 5% CO 2 incubator for 5 days, 4% paraformaldehyde The solution was fixed for 5 min, and the cells were permeabilized with 0.1% Triton x-100 for 10 min. The cells were blocked with 1% bovine serum white egg (BSA) for 1 h at room temperature.
  • BSA bovine serum white egg
  • the cytoskeletal microfilaments (green) were labeled with FITC-Phalloidin, and the nucleus (red) was labeled with PI, observed under a fluorescent confocal microscope, see Figure 7.
  • mesenchymal stem cells and fusion protein microparticles were prepared by basalizing and immobilizing the human epithelial cell adhesion factor hE-cad-Fc fusion protein on the surface of PLGA microspheres (hE-cad-Fc-PLGA).
  • the spheres are mixed to form cell aggregates, forming a three-dimensional culture model of the composite cells.
  • the specific construction method of this model is as follows:
  • the microspheres prepared by chemically synthesizing polymer PLGA have a particle size ranging from 15 ⁇ 5 ⁇ m;
  • the hE-cad-Fc fusion protein purified by the eukaryotic protein expression system was diluted to 10 ⁇ g/ml, the microspheres were immersed, and incubated at 37 ° C for 2 hours;
  • the sphere/cell forms a composite three-dimensional aggregate.
  • the morphology of the cells was observed by phase contrast microscopy. As shown in Fig. 9, the results showed that MSC cells were aggregated with hE-cad-Fc-PLGA microspheres compared with MSC cell aggregates and MSC cells and PLGA microsphere composite aggregates.
  • the body is uniform in size, complete in shape, and has a smooth and dense cell layer on the edge, which has high morphological stability.
  • the mesenchymal stem cells and the hE-cad-Fc-PLGA three-dimensional composite cell aggregate model were constructed by the present invention, and the control group was MSC cell aggregates, MSC cells and PLGA microsphere composite aggregates (preparation method is the same as in Example 5).
  • the above cell aggregates were transferred to an ultra-low adhesion cell culture plate, and cell viability was measured by the CCK-8 method on day 1, day 2, day 3, day 4, day 5, and the detection method was the same as in Example 4.
  • Cell aggregates were stained for life and death (FDA/PI) on day 1 and day 5 to characterize cell survival within the aggregate.
  • the specific steps were as follows: FDA (diluted 1:1000 in PBS) and PI (diluted 1:100 in PBS) were added to the medium, and incubated for 10 min, and observed under a fluorescence microscope.
  • the hE-cad-Fc-PLGA microsphere composite cell was cultured in a three-dimensional culture model.
  • the mesenchymal stem cells exhibit better proliferation characteristics.
  • the results of dead and live cell staining showed that it was cultured for 5 days. After that, compared with the control group, the cell activity of the hE-cad-Fc-PLGA microsphere composite cell aggregate was significantly improved (green spot was a living cell).
  • the invention detects the change of intracellular signal pathway by western blotting method.
  • E-cadherin, ⁇ -catenin, p120-catenin, ⁇ -catenin membrane protein complexes, and phosphorylation of AKT-t308 and ERK were selected to reveal the membrane-mediated pathway of hE-cad-Fc fusion protein surface-modified microspheres. Impact.
  • MSC cell membrane protein pathways were detected in MSC cell aggregates, MSC cells and PLGA microsphere complex aggregates, and MSC cells and hE-cad-Fc-PLGA microsphere complex aggregates. Same as Embodiment 3. After cell aggregate formation, RAPI lyses the cells and collects total protein.
  • hE-cad-Fc fusion protein matrixing can transform cell-cell adhesion factor interactions between cells and matrix, and it regulates cell biological properties by activating related membrane protein molecular pathways. Furthermore, the proliferation of mesenchymal stem cells is promoted efficiently and permanently.
  • the base sequence of the fusion protein molecule hE-cad-Fc is the sequence of the sequence ⁇ 400>1:

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Abstract

提供了一种三维复合细胞聚集体模型，其制备方法和应用。该模型是通过以下方式制备的：以天然或化学合成聚合物为基质材料制备亚微米级微球，并在微球表面固定化由钙粘素蛋白胞外域和FC结构域构成的融合蛋白，与干细胞混合后形成微球/细胞复合聚集体。

Description

一种三维复合细胞聚集体模型及其制备方法与应用 技术领域
本发明涉及细胞体外培养技术领域，尤其是一种三维复合细胞聚集体模型及其制备方法与应用。
背景技术
在体内，细胞处于一个高度信息化的微环境中，其中包括各种时间/空间动态变化的物理化学信号，细胞受各种信号刺激调控，以实现其特定的生物学功能。这种细胞与微环境间的相互作用包括来自周围细胞、细胞外基质和可溶性因子的各种复杂的生物化学、生物力学和生物电学等信号刺激响应。其中，细胞与细胞之间是通过直接接触或旁分泌可溶性因子而发生相互作用。这种细胞间的通讯作用是维持细胞、组织及器官结构与功能的重要环节，同时也是调节体内外组织修复与重建的关键因素。如何有效模拟体内微环境、调控细胞与外界微环境间的相互作用，对于细胞形态与功能的维持以及体外组织的仿生重建至关重要。细胞三维培养(three-dimensional cell culture,TDCC)是指将具有三维结构的材料载体与各种不同种类的细胞在体外共同培养，使细胞能够在载体的三维立体空间结构中迁移、生长,构成三维的细胞-载体复合物。该培养方式中通过载体材料的设计与制备，既能最大程度的模拟体内环境使细胞功能稳定表达，又能展现细胞培养的直观性及条件可控性的优势，是目前体内外组织结构及其功能重建的最常用手段之一。
近年来，利用干细胞体外三维扩增及诱导分化的方法，进行体内 组织修复的策略已成为生命科学、再生医学等领域的研究热点。其中，胚胎干细胞(ESCs)由于其具有在体内、外向多种类型组织细胞分化的潜能，具有广阔的应用前景。间充质干细胞(MSCs)是干细胞家族的重要成员，连续传代培养和冷冻保存后仍具有多向分化潜能，且在体内或体外特定的诱导条件下，可分化为脂肪、骨、软骨、肌肉、肌腱、韧带、神经、肝、心肌、内皮等多种组织细胞，能够作为理想的种子细胞用于衰老和病变引起的组织器官损伤修复。
胚胎干细胞的分化应首先在无血清的培养基中诱导其聚集形成细胞聚集体，即拟胚体(EB)，此后模仿体内胚胎发生的条件，依次改变拟胚体的生长环境，诱导其分化出特定细胞。而现有的MSCs的扩增及诱导分化方法主要为二维平板培养。由于在三维培养体系中细胞的生长方式更接近体内，能够更为真实的模拟复杂的体内细胞微环境，促进干细胞体外诱导分化，有学者采用模拟胚胎干细胞拟胚体(EB)的形态结构制备间充质干细胞聚集体，发现该培养方式能够提高间充质干细胞中细胞因子的分泌，还能显著提高细胞诱导分化效率。但是，目前以上二者的培养及定向诱导分化过程中均存在有长期培养细胞活性降低，诱导分化效率较低，诱导效果不稳定，以及体内诱导效果不确切等问题亟待解决，限制其进一步向临床应用转化。
借助工程学、材料学等相关学科的发展，采取在细胞聚集体中加入微球的方式，有望改善聚集体内部的结构，进而提高三维细胞聚集体中细胞活性和定向诱导分化效率。但目前学者采用的微球材质主要为常见的天然细胞外基质或合成化学材料，并没有脱离传统细胞培养 中基于整合素家族信号分子改善材料与细胞相互作用的设计思路。
钙黏素蛋白是细胞间黏着连接的重要组成成分，具有介导细胞-细胞间特异性链接的功能。有文献表明，E-钙黏素融合蛋白能够促进多种细胞增殖并提高细胞活性。如T.AKaike课题组率先构建了鼠源E-cadherin胞外域和IgG蛋白Fc端重组蛋白mE-cadherin-Fc。实验结果表明，以胶原对照，鼠肝原代细胞能够在细胞膜蛋白E-cadherin介导下更好地粘附于mE-Cadherin-Fc涂膜的平板表面，且细胞DNA合成活性降低和色氨酸合酶的表达保持不变，同时，该课题组也将mE-cadherin-Fc应用于鼠源胚胎干细胞的培养，发现胚胎干细胞在该平板表面不形成集落，能够大量扩增并且显著抑制细胞自分化。目前有关人源钙黏素蛋白的相关研究则少见报道。本发明人先前曾在包被有人源细胞膜蛋白E-钙黏素的培养平板上二维培养人骨髓间充质干细胞，将细胞与细胞间相互作用转化到细胞与基质之间，发现在hE-Cad-Fc融合蛋白基质显著改善二维培养间充质干细胞的粘附、增殖及肝定向诱导分化效率。
在此背景下，通过基因工程技术制备具有E-钙黏素功能的融合蛋白，并用于生物材料微球的表面改性，制备E-钙黏素介导的生物材料微球与干细胞的复合细胞聚集体，建立更加有效促进间充质干细胞扩增及定向诱导分化的细胞三维培养技术，对于细胞-细胞、细胞-细胞外基质以及细胞-可溶性因子间的相互作用、以及体外构建间充质干细胞组织工程化各类组织器官类似物或等同物的研究具有重要的理论研究意义和应用开发价值。
发明内容
本发明所要解决的技术问题在于提供一种三维复合细胞聚集体模型，该模型充分考虑三维聚集体微环境中细胞间相互接触较强的特点，首次提出以细胞间黏附因子表面改性控释细胞生长因子的微球，通过钙黏素和黏着连接信号分子促进微球与细胞共培养形成微球/干细胞复合微球，是一种实现从聚集体内部调控聚集体细胞生物学活性的干细胞培养技术。
本发明所要解决的另一技术问题在于提供上述三维复合细胞聚集体模型的制备方法。
本发明所要解决的另一技术问题在于提供上述三维复合细胞聚集体模型的应用。
为解决上述技术问题，本发明的技术方案是：
一种三维复合细胞聚集体模型，是内部含有微球的干细胞聚集体，由所述微球表面固定化基因工程钙黏素蛋白家族的融合蛋白分子，该融合蛋白分子与干细胞通过干细胞自有的钙黏素粘附因子进行构建，进而使所述微球与干细胞共培养而获得。
优选的，上述三维复合细胞聚集体模型，所述微球是通过天然或化学合成聚合物制备而成的，其粒径范围在0.5-40μm。
优选的，上述三维复合细胞聚集体模型，所述聚合物为天然聚合物，包括胶原、明胶、海藻酸、壳聚糖、透明质酸；或为化学合成聚合物，包括聚乳酸、聚乙醇酸、聚乳酸-羟基乙酸、聚乙二醇或聚苯乙烯。
优选的，上述三维复合细胞聚集体模型，所述微球内部或表面可缓释或固定细胞活性因子。
优选的，上述三维复合细胞聚集体模型，所述干细胞为胚胎干细胞或间充质干细胞。
优选的，上述三维复合细胞聚集体模型，所述基因工程钙黏素蛋白家族的融合蛋白分子，包括钙黏素蛋白的胞外域和免疫球蛋白的Fc结构域，并通过Fc介导的物理或化学结合，用于各种形态的材料本体或/及材料表面修饰，提高材料的亲水性和钙黏素蛋白介导的细胞特异性粘附，改善材料的生物相容性。
优选的，上述三维复合细胞聚集体模型，所述融合蛋白分子的种类是E-钙黏素蛋白。
优选的，上述三维复合细胞聚集体模型，所述融合蛋白分子是利用基因工程技术将E-钙黏着蛋白胞外域和免疫球蛋白Fc结构域基因重组，构建含有E-钙黏着蛋白和Fc双功能融合蛋白基因序列，经基因转染、蛋白表达及分离纯化而制得。
优选的，上述三维复合细胞聚集体模型，所述融合蛋白分子通过Fc介导固定于微球表面，与干细胞混合均匀后以离心或悬滴法制备含有微球的微球/细胞复合聚集体，随后保持其三维结构进行长期培养，以达成通过组织工程学原理和技术实现三维细胞-基质间的相互作用与细胞-细胞间相互作用的同效替代，有效改善细胞聚集体内部结构及细胞功能调控。
优选的，上述三维复合细胞聚集体模型，所述融合蛋白分子是含 有目的基因人上皮细胞钙黏素粘附因子hE-cadherin的胞外域hE-cad、人免疫球蛋白IgG 1的Fc段结构域Fc基因重组构建的真核细胞表达基因hE-cad-Fc，其碱基序列为序列表<400>1所述序列。
优选的，上述三维复合细胞聚集体模型，所述融合蛋白分子是由基因质粒经真核细胞基因转染、蛋白表达及蛋白A亲和层析柱分离纯化而制得的hE-cad-Fc融合蛋白，所得蛋白进行SDS-PAGE和Western blot分析，其碱基序列为序列表<400>1所述序列。
优选的，上述三维复合细胞聚集体模型，所述基因质粒获得的具体步骤为：采用基因重组PCR技术扩增目的基因hE-cad和Fc序列，并分别通过双酶切连接并嵌入真核细胞表达基因质粒载体pcDNA3.1中，得到含有双功能目的基因hE-cad-Fc片段的真核细胞表达基因质粒pcDNA3.1-E-cad-Fc，其碱基序列为序列表<400>2所述序列。
优选的，上述三维复合细胞聚集体模型，所述融合蛋白分子的使用浓度范围为0.1-20μg/ml。
上述三维复合细胞聚集体模型的制备方法，具体步骤如下：
第一、使用天然或化学合成聚合物制备缓慢释放生长因子的亚微米级微球；
第二、将经过真核细胞蛋白表达体系纯化的hE-cad-Fc融合蛋白稀释，浸泡微球，在4℃至37℃，孵育0.5-24小时；
第三、弃上清，用PBS淋洗1-3遍，除去未稳定固定的融合蛋白hE-cad-Fc，获得hE-cad-Fc基质化的微球；
第四、将干细胞与上述hE-cad-Fc基质化微球混匀，通过悬滴 法、离心法等方法诱导干细胞形成三维聚集体，或将细胞包裹于水凝胶，多孔支架等三维培养环境中，使其自发形成细胞聚集体。
优选的，上述三维复合细胞聚集体模型的制备方法，所述天然聚合物为胶原(Collagen)、明胶(Gelatin)、海藻酸(Alginate)、壳聚糖(Chitosan)、透明质酸(Hyaturonic acid)或对以上材料进行化学修饰而成的聚合物。
优选的，上述三维复合细胞聚集体模型的制备方法，所述化学合成聚合物为聚乳酸(PLA)、聚乙醇酸(PGA)、聚乳酸-羟基乙酸(PLGA)、聚乙二醇(PEG)或聚苯乙烯(PS)或对以上材料进行化学修饰而成的聚合物。
优选的，上述三维复合细胞聚集体模型的制备方法，所得到的微球的粒径范围在0.5-40μm。
优选的，上述三维复合细胞聚集体模型的制备方法，所述hE-cad-Fc融合蛋白使用浓度为0.1-20μg/ml。
优选的，上述三维复合细胞聚集体模型的制备方法，所述细胞与hE-cad-Fc基质化微球混合比例为1:1-10:1。
优选的，上述三维复合细胞聚集体模型的制备方法，所述细胞聚集体制备方法为悬滴培养48h-72h，或离心后培养12-24h。
上述三维复合细胞聚集体模型的应用，能够用于提高间充质干细胞黏附、增殖、批量扩增以及经时、定向调控间充质干细胞诱导分化及分化细胞的功能表达。
优选的，上述三维复合细胞聚集体模型的应用，能够用于研究细 胞-细胞、细胞-细胞外基质以及细胞-可溶性因子间的相互作用，以及体外间充质干细胞扩增培养、构建组织工程化各类组织器官类似物或等同物。
本发明的有益效果是：
一、本发明利用亚微米级微球缓释细胞因子、并以人E-钙粘素介导生物材料与细胞间相互作用形成可由内而外调控细胞生物学活性的三维复合细胞聚集体模型。该模型可用于干细胞三维状态下增殖、分化的研究，克服了传统干细胞三维培养由外向内调控受传质阻力影响引起细胞非均质生长、以及接触抑制增殖等的不足，为体外构建和优化干细胞三维仿生培养技术，以及组织工程化构建各类组织器官类似物或等同物提供理论和技术支持。
二、本发明利用基因工程原理和技术，将人上皮细胞E-钙黏素的胞外域与人免疫球蛋白IgG 1Fc段融合，制备了具有E-钙黏素蛋白和Fc双功能的融合蛋白。
三、本发明的融合蛋白通过Fc介导在材料表面自组装形成单分子层，实现材料表面改性，提高材料的亲水性，改善材料的生物相容性，加强了细胞与材料的特异性相互作用，并进一步调控细胞内信号通路。
四、本发明的hE-cad-Fc融合蛋白通过在微球表面基质化固定，在三维聚集体培养条件下有效的促进干细胞增殖和分化功能表达，促进实现组织工程、再生医学相关种子细胞的批量扩增及诱导分化。
五、本发明的融合蛋白基质化固定方法简单易行，修饰效率高且 稳定性好，降低了实验成本。利用本发明构建的三维聚集体模型可更有效实现细胞三维仿生培养，为考察细胞-细胞、细胞-细胞外基质和细胞-可溶性因子间的相互作用、考察特定细胞因子在三维聚集体中对细胞的生长、迁移、增殖与分化等生物学行为的调控规律与机制、揭示干细胞定向分化的分子调控机制等基础与应用研究提供干细胞培养技术。
附图说明
图1是重组质粒构建示意图；
图2是重组质粒鉴定；
图3是纯化融合蛋白鉴定；
图4是不同浓度hE-cad-Fc融合蛋白对其在PLGA微球表面基质化的蛋白固定量的定量检测；
图5是在PLGA微球表面基质化固定hE-cad-Fc融合蛋白的免疫荧光检测；
图6是hE-cad-Fc融合蛋白基质化表面改性PLGA微球对间充质干细胞增殖的影响；
图7是hE-cad-Fc融合蛋白基质化表面改性PLGA微球对间充质干细胞形态的影响；
图8是三维复合细胞聚集体模型示意图；
图9是单纯细胞聚集体以及微球/细胞复合聚集体形态的光镜照片；
图10是hE-cad-Fc融合蛋白基质化表面改性PLGA微球对三维复 合间充质干细胞聚集体细胞增殖的影响；
图11是hE-cad-Fc融合蛋白基质化表面改性PLGA微球对三维复合间充质干细胞聚集体细胞活性的影响。
图12是hE-cad-Fc融合蛋白基质化表面改性PLGA微球对三维复合间充质干细胞聚集体中细胞膜信号通路的影响。
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。
实施例1  获得pcDNA3.1-hE-cad-Fc基因质粒
利用基因重组技术将人上皮细胞黏附因子(hE-cadherin)胞外域(hE-cad)和人免疫球蛋白(IgG 1)Fc段(Fc)进行融合,得到含有目的基因hE-cad和Fc的质粒pcDNA3.1-hE-cad-Fc。即：以人E-cadherin mRNA(Gene bank:NM 004360.3)全长为模版，利用合成的引物特异性扩增hE-cadherin胞外域片段。引物序列为：
5’-CGCAAGCTTATGGGCCCTTG-GAGCCGCAGC-3’；
5’-TTGCGGCCGCAGGCAGGAATTTGCAATCCTGC-3’。
如图1所示，1％的琼脂糖凝胶电泳分析PCR产物，将胶回收PCR产物经Hind III和Not I双酶切后，插入pcDNA3.1-Fc的Hind III和Not I位点之间。质粒pcDNA3.1-Fc由东京工业大学赤池敏宏教授惠赠。
如图2所示，重组表达质粒经Hind III和Not I双酶切电泳鉴定，显示切出与目的基因大小一致的DNA片段，将此质粒载体命名为pcDNA3.1-hE-cad-Fc。
测序检测表明pcDNA3.1-hE-cad-Fc全长序列共计3020个碱基，该基因质粒的碱基序列为序列表中<400>2所示序列。
实施例2  获得hE-cad-Fc融合蛋白
将实施例1构建的含有目的基因的质粒pcDNA3.1-hE-cad-Fc转染293F细胞，悬浮培养72h，收集细胞培养上清液，利用rProtein AFF柱对hE-cad-Fc融合蛋白进行纯化。纯化的融合蛋白利用抗E-cadherin抗体通过免疫印迹检测，如图3所示，图中没有其他非特异性曝光条带出现，分子量大小约为120KD的条带为还原态E-cadherin(图3中1)，240KD处条带为非还原E-cadherin(图3中2)。此结果说明，本发明制备得到了hE-cad-Fc融合蛋白，其碱基序列为序列表中<400>1所示序列，该融合蛋白非还原态能够形成稳定二聚体结构。
实施例3  hE-cad-Fc融合蛋白基质化表面改性PLGA微球的制备
首先通过化学合成聚合物PLGA制备获得粒径范围在15±5μm的微球置于EP管中；随后将上述hE-cad-Fc融合蛋白溶液稀释至10μg/ml，浸泡微球，37℃孵育2小时；离心弃上清，用PBS淋洗3遍除去未被固定的融合蛋白hE-cad-Fc，获得hE-cad-Fc基质化表面改性PLGA微球，并将其命名为hE-cad-Fc-PLGA。
本发明通过Elisa法检测hE-cad-Fc溶液的蛋白浓度对PLGA微 球基质化表面改性的影响。hE-cad-Fc基质化方法同上。即：获得hE-cad-Fc-PLGA后，加入1％BSA室温封闭1h，加入抗E-cadherin抗体37℃孵育1h，PBST清洗3次后加入生物素标记二抗37℃孵育30min，PBS清洗3次，而后加入TBA显色液于37℃避光孵育10min，加入终止液，离心取上清100μl加入96孔板中，于10min内使用酶标仪在450nm波长下读板取得结果。如图4所示，随着hE-cad-Fc溶液浓度的提高，PLGA微球表面基质化hE-cad-Fc蛋白量逐渐升高。
如图5所示，通过对PLGA和hE-cad-Fc-PLGA进行E-cadherin免疫荧光染色观察检测hE-cad-Fc融合蛋白在微球表面的分布。在融合蛋白固定后，以1％BSA室温封闭1h，加入抗E-cadherin抗体4℃孵育过夜，PBST清洗3次后加入荧光标记二抗37℃孵育1h，PBS清洗3次，吸取微球滴加至载玻片上，封片后在激光共聚焦显微镜下观察。实验结果表明，hE-cad-Fc-PLGA表面均匀覆盖有hE-cad-Fc融合蛋白。
实施例4  hE-cad-Fc-PLGA对间充质干细胞增殖及形态的影响
本发明采用CCK-8法检测PLGA微球、hE-cad-Fc-PLGA对脐带间充质干细胞增殖的影响。首先将hE-cad-Fc-PLGA、未固定蛋白的PLGA微球(对照组)与间充质干细胞以1:1的数量比混匀，取每孔5000个间充质细胞、每孔5000个间充质细胞+5000个PLGA微球、及每孔5000个间充质细胞+5000h个hE-cad-Fc-PLGA微球加入96孔板，37℃5％CO2恒温细胞培养箱中培养5天，每天测定细胞活力，具体方法 为：每孔加入10ulCCK-8，孵育4h后，酶联免疫监测仪检测OD450nm，记录结果，以时间为横坐标，吸光值为纵坐标做图，见图6。
利用细胞骨架免疫荧光染色方法观察细胞的形态，实验分组处理同上。以每孔2*104个间充质细胞，细胞与微球1:1的比例铺种于24孔板，37℃5％CO2恒温细胞培养箱中培养5天，4％的多聚甲醛溶液进行细胞固定5min，用0.1％Triton x-100进行细胞透膜处理10min。细胞用1％牛血清白蛋(BSA)室温封闭1h。用FITC-鬼笔环肽标记细胞骨架微丝(绿)，PI标记细胞核(红)，荧光共聚焦显微镜下观察，见图7。
以上结果显示，hE-cad-Fc基质化微球能够募集间充质干细胞粘附，并促进细胞增殖。
实施例5  三维复合细胞聚集体模型构建
如图8所示，通过将人上皮细胞粘附因子hE-cad-Fc融合蛋白基质化固定于PLGA的微球(hE-cad-Fc-PLGA)表面，制备由间充质干细胞以及融合蛋白微球混合形成细胞聚集体，形成复合细胞三维培养模型。该模型的具体构建方法如下：
第一、化学合成聚合物PLGA制备而成的微球，粒径范围在15±5μm；
第二、将经过真核细胞蛋白表达体系纯化的hE-cad-Fc融合蛋白稀释至10μg/ml，浸泡微球，37℃孵育2小时；
第三、弃上清，用PBS淋洗3遍，除去未稳定固定的融合蛋白 hE-cad-Fc；
第四、将细胞与hE-cad-Fc基质化微球以1:1的数量比混匀，6*105cells/well培养于Stem Cell AggrewellTM，1000rpm离心5min后静置培养16h，诱导微球/细胞形成复合三维聚集体。
以相差显微镜经时观察细胞形态，如图9所示，结果表明：与MSC细胞聚集体和MSC细胞与PLGA微球复合聚集体相比，MSC细胞与hE-cad-Fc-PLGA微球复合聚集体大小均一、形状完整、边缘有光滑致密的细胞层包裹，形态稳定性较高。
实施例6  三维复合细胞聚集体中间充质干细胞的增殖活性检测
利用本发明构建间充质干细胞与hE-cad-Fc-PLGA三维复合细胞聚集体模型，对照组为MSC细胞聚集体、MSC细胞与PLGA微球复合聚集体(制备方法同实施例5)。将以上细胞聚集体移入超低粘附细胞培养平板中，并于day1，day2，day3，day4，day5以CCK-8法测定细胞活力，检测方法同实施例4。在培养day1及day 5对细胞聚集体进行死活细胞染色(FDA/PI)，以表征聚集体内部细胞存活情况。具体步骤为：在培养基中分别加入FDA(用PBS 1：1000稀释)和PI(用PBS 1:100稀释)，孵育10min，荧光显微镜下观察。
如图10所示，相较于对照组(纯间充质干细胞聚集体，间充质干细胞与微球复合聚集体)，该hE-cad-Fc-PLGA微球复合细胞三维培养模型中，间充质干细胞表现出更良好的增殖特性。
如图11所示，死活细胞染色(FDA/PI)结果表明，在培养5天 后，相较于对照组，该hE-cad-Fc-PLGA微球复合细胞聚集体三维培养模型中，细胞活性得到了显著提高(绿色光点为活细胞)。
实施例7  三维复合细胞聚集体中间充质细胞E-cadherin相关通路检测
本发明通过western blotting法检测细胞内信号通路变化。选取细胞内E-cadherin，β-catenin，p120-catenin，α-catenin膜蛋白复合物，以及AKT-t308及ERK磷酸化表达，初步揭示hE-cad-Fc融合蛋白表面改性微球对细胞膜通路的影响。
如图12所示，对MSC细胞聚集体、MSC细胞与PLGA微球复合聚集体以及MSC细胞与hE-cad-Fc-PLGA微球复合聚集体中相关MSC细胞膜蛋白通路进行检测，聚集体构建方法同实施例3。细胞聚集体形成后RAPI裂解细胞并收取总蛋白。配置10％SDS-PAGE凝胶，电泳转膜，5％脱脂牛奶室温封闭1h，加入抗E-cadherin抗体4℃孵育过夜，PBST清洗3次后加入HRP标记二抗37℃孵育1min，PBS清洗3次，加入ECL发光液曝光，暗室拍摄。与对照相比，添加有hE-cad-Fc-PLGA的复合聚集体中膜蛋白β-catenin上调，且AKT-t308及ERK磷酸化水平显著提高。
因此，可以得出结论：hE-cad-Fc融合蛋白基质化能够将细胞与细胞间的黏附因子相互作用转化到细胞与基质之间，且其通过激活相关膜蛋白分子通路调控细胞生物学特性，进而有效且持久地促进间充质干细胞增殖。
上述参照实施例对该一种三维复合细胞聚集体模型及其制备方法与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
备注：基因序列
融合蛋白分子hE-cad-Fc的碱基序列为序列表<400>1所述序列：
Figure PCTCN2016073472-appb-000001
Figure PCTCN2016073472-appb-000002
Figure PCTCN2016073472-appb-000003
测序检测表明pcDNA3.1-E-cadherin-Fc全长序列为，共计3020个碱基：(该基因质粒的碱基序列为序列表中<400>2所示序列)
Figure PCTCN2016073472-appb-000004
Figure PCTCN2016073472-appb-000005

Claims (10)

  1. 一种三维复合细胞聚集体模型，其特征在于：是内部含有微球的干细胞聚集体，由所述微球表面固定化基因工程钙黏素蛋白家族的融合蛋白分子，该融合蛋白分子与干细胞通过干细胞自有的钙黏素粘附因子进行构建，进而使所述微球与干细胞共培养而获得。
  2. 根据权利要求1所述的三维复合细胞聚集体模型，其特征在于：所述聚合物为天然聚合物，包括胶原、明胶、海藻酸、壳聚糖、透明质酸；或为化学合成聚合物，包括聚乳酸、聚乙醇酸、聚乳酸-羟基乙酸、聚乙二醇或聚苯乙烯；所示聚合物的粒径范围在0.5-40μm。
  3. 根据权利要求1所述的三维复合细胞聚集体模型，其特征在于：所述干细胞为胚胎干细胞或间充质干细胞。
  4. 根据权利要求1所述的三维复合细胞聚集体模型，其特征在于：所述融合蛋白分子是利用基因工程技术将E-钙黏素蛋白胞外域和免疫球蛋白Fc结构域基因重组，构建含有E-钙黏素蛋白和Fc双功能融合蛋白基因序列，经基因转染、表达及分离纯化而制得，所述融合蛋白分子通过Fc介导固定于微球表面。
  5. 根据权利要求1所述的三维复合细胞聚集体模型，其特征在于：所述融合蛋白分子hE-cad-Fc的碱基序列为序列表<400>1所述序列。
  6. 权利要求1-5之一所述三维复合细胞聚集体模型的制备方法，其特征在于：具体步骤如下：
    (1)使用天然或化学合成聚合物制备缓慢释放生长因子的亚微 米级微球；
    (2)将经过真核细胞蛋白表达体系表达并纯化的hE-cad-Fc融合蛋白稀释，浸泡微球，在4℃至37℃，孵育0.5-24小时；
    (3)弃上清，用PBS淋洗1-3遍，除去未稳定固定的融合蛋白hE-cad-Fc，获得hE-cad-Fc基质化的微球；
    (4)将干细胞与上述hE-cad-Fc基质化微球混匀，通过悬滴法、离心法等方法诱导干细胞形成三维聚集体，或将细胞包裹于水凝胶、多孔支架等三维培养环境中，使其自发形成细胞聚集体。
  7. 根据权利要求6所述的三维复合细胞聚集体模型的制备方法，其特征在于：所述hE-cad-Fc融合蛋白使用浓度为0.1-20μg/ml。
  8. 根据权利要求6所述的三维复合细胞聚集体模型的制备方法，其特征在于：所述细胞与hE-cad-Fc基质化微球混合比例为1:1-10:1。
  9. 根据权利要求6所述的三维复合细胞聚集体模型的制备方法，其特征在于：所述细胞聚集体制备方法为悬滴培养48h-72h，或离心后培养12-24h。
  10. 权利要求1-5之一所述三维复合细胞聚集体模型的应用，其特征在于：能够用于提高材料的干细胞黏附、增殖以及批量扩增及经时、定向调控诱导干细胞分化、及分化细胞功能的表达和维持。
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