WO2024082430A1 - Tumor microfluidic chip for screening mesenchymal stem cell migration and preparation and use method therefor - Google Patents
Tumor microfluidic chip for screening mesenchymal stem cell migration and preparation and use method therefor Download PDFInfo
- Publication number
- WO2024082430A1 WO2024082430A1 PCT/CN2022/140871 CN2022140871W WO2024082430A1 WO 2024082430 A1 WO2024082430 A1 WO 2024082430A1 CN 2022140871 W CN2022140871 W CN 2022140871W WO 2024082430 A1 WO2024082430 A1 WO 2024082430A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- channel
- migration
- cell
- microfluidic chip
- cell culture
- Prior art date
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 95
- 230000012292 cell migration Effects 0.000 title claims abstract description 75
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 41
- 238000012216 screening Methods 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 238000004113 cell culture Methods 0.000 claims abstract description 113
- 239000011159 matrix material Substances 0.000 claims abstract description 81
- 230000005012 migration Effects 0.000 claims abstract description 65
- 238000013508 migration Methods 0.000 claims abstract description 65
- 230000010412 perfusion Effects 0.000 claims abstract description 52
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims description 98
- 239000000499 gel Substances 0.000 claims description 55
- 210000004881 tumor cell Anatomy 0.000 claims description 37
- 239000006285 cell suspension Substances 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 19
- 239000000700 radioactive tracer Substances 0.000 claims description 15
- 210000000056 organ Anatomy 0.000 claims description 12
- 238000002474 experimental method Methods 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- 238000002372 labelling Methods 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 6
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 5
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 5
- 238000005516 engineering process Methods 0.000 claims description 4
- 108010082117 matrigel Proteins 0.000 claims description 4
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 4
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 229920001661 Chitosan Polymers 0.000 claims description 3
- 108010035532 Collagen Proteins 0.000 claims description 3
- 102000008186 Collagen Human genes 0.000 claims description 3
- 108010080379 Fibrin Tissue Adhesive Proteins 0.000 claims description 3
- 108010022355 Fibroins Proteins 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 3
- 229920001436 collagen Polymers 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 230000003993 interaction Effects 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 229920000515 polycarbonate Polymers 0.000 claims description 3
- 239000004417 polycarbonate Substances 0.000 claims description 3
- -1 polydimethylsiloxane Polymers 0.000 claims description 3
- 230000008054 signal transmission Effects 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- 238000007711 solidification Methods 0.000 claims description 3
- 230000008023 solidification Effects 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000003399 chemotactic effect Effects 0.000 claims description 2
- 239000003292 glue Substances 0.000 claims description 2
- 238000005459 micromachining Methods 0.000 claims description 2
- 210000001519 tissue Anatomy 0.000 description 19
- 102100023600 Fibroblast growth factor receptor 2 Human genes 0.000 description 17
- 101000827688 Homo sapiens Fibroblast growth factor receptor 2 Proteins 0.000 description 17
- 230000035605 chemotaxis Effects 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- IPJDHSYCSQAODE-UHFFFAOYSA-N 5-chloromethylfluorescein diacetate Chemical compound O1C(=O)C2=CC(CCl)=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 IPJDHSYCSQAODE-UHFFFAOYSA-N 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 206010008342 Cervix carcinoma Diseases 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 4
- 201000010881 cervical cancer Diseases 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000001360 synchronised effect Effects 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 102000012422 Collagen Type I Human genes 0.000 description 3
- 108010022452 Collagen Type I Proteins 0.000 description 3
- 101001121408 Homo sapiens L-amino-acid oxidase Proteins 0.000 description 3
- 102100026388 L-amino-acid oxidase Human genes 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 229940096422 collagen type i Drugs 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 208000037887 cell injury Diseases 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000000206 photolithography Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100012902 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FIG2 gene Proteins 0.000 description 1
- 101100233916 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) KAR5 gene Proteins 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004791 biological behavior Effects 0.000 description 1
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012806 monitoring device Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
Abstract
A tumor microfluidic chip for screening mesenchymal stem cell migration, and a preparation and use method therefor. The microfluidic chip comprises a chip main body having a microstructure channel machined on the surface, and a substrate tightly attached to the chip main body. The microstructure channel on the chip main body comprises a central cell culture channel, and a plurality of independent cell migration units which are symmetrically arranged on the two sides of the central cell culture channel. Each cell migration unit comprises a matrix gel perfusion channel on the inner side, and a migration cell culture channel on the outer side. Intercepting structures are arranged between the central cell culture channel and the matrix gel perfusion channel of each cell migration unit and between the matrix gel perfusion channel and the migration cell culture channel of the same cell migration unit.
Description
本发明属于生物技术领域，涉及一种间充质干细胞的肿瘤趋化性的监测装置，尤其涉及一种用于间充质干细胞迁移筛选的肿瘤微流控芯片及其制备和应用方法。The invention belongs to the field of biotechnology and relates to a monitoring device for tumor chemotaxis of mesenchymal stem cells, and in particular to a tumor microfluidic chip for mesenchymal stem cell migration screening and a preparation and application method thereof.
恶性肿瘤是目前严重威胁全人类健康的世界性难题，据美国癌症协会报道的数据显示2020年全球分别有将近1930万新确诊肿瘤患者和1000万肿瘤死亡的患者。铂类等化疗药物已广泛用于肿瘤治疗，但易引起全身毒性和耐药，因此，建立一种精准靶向地药物递送平台对实现肿瘤精准治疗具有重要意义。Malignant tumors are a global problem that seriously threatens the health of all mankind. According to data reported by the American Cancer Society, there will be nearly 19.3 million newly diagnosed cancer patients and 10 million cancer deaths in the world in 2020. Chemotherapy drugs such as platinum have been widely used in tumor treatment, but they are prone to cause systemic toxicity and drug resistance. Therefore, establishing a precise and targeted drug delivery platform is of great significance to achieve precise tumor treatment.
间充质干细胞(Mesenchymal stem cells,MSCs)是一类具有自我更新和多向分化潜能的细胞群。MSCs组织来源广泛，易扩增，免疫原性低，且研究已证明MSCs对恶性肿瘤组织具有独特的迁移趋化性，表明MSCs是一种理想的药物靶向递送的载体。然而，尽管不同组织来源的MSCs具有相似的形态和免疫表型，它们在增殖、细胞因子分泌以及细胞迁移能力等生物学行为上存在差异。因此，比较并筛选出拥有最强肿瘤迁移趋化能力的MSCs尤为重要。Mesenchymal stem cells (MSCs) are a type of cell population with the potential for self-renewal and multidirectional differentiation. MSCs have a wide range of tissue sources, are easy to proliferate, have low immunogenicity, and studies have shown that MSCs have unique migration chemotaxis for malignant tumor tissues, indicating that MSCs are an ideal carrier for targeted drug delivery. However, although MSCs from different tissue sources have similar morphology and immune phenotypes, they differ in biological behaviors such as proliferation, cytokine secretion, and cell migration ability. Therefore, it is particularly important to compare and screen out MSCs with the strongest tumor migration chemotaxis.
评价细胞迁移特性的模型分为体内和体外模型。体外细胞迁移模型较体内模型成本低、操作简单，可避免动物实验涉及的伦理问题，也可避免人类与实验动物种属特异性差异造成实验结果不一致的现象。传统的体外模型主要依赖于划痕实验法和transwell实验法，但划痕实验重复性较差，且易损伤细胞，Transwell实验操作复杂且不适合实时动态监测。Models for evaluating cell migration characteristics are divided into in vivo and in vitro models. In vitro cell migration models are lower in cost and simpler to operate than in vivo models. They can avoid ethical issues involved in animal experiments and the phenomenon of inconsistent experimental results caused by species-specific differences between humans and experimental animals. Traditional in vitro models mainly rely on scratch experiments and transwell experiments, but scratch experiments have poor reproducibility and are prone to cell damage. Transwell experiments are complex to operate and are not suitable for real-time dynamic monitoring.
微流控芯片技术因其结构设计灵活和规模集成的优势，已在细胞操控和分析领域得到迅速发展。微流控芯片的管道尺寸在微米量级，细胞和试剂消耗少，且能较好地模拟体内微环境，不损伤细胞，适合动态观察。然而，目前用于监测细胞迁移的多为单通道芯片，具有通量低，集成度差，实验重复性差等缺陷。此外，在同一块微流控芯片上同时实现多种MSCs对肿瘤细胞迁移趋化性的监测和筛选的研究分析还处于空白阶段。Microfluidic chip technology has been rapidly developed in the field of cell manipulation and analysis due to its flexible structural design and large-scale integration advantages. The channel size of the microfluidic chip is in the micrometer range, which consumes less cells and reagents, and can better simulate the in vivo microenvironment without damaging cells, making it suitable for dynamic observation. However, most of the chips currently used to monitor cell migration are single-channel chips, which have defects such as low throughput, poor integration, and poor experimental repeatability. In addition, the research and analysis of the monitoring and screening of the chemotaxis of tumor cell migration by multiple MSCs on the same microfluidic chip is still in a blank stage.
发明内容Summary of the invention
本发明针对上述现有技术的不足，提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片及其制备和应用方法，该肿瘤微流控芯片实现了多种类型间充质干细胞和肿瘤细胞的同步共培养，操作简单，降低了实际样品的用量，并模拟人间充质干细胞对肿瘤细胞的趋化性并同步比较差异，能够为筛选肿瘤靶向治疗高效药物载体提供理论依据。In view of the deficiencies of the above-mentioned prior art, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening and a preparation and application method thereof. The tumor microfluidic chip realizes the synchronous co-culture of various types of mesenchymal stem cells and tumor cells, is simple to operate, reduces the amount of actual samples, simulates the chemotaxis of human mesenchymal stem cells to tumor cells and synchronously compares the differences, and can provide a theoretical basis for screening efficient drug carriers for tumor targeted therapy.
为实现上述目的，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，具有这样的特征：包括表面加工出微结构通道的芯片主体和紧密贴合在芯片主体上的基板；芯片主体上的微结构通道包括一条中央细胞培养通道和若干个对称排列在中央细胞培养通道两侧的相互独立的细胞迁移单元；所述细胞迁移单元包括一条内侧的基质胶灌注通道和一条外侧的迁移细胞培养通道；中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道的两端均具有进样口和出样口；所述中央细胞培养通道用于肿瘤细胞的培养；基质胶灌注通道用于基质胶的填充；若干迁移细胞培养通道用于不同组织来源间充质干细胞的培养；所述中央细胞培养通道与各细胞迁移单元的基质胶灌注通道之间、以及同一细胞迁移单元的 基质胶灌注通道与迁移细胞培养通道之间均设有截流结构，截流结构由若干间隔设置的柱状结构构成，中央细胞培养通道与各细胞迁移单元的基质胶灌注通道、以及同一细胞迁移单元的基质胶灌注通道与迁移细胞培养通道通过截流结构进行细胞之间的信号传输和相互作用以及细胞迁移。To achieve the above-mentioned purpose, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening, which has the following characteristics: it comprises a chip body with a microstructure channel processed on the surface and a substrate tightly fitted on the chip body; the microstructure channel on the chip body comprises a central cell culture channel and a plurality of independent cell migration units symmetrically arranged on both sides of the central cell culture channel; the cell migration unit comprises an inner matrix gel perfusion channel and an outer migration cell culture channel; both ends of the central cell culture channel and each matrix gel perfusion channel and the migration cell culture channel are provided with an inlet and an outlet; the central cell culture channel is used for culturing tumor cells; the matrix gel perfusion channel is used for filling matrix gel; a plurality of migration cell culture channels are used for culturing mesenchymal stem cells from different tissue sources; the central cell culture channel and the matrix gel perfusion channel of each cell migration unit, as well as the matrix gel perfusion channel of the same cell migration unit, are connected to each other. An interception structure is provided between the matrix gel perfusion channel and the migration cell culture channel. The interception structure is composed of a number of columnar structures arranged at intervals. The central cell culture channel and the matrix gel perfusion channel of each cell migration unit, as well as the matrix gel perfusion channel of the same cell migration unit and the migration cell culture channel, carry out signal transmission and interaction between cells and cell migration through the interception structure.
进一步，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，还可以具有这样的特征：其中，所述细胞迁移单元中，基质胶灌注通道和迁移细胞培养通道的两端均向外侧方向弯折。Furthermore, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening, which may also have the following characteristics: wherein, in the cell migration unit, both ends of the matrix gel perfusion channel and the migration cell culture channel are bent outward.
进一步，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，还可以具有这样的特征：其中，所述芯片主体的材料为聚二甲基硅氧烷、聚甲基丙烯酸甲酯或聚碳酸酯；所述基板的材料为玻璃或硅片。Furthermore, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening, which may also have the following characteristics: wherein the material of the chip body is polydimethylsiloxane, polymethyl methacrylate or polycarbonate; the material of the substrate is glass or silicon wafer.
进一步，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，还可以具有这样的特征：其中，所述芯片主体与基板通过等离子体键合工艺实现两者之间的紧密贴合。Furthermore, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening, which may also have the following characteristics: wherein the chip body and the substrate are tightly fitted together by a plasma bonding process.
进一步，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，还可以具有这样的特征：其中，所述中央细胞培养通道和迁移细胞培养通道培养肿瘤细胞和间充质干细胞前涂覆有培养基质，培养基质为明胶、壳聚糖、丝素蛋白、鼠尾胶原蛋白、纤维蛋白胶、基质胶中的一种或几种。Furthermore, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening, which may also have the following characteristics: wherein, the central cell culture channel and the migration cell culture channel are coated with a culture matrix before culturing tumor cells and mesenchymal stem cells, and the culture matrix is one or more of gelatin, chitosan, silk fibroin, rat tail collagen, fibrin glue, and matrix glue.
进一步，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，还可以具有这样的特征：其中，所述芯片主体的中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道均位于同一平面，且宽度和高度均相同，分别为1000μm和100μm；基质胶灌注通道的长度为1.0cm，迁移细胞培养通的长度为0.8cm， 中央细胞培养通道的长度根据细胞迁移单元的数量而定；所述截流结构由5个六棱柱结构等间距排列而成，各六棱柱结构的边长为100μm，六棱柱结构之间的间距为50μm。Furthermore, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening, which may also have the following characteristics: wherein, the central cell culture channel of the chip body and each matrix gel perfusion channel and migration cell culture channel are all located in the same plane, and have the same width and height, which are 1000 μm and 100 μm respectively; the length of the matrix gel perfusion channel is 1.0 cm, the length of the migration cell culture channel is 0.8 cm, and the length of the central cell culture channel is determined according to the number of cell migration units; the intercepting structure is composed of 5 hexagonal prism structures arranged at equal intervals, the side length of each hexagonal prism structure is 100 μm, and the spacing between the hexagonal prism structures is 50 μm.
本发明还提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片的制备方法，具有这样的特征：包括以下步骤：步骤一、用计算机辅助软件设计和绘制所述芯片主体的微结构通道图形；步骤二、通过微加工技术制备具有微结构通道的芯片主体，通过手动打孔器制备中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道的进样口及出样口；步骤三、将芯片主体和基板用无水乙醇清洗后，进行高压灭菌处理，最后进行等离子体键合，得到微流控芯片。The present invention also provides a method for preparing a tumor microfluidic chip for mesenchymal stem cell migration screening, which has the following characteristics: comprising the following steps: step 1, designing and drawing the microstructure channel graphics of the chip body using computer-aided software; step 2, preparing a chip body with a microstructure channel using micromachining technology, and preparing a central cell culture channel and each matrix gel perfusion channel, and an inlet and outlet for a migration cell culture channel using a manual puncher; step 3, cleaning the chip body and substrate with anhydrous ethanol, performing high-pressure sterilization, and finally performing plasma bonding to obtain a microfluidic chip.
本发明还提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片的应用方法，具有这样的特征：包括以下步骤：The present invention also provides an application method of a tumor microfluidic chip for mesenchymal stem cell migration screening, which has the following characteristics: comprising the following steps:
步骤一、微流控芯片的预处理：取三个微流控芯片，分别为实验用微流控芯片、正常细胞对照用微流控芯片和空白对照用微流控芯片；涂覆培养基质：对于各微流控芯片，向所述中央细胞培养通道和各迁移细胞培养通道填充培养基质溶液，然后将微流控芯片置于37℃恒温箱中1小时，使中央细胞培养通道和各迁移细胞培养通道的表面涂覆上培养基质；恢复疏水性：对于各微流控芯片，用无菌水冲洗中央细胞培养通道和各迁移细胞培养通道中多余的培养基质溶液，将微流控芯片置于80℃的烘箱中1-2小时，使微流控芯片干燥并恢复疏水性； Step 1, pretreatment of microfluidic chips: take three microfluidic chips, namely, a microfluidic chip for experiment, a microfluidic chip for normal cell control and a microfluidic chip for blank control; coating culture matrix: for each microfluidic chip, fill the central cell culture channel and each migration cell culture channel with culture matrix solution, and then place the microfluidic chip in a 37°C constant temperature box for 1 hour to coat the surface of the central cell culture channel and each migration cell culture channel with culture matrix; restore hydrophobicity: for each microfluidic chip, rinse the excess culture matrix solution in the central cell culture channel and each migration cell culture channel with sterile water, and place the microfluidic chip in an oven at 80°C for 1-2 hours to dry the microfluidic chip and restore hydrophobicity;
步骤二、荧光标记细胞：取生长状态良好的肿瘤细胞株、与肿瘤细胞同器官来源的正常细胞株和不同组织来源间充质干细胞，用活细胞示踪剂分别标记肿瘤细胞、与肿瘤细胞同器官来源的正常细胞和各间充质干细胞，其中，标记肿瘤细 胞、与肿瘤细胞同器官来源的正常细胞的活细胞示踪剂的颜色与标记间充质干细胞的活细胞示踪剂的颜色不同； Step 2, fluorescently labeling cells: taking tumor cell lines with good growth status, normal cell lines derived from the same organ as the tumor cells, and mesenchymal stem cells derived from different tissues, and labeling the tumor cells, normal cells derived from the same organ as the tumor cells, and mesenchymal stem cells with live cell tracers, respectively, wherein the color of the live cell tracer labeling the tumor cells and the normal cells derived from the same organ as the tumor cells is different from the color of the live cell tracer labeling the mesenchymal stem cells;
步骤三、接种及培养细胞：对于各微流控芯片，将基质胶注入基质胶灌注通道，将微流控芯片放入37℃培养箱30分钟，促进基质胶固化；将活细胞示踪剂标记的肿瘤细胞和与肿瘤细胞同器官来源的正常细胞分别加入培养基配制成细胞悬液，然后分别种植于实验用微流控芯片和正常细胞对照用微流控芯片的中央细胞培养通道；将培养基灌入空白对照用微流控芯片的中央细胞培养通道；将各活细胞示踪剂标记的间充质干细胞分别加入培养基配制成细胞悬液，然后分别种植于各微流控芯片的不同细胞迁移单元的移细胞培养通道，三个微流控芯进行相同的种植；将三个微流控芯片放入培养箱孵育； Step 3, inoculating and culturing cells: for each microfluidic chip, inject the matrix gel into the matrix gel perfusion channel, and place the microfluidic chip in a 37°C incubator for 30 minutes to promote the solidification of the matrix gel; add the tumor cells labeled with the live cell tracer and the normal cells from the same organ as the tumor cells into the culture medium to prepare a cell suspension, and then plant them in the central cell culture channel of the experimental microfluidic chip and the normal cell control microfluidic chip respectively; perfuse the culture medium into the central cell culture channel of the blank control microfluidic chip; add the mesenchymal stem cells labeled with each live cell tracer into the culture medium to prepare a cell suspension, and then plant them in the cell culture channels of different cell migration units of each microfluidic chip, and perform the same planting on the three microfluidic cores; put the three microfluidic chips into the incubator for incubation;
步骤四、观察和监测细胞的迁移：待间充质干细胞贴壁后，取出微流控芯片在倒置荧光显微镜下观察并记录不同间充质干细胞在各基质胶灌注通道的迁移情况，记为0小时；继续培养n小时后，取出微流控芯片在倒置荧光显微镜下观察并记录不同间充质干细胞在相应基质胶灌注通道的迁移情况，记为n小时；用软件测量并比较n小时内不同间充质干细胞向肿瘤细胞、与肿瘤细胞同器官来源的正常细胞及培养基的趋化迁移的迁移面积和最大迁移距离。Step 4, observe and monitor cell migration: after the mesenchymal stem cells adhere to the wall, take out the microfluidic chip and observe and record the migration of different mesenchymal stem cells in each matrix gel perfusion channel under an inverted fluorescence microscope, which is recorded as 0 hour; after continuing to culture for n hours, take out the microfluidic chip and observe and record the migration of different mesenchymal stem cells in the corresponding matrix gel perfusion channel under an inverted fluorescence microscope, which is recorded as n hours; use software to measure and compare the migration area and maximum migration distance of the chemotactic migration of different mesenchymal stem cells to tumor cells, normal cells from the same organ as the tumor cells, and culture medium within n hours.
进一步，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片的应用方法，还可以具有这样的特征：其中，涂覆培养基质前，对微流控芯片进行无菌处理；无菌处理方法为：用75％酒精(体积分数)润洗中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道，然后将微流控芯片置于紫外光照射下灭菌过夜，待75％酒精完全挥发后再涂覆培养基质。Furthermore, the present invention provides an application method of a tumor microfluidic chip for mesenchymal stem cell migration screening, which may also have the following characteristics: wherein, before coating the culture matrix, the microfluidic chip is aseptically treated; the aseptic treatment method is: rinsing the central cell culture channel and each matrix gel perfusion channel and the migration cell culture channel with 75% alcohol (volume fraction), and then placing the microfluidic chip under ultraviolet light for sterilization overnight, and then coating the culture matrix after the 75% alcohol is completely evaporated.
进一步，本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片的应用方法，还可以具有这样的特征：其中，步骤三中，种植于中央细胞培养通道的肿瘤细胞和与肿瘤细胞同器官来源的正常细胞的细胞悬液的细胞密度为2×10
6/ml；种植于移细胞培养通道的间充质干细胞的细胞悬液的细胞密度为1×10
6/ml。
Furthermore, the present invention provides an application method of a tumor microfluidic chip for mesenchymal stem cell migration screening, which may also have the following characteristics: wherein, in step three, the cell density of the cell suspension of tumor cells and normal cells from the same organ as the tumor cells planted in the central cell culture channel is 2×10 6 /ml; the cell density of the cell suspension of mesenchymal stem cells planted in the migration cell culture channel is 1×10 6 /ml.
本发明的有益效果在于：本发明从MSCs特异的肿瘤趋化性出发，依托微流控芯片技术，提供了一种用于间充质干细胞迁移筛选的肿瘤微流控芯片及其制备和应用方法，该微流控芯片实现了多种类型MSCs和肿瘤细胞的同步共培养，并可实现同步比较不同类型MSCs趋化性的差异。具体的：The beneficial effects of the present invention are as follows: Based on the specific tumor chemotaxis of MSCs and relying on microfluidic chip technology, the present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening and its preparation and application methods, and the microfluidic chip realizes the synchronous co-culture of multiple types of MSCs and tumor cells, and can realize the synchronous comparison of the differences in the chemotaxis of different types of MSCs. Specifically:
一、本发明的微流控芯片包含多个通道，所有通道尺寸均在微米量级，细胞和试剂消耗量极少，降低成本，减少细胞损伤；1. The microfluidic chip of the present invention comprises multiple channels, all of which are in the micrometer range in size, consumes very few cells and reagents, reduces costs, and reduces cell damage;
二、本发明涉及的微流控芯片包含一条用于肿瘤细胞培养的中央细胞培养通道和多个对称排列在中央细胞培养通道两侧的独立的细胞迁移单元集成度高，能够同时实现对多种组织来源MSCs迁移特性的观察和监测，和划痕实验及transwell实验相比，降低了成本，提高实验效率，且实验重复性更好；Second, the microfluidic chip of the present invention comprises a central cell culture channel for tumor cell culture and a plurality of independent cell migration units symmetrically arranged on both sides of the central cell culture channel. It has a high degree of integration and can simultaneously observe and monitor the migration characteristics of MSCs from multiple tissue sources. Compared with the scratch experiment and the transwell experiment, it reduces the cost, improves the experimental efficiency, and has better experimental repeatability.
三、本发明的微流控芯片的各细胞迁移单元均包含一条基质胶灌注通道，可在三维层面观测不同MSCs的迁移能力；3. Each cell migration unit of the microfluidic chip of the present invention comprises a matrix gel perfusion channel, which can observe the migration ability of different MSCs in three dimensions;
四、本发明的微流控芯片实现了多种类型MSCs和肿瘤细胞的同步共培养，操作简单，降低了实际样品的用量，并模拟人间充质干细胞对肿瘤细胞的趋化性并同步比较差异，能够为筛选肿瘤靶向治疗高效药物载体提供理论依据。4. The microfluidic chip of the present invention realizes the synchronous co-culture of various types of MSCs and tumor cells, is simple to operate, reduces the amount of actual samples used, and simulates the chemotaxis of human mesenchymal stem cells to tumor cells and compares the differences simultaneously, which can provide a theoretical basis for screening efficient drug carriers for tumor targeted therapy.
图1是本发明微流控芯片的结构示意图；FIG1 is a schematic diagram of the structure of a microfluidic chip of the present invention;
图2是图1中A区域的放大图；FIG2 is an enlarged view of area A in FIG1 ;
图3是本发明微流控芯片的生物相容性检测结果图；FIG3 is a diagram showing the biocompatibility test results of the microfluidic chip of the present invention;
图4是不同组织来源的MSCs在微流控芯片上对宫颈癌细胞趋化性的观察和比较结果图。FIG. 4 is a diagram showing the observation and comparison of the chemotaxis of MSCs from different tissue sources to cervical cancer cells on a microfluidic chip.
为了使本领域技术领域人员更好地理解本发明的技术方案，下面结合具体实施例对本发明的上述方案作进一步说明。In order to enable those skilled in the art to better understand the technical solution of the present invention, the above solution of the present invention is further described below in conjunction with specific embodiments.
下述实施例中所使用的实验方法，如无特殊说明，均为常规方法，所用的试剂、方法和设备，如无特殊说明，均为本技术领域常规试剂、方法和设备。The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.
本发明提供一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，包括表面加工出微结构通道的芯片主体和紧密贴合在芯片主体上的基板，微结构通道形成于芯片主体与基板间。The present invention provides a tumor microfluidic chip for mesenchymal stem cell migration screening, comprising a chip body with a microstructure channel processed on the surface and a substrate tightly attached to the chip body, wherein the microstructure channel is formed between the chip body and the substrate.
如图1所示，芯片主体上的微结构通道包括一条中央细胞培养通道1和四个对称排列在中央细胞培养通道1两侧的相互独立的细胞迁移单元2，细胞迁移单元2包括一条内侧的基质胶灌注通道21和一条外侧的迁移细胞培养通道22。其中，“内”和“外”分别是指相对靠近和远离中央细胞培养通道1的方向。细胞迁移单元2也可以设置若干个，对称排列在中央细胞培养通道1两侧即可。As shown in FIG1 , the microstructure channel on the chip body includes a central cell culture channel 1 and four independent cell migration units 2 symmetrically arranged on both sides of the central cell culture channel 1, and the cell migration unit 2 includes an inner matrix gel perfusion channel 21 and an outer migration cell culture channel 22. Here, "inside" and "outside" refer to the directions relatively close to and away from the central cell culture channel 1, respectively. A plurality of cell migration units 2 can also be provided, and they can be symmetrically arranged on both sides of the central cell culture channel 1.
中央细胞培养通道1和各基质胶灌注通道21、迁移细胞培养通道22的两端均具有沿芯片主体厚度方向的进样口和出样口。Both ends of the central cell culture channel 1 and each of the matrix gel perfusion channels 21 and the migration cell culture channels 22 are provided with a sample inlet and a sample outlet along the thickness direction of the chip body.
中央细胞培养通道1用于肿瘤细胞的培养，基质胶灌注通道21用于基质胶的填充，若干迁移细胞培养通道22用于不同组织来源间充质干细胞的培养。The central cell culture channel 1 is used for the culture of tumor cells, the matrix gel perfusion channel 21 is used for filling with matrix gel, and the plurality of migration cell culture channels 22 are used for the culture of mesenchymal stem cells from different tissue sources.
中央细胞培养通道1与各细胞迁移单元2的基质胶灌注通道21之间、以及同一细胞迁移单元2的基质胶灌注通道21与迁移细胞培养通道22之间均设有截流结构3，截流结构3由若干间隔设置的柱状结构31构成，中央细胞培养通道1与各细胞迁移单元2的基质胶灌注通道21、以及同一细胞迁移单元2的基质胶灌注通道21与迁移细胞培养通道22通过截流结构3进行细胞之间的信号传输和相互作用以及细胞迁移。An interception structure 3 is provided between the central cell culture channel 1 and the matrix gel perfusion channel 21 of each cell migration unit 2, and between the matrix gel perfusion channel 21 of the same cell migration unit 2 and the migration cell culture channel 22. The interception structure 3 is composed of a plurality of columnar structures 31 arranged at intervals. The central cell culture channel 1 and the matrix gel perfusion channel 21 of each cell migration unit 2, and between the matrix gel perfusion channel 21 of the same cell migration unit 2 and the migration cell culture channel 22 carry out signal transmission and interaction between cells and cell migration through the interception structure 3.
在一优选的实施例中，细胞迁移单元2中的基质胶灌注通道21和迁移细胞培养通道22的两端均向外侧方向弯折，该弯折结构可以增加各通道进样口和出样口的距离，一方面便于加工各通道的进样口和出样口，另一方面便于各通道细胞的接种或基质胶的灌注。In a preferred embodiment, both ends of the matrix gel perfusion channel 21 and the migration cell culture channel 22 in the cell migration unit 2 are bent outward. The bent structure can increase the distance between the sample inlet and the sample outlet of each channel, which is convenient for processing the sample inlet and the sample outlet of each channel on the one hand, and is convenient for inoculation of cells in each channel or perfusion of matrix gel on the other hand.
芯片主体的材料为聚二甲基硅氧烷(PDMS)，也可以为聚甲基丙烯酸甲酯(PMMA)或聚碳酸酯(PC)；基板的材料为玻璃，也可以为硅片。芯片主体与基板通过等离子体键合工艺实现两者之间的紧密贴合。The chip body is made of polydimethylsiloxane (PDMS), or polymethyl methacrylate (PMMA) or polycarbonate (PC); the substrate is made of glass or silicon wafer. The chip body and the substrate are closely attached to each other through a plasma bonding process.
中央细胞培养通道1和迁移细胞培养通道22培养肿瘤细胞和间充质干细胞前涂覆有培养基质；培养基质为明胶、壳聚糖、丝素蛋白、鼠尾胶原蛋白、纤维蛋白胶、基质胶(Matrigel)中的一种或几种。The central cell culture channel 1 and the migration cell culture channel 22 are coated with a culture matrix before culturing tumor cells and mesenchymal stem cells; the culture matrix is one or more of gelatin, chitosan, silk fibroin, rat tail collagen, fibrin glue, and Matrigel.
芯片主体的中央细胞培养通道1和各基质胶灌注通道21、迁移细胞培养通道22均位于同一平面，且宽度和高度均相同，分别为1000μm和100μm；基质胶灌注通道21的长度为1.0cm，迁移细胞培养通22的长度为0.8cm，中央细胞培 养通道1的长度根据细胞迁移单元2的数量而定，本实施例在中央细胞培养通道1的长度方向上排列有两个细胞迁移单元2，中央细胞培养通道1的长度为2.6cm。优选的，截流结构3由5个六棱柱等间距排列而成，各六棱柱结构的边长为100μm，六棱柱结构之间的间距为50μm。The central cell culture channel 1 of the chip body and each matrix gel perfusion channel 21 and migration cell culture channel 22 are all located in the same plane, and the width and height are the same, which are 1000 μm and 100 μm respectively; the length of the matrix gel perfusion channel 21 is 1.0 cm, the length of the migration cell culture channel 22 is 0.8 cm, and the length of the central cell culture channel 1 is determined according to the number of cell migration units 2. In this embodiment, two cell migration units 2 are arranged in the length direction of the central cell culture channel 1, and the length of the central cell culture channel 1 is 2.6 cm. Preferably, the intercepting structure 3 is composed of 5 hexagonal prisms arranged at equal intervals, the side length of each hexagonal prism structure is 100 μm, and the spacing between the hexagonal prism structures is 50 μm.
本发明用于间充质干细胞迁移筛选的肿瘤微流控芯片的制备方法，包括以下步骤：The method for preparing a tumor microfluidic chip for mesenchymal stem cell migration screening of the present invention comprises the following steps:
步骤一、用计算机辅助软件(CAD)设计和绘制芯片主体的微结构通道图形；Step 1: Design and draw the microstructure channel graphics of the chip body using computer-aided software (CAD);
步骤二、依次通过光刻、软光刻法、模塑法等微加工技术制备具有微结构通道的芯片主体，通过手动打孔器制备中央细胞培养通道1和各基质胶灌注通道21、迁移细胞培养通道22的进样口及出样口；Step 2: Prepare a chip body with microstructure channels by microfabrication techniques such as photolithography, soft photolithography, and molding, and prepare the inlet and outlet of the central cell culture channel 1 and each matrix gel perfusion channel 21 and the migration cell culture channel 22 by a manual puncher;
步骤三、将芯片主体和基板用无水乙醇清洗后，进行高压灭菌处理，最后进行等离子体键合，得到微流控芯片。Step 3: Clean the chip body and substrate with anhydrous ethanol, sterilize them under high pressure, and finally perform plasma bonding to obtain a microfluidic chip.
本发明用于间充质干细胞迁移筛选的肿瘤微流控芯片的生物相容性检测：采用三个微流控芯片分别对与宫颈癌细胞(SiHa细胞)、正常宫颈上皮细胞(ECT1/E6E7细胞)和脂肪间充质干细胞(Adipose tissue-derived MSCs,AT-MSCs)的生物相容性进行检测。The present invention is used for biocompatibility testing of the tumor microfluidic chip for mesenchymal stem cell migration screening: three microfluidic chips are used to respectively test the biocompatibility with cervical cancer cells (SiHa cells), normal cervical epithelial cells (ECT1/E6E7 cells) and adipose tissue-derived MSCs (AT-MSCs).
首先，依次对三个微流控芯片进行无菌处理、涂覆培养基质及恢复疏水性的处理。然后，采用红色荧光活细胞示踪剂(CellTracker
TM Red CMTPX)标记SiHa细胞和ECT1/E6E7细胞，采用绿色荧光活细胞示踪剂(CellTracker
TM Green CMFDA)标记MSCs。最后，取经CellTracker
TM Red CMTPX标记后的SiHa细胞和ECT1/E6E7细胞，经胰酶消化离心后，加入培养基(DMEM培养基，下同) 配制成密度为2×10
6/ml的细胞悬液；用移液枪将配制好的SiHa细胞和ECT1/E6E7细胞的细胞悬液分别加入两个微流控芯片的中央细胞培养通道中，控制加样速度，使细胞悬液缓慢注入中央细胞培养通道。取经CellTracker
TM Green CMFDA标记后的AT-MSCs，经胰酶消化离心后，加入培养基配制成密度为1×10
6/ml的细胞悬液；用移液枪将配制好的AT-MSCs的细胞悬液加入另一个微流控芯片的迁移细胞培养通道，控制加样速度，使细胞悬液缓慢注入迁移细胞培养通道。将三个微流控芯片重新放入培养皿，用PBS填充芯片周围的培养皿，放入37℃恒温培养箱培养。待细胞贴壁后，分别于孵育0、24、和48h后，取出芯片，在倒置荧光显微镜下观察记录SiHa细胞、ECT1/E6E7细胞和AT-MSCs的生长状况，结果均显示SiHa细胞、ECT1/E6E7细胞和AT-MSCs在微流控芯片上生长良好，如图3所示，结果表明该微流控芯片具有良好的生物相容性。在其他实施例中，微流控芯片的生物相容性的检测也可通过DAPI/PI，Calcein-AM/PI等活细胞、死细胞原位双染法进行。
First, the three microfluidic chips were sterilized, coated with culture matrix, and treated to restore hydrophobicity. Then, SiHa cells and ECT1/E6E7 cells were labeled with red fluorescent live cell tracer (CellTracker TM Red CMTPX), and MSCs were labeled with green fluorescent live cell tracer (CellTracker TM Green CMFDA). Finally, SiHa cells and ECT1/E6E7 cells labeled with CellTracker TM Red CMTPX were taken, digested with trypsin, centrifuged, and added with culture medium (DMEM culture medium, the same below) to prepare a cell suspension with a density of 2×10 6 /ml; the prepared cell suspensions of SiHa cells and ECT1/E6E7 cells were added to the central cell culture channels of the two microfluidic chips respectively with a pipette, and the sample addition speed was controlled to slowly inject the cell suspension into the central cell culture channel. Take the AT-MSCs labeled with CellTracker TM Green CMFDA, digest them with trypsin and centrifuge them, then add culture medium to prepare a cell suspension with a density of 1×10 6 /ml; use a pipette to add the prepared AT-MSCs cell suspension to the migration cell culture channel of another microfluidic chip, control the sample addition speed, and slowly inject the cell suspension into the migration cell culture channel. Put the three microfluidic chips back into the culture dish, fill the culture dish around the chip with PBS, and put it into a 37℃ constant temperature incubator for culture. After the cells adhere to the wall, take out the chip after incubation for 0, 24, and 48 hours, and observe and record the growth status of SiHa cells, ECT1/E6E7 cells and AT-MSCs under an inverted fluorescence microscope. The results all show that SiHa cells, ECT1/E6E7 cells and AT-MSCs grow well on the microfluidic chip, as shown in Figure 3. The results show that the microfluidic chip has good biocompatibility. In other embodiments, the biocompatibility of the microfluidic chip can also be detected by in situ double staining of live cells and dead cells such as DAPI/PI and Calcein-AM/PI.
本发明用于间充质干细胞迁移筛选的肿瘤微流控芯片的应用方法，包括以下步骤：The application method of the tumor microfluidic chip for mesenchymal stem cell migration screening of the present invention comprises the following steps:
步骤一、微流控芯片的预处理：Step 1: Pretreatment of microfluidic chip:
取三个微流控芯片，分别为实验用微流控芯片、正常细胞对照用微流控芯片和空白对照用微流控芯片。Take three microfluidic chips, namely, a microfluidic chip for experiment, a microfluidic chip for normal cell control and a microfluidic chip for blank control.
无菌处理：对于各微流控芯片，用75％酒精(体积分数)润洗中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道，然后将微流控芯片置于紫外光照射下灭菌过夜，待75％酒精完全挥发后再涂覆培养基质。Sterile treatment: For each microfluidic chip, rinse the central cell culture channel and each matrix gel perfusion channel and migration cell culture channel with 75% alcohol (volume fraction), then sterilize the microfluidic chip under ultraviolet light overnight, and then coat the culture matrix after the 75% alcohol is completely evaporated.
涂覆培养基质：对于各微流控芯片，向中央细胞培养通道和各迁移细胞培养通道填充稀释后的鼠尾胶原蛋白I型溶液，然后将微流控芯片置于37℃含5v/v％CO
2恒温箱中1小时，使中央细胞培养通道和各迁移细胞培养通道的表面涂覆上鼠尾胶原蛋白I型，以促进细胞贴壁。
Coating the culture matrix: For each microfluidic chip, fill the central cell culture channel and each migration cell culture channel with diluted rat tail collagen type I solution, and then place the microfluidic chip in a 37°C incubator containing 5v/v% CO2 for 1 hour to coat the surface of the central cell culture channel and each migration cell culture channel with rat tail collagen type I to promote cell adhesion.
恢复疏水性：对于各微流控芯片，用无菌水冲洗中央细胞培养通道和各迁移细胞培养通道中多余的鼠尾胶原蛋白I型溶液，将微流控芯片置于80℃的烘箱中1-2小时，使微流控芯片干燥并恢复PDMS疏水性。Restoring hydrophobicity: For each microfluidic chip, rinse the excess rat tail collagen type I solution in the central cell culture channel and each migration cell culture channel with sterile water, and place the microfluidic chip in an oven at 80°C for 1-2 hours to dry the microfluidic chip and restore the hydrophobicity of PDMS.
步骤二、荧光标记宫颈癌细胞(SiHa细胞)、正常宫颈上皮细胞(ECT1/E6E7细胞)和不同组织来源的间充质干细胞(MSCs)：Step 2: Fluorescently label cervical cancer cells (SiHa cells), normal cervical epithelial cells (ECT1/E6E7 cells) and mesenchymal stem cells (MSCs) from different tissue sources:
进行荧光标记处理前，用二甲基亚砜(dimethyl sulfoxide，DMSO)将绿色荧光活细胞示踪剂(CellTracker
TM Green CMFDA)和红色荧光活细胞示踪剂(CellTracker
TM Red CMTPX)配制成10mM的原始溶液，再将原始溶液加入培养基中，配置成终浓度为5μM的活细胞示踪剂工作液。取处于对数生长期且生长状态良好的SiHa细胞、ECT1/E6E7细胞和各组织来源的MSCs，弃去培养液，用PBS缓冲液清洗2次。用移液枪分别向SiHa细胞和ECT1/E6E7细胞的培养皿中缓慢加入6ml的CellTracker
TM Red CMTPX，用移液枪向各组织来源的MSCs的培养皿中缓慢加入6ml的CellTracker
TM Green CMFDA工作液，将上述培养皿置于37℃含5％CO
2的培养箱孵育1h。孵育结束后，取出培养皿，置于倒置荧光显微镜下观察荧光标记情况，结果显示SiHa细胞、ECT1/E6E7细胞和各组织来源的MSCs的均成功携带了荧光示踪标记。随后，弃去示踪剂工作液，用PBS缓冲液清洗细胞1次后，加入相应的培养基，放入37℃含5％CO
2的培养箱继续 培养。
Before fluorescent labeling, dimethyl sulfoxide (DMSO) was used to prepare a 10 mM original solution of green fluorescent live cell tracer (CellTracker TM Green CMFDA) and red fluorescent live cell tracer (CellTracker TM Red CMTPX), and the original solution was added to the culture medium to prepare a live cell tracer working solution with a final concentration of 5 μM. SiHa cells, ECT1/E6E7 cells and MSCs from various tissues that were in logarithmic growth phase and in good growth state were taken, the culture medium was discarded, and the cells were washed twice with PBS buffer. 6 ml of CellTracker TM Red CMTPX was slowly added to the culture dishes of SiHa cells and ECT1/E6E7 cells with a pipette, and 6 ml of CellTracker TM Green CMFDA working solution was slowly added to the culture dishes of MSCs from various tissues with a pipette, and the above culture dishes were placed in a 37°C incubator containing 5% CO 2 and incubated for 1 hour. After the incubation, the culture dish was removed and placed under an inverted fluorescence microscope to observe the fluorescent labeling. The results showed that SiHa cells, ECT1/E6E7 cells and MSCs from various tissues all successfully carried fluorescent tracer labels. Subsequently, the tracer working solution was discarded, the cells were washed once with PBS buffer, and the corresponding culture medium was added and placed in a 37°C incubator containing 5% CO 2 for continued culture.
步骤三、接种及培养细胞：对于各微流控芯片，用移液枪分别从各基质胶灌注通道的进样口处缓慢注入冰上预冷的基质胶∶培养基(体积比1∶1)稀释液，将微流控芯片置于培养皿中，放入37℃含5％CO
2的培养箱30分钟，促进基质胶固化。
Step 3: Inoculation and culture of cells: For each microfluidic chip, use a pipette to slowly inject ice-precooled matrix gel: culture medium (volume ratio 1:1) dilution solution from the injection port of each matrix gel perfusion channel, place the microfluidic chip in a culture dish, and put it in a 37°C incubator containing 5% CO2 for 30 minutes to promote the solidification of the matrix gel.
取经CellTracker
TM Red CMTPX标记后的SiHa细胞和ECT1/E6E7细胞，经胰酶消化离心后，加入培养基配制成密度为2×10
6/ml的细胞悬液。取出各微流控芯片，用移液枪将SiHa细胞的细胞悬液、ECT1/E6E7细胞的细胞悬液和不含细胞的DMEM培养基分别加入实验用微流控芯片、正常细胞对照用微流控芯片和空白对照用微流控芯片的中央细胞培养通道中，控制加样速度，使SiHa细胞的细胞悬液、ECT1/E6E7细胞的细胞悬液和不含细胞的DMEM培养基缓慢流入相应的中央细胞培养通道。将各微流控芯片重新放入培养皿，用PBS填充芯片周围的培养皿，放入37℃恒温培养箱孵育6～8h。待SiHa细胞和ECT1/E6E7细胞贴壁后，取经CellTracker
TM Green CMFDA标记后的不同组织来源的MSCs，经胰酶消化离心后，加入培养基配制成密度为1×10
6/ml的细胞悬液。取出各微流控芯片，用移液枪将不同组织来源的MSCs的细胞悬液分别加入各微流控芯片的不同细胞迁移单元的移细胞培养通道中，控制加样速度，使各种MSCs的细胞悬液缓慢注入各移细胞培养通道。将各微流控芯片重新放入培养皿，放入37℃含5％CO
2的培养箱继续孵育。
SiHa cells and ECT1/E6E7 cells labeled with CellTracker TM Red CMTPX were taken out, digested with trypsin and centrifuged, and then added to the culture medium to prepare a cell suspension with a density of 2×10 6 /ml. Each microfluidic chip was taken out, and the cell suspension of SiHa cells, the cell suspension of ECT1/E6E7 cells and the DMEM culture medium without cells were added to the central cell culture channel of the experimental microfluidic chip, the normal cell control microfluidic chip and the blank control microfluidic chip respectively with a pipette, and the sample addition speed was controlled so that the cell suspension of SiHa cells, the cell suspension of ECT1/E6E7 cells and the DMEM culture medium without cells slowly flowed into the corresponding central cell culture channel. Each microfluidic chip was put back into the culture dish, and the culture dish around the chip was filled with PBS, and placed in a 37°C constant temperature incubator for incubation for 6 to 8 hours. After SiHa cells and ECT1/E6E7 cells adhered to the wall, MSCs from different tissues were obtained after being labeled with CellTracker TM Green CMFDA. After trypsin digestion and centrifugation, culture medium was added to prepare a cell suspension with a density of 1×10 6 /ml. Each microfluidic chip was taken out, and the cell suspensions of MSCs from different tissues were added to the cell culture channels of different cell migration units of each microfluidic chip with a pipette. The sample addition speed was controlled to slowly inject the cell suspensions of various MSCs into each cell culture channel. Each microfluidic chip was put back into the culture dish and placed in a 37°C incubator containing 5% CO 2 for further incubation.
步骤四、观察和监测细胞的迁移(间充质干细胞肿瘤趋化性的观察和比较)：Step 4: Observe and monitor cell migration (observation and comparison of mesenchymal stem cell tumor chemotaxis):
MSCs接种结束后，将各微流控芯片放入37℃含5％CO
2的培养箱中孵育 6～8h，待各种MSCs贴壁后，取出微流控芯片，在倒置荧光显微镜下观察并记录SiHa细胞、ECT1/E6E7细胞和各组织来源的MSCs的生长状态，以及观察并记录各基质胶灌注通道中不同组织来源的MSCs的迁移行为，记为0h。将各微流控芯片放入37℃含5％CO
2的培养箱中继续培养，待孵育24h后，取出微流控芯片，在倒置荧光显微镜下观察并记录SiHa细胞、ECT1/E6E7细胞和各组织来源的MSCs的生长状态，观察并记录各基质胶灌注通道中不同组织来源的MSCs的迁移行为，记为24h。用Image-Pro Plus软件测量并比较24小时内不同MSCs在基质胶灌注通道内向SiHa细胞、ECT1/E6E7细胞及不含细胞的DMEM培养基趋化迁移的迁移面积和最大迁移距离。
After the MSCs inoculation, each microfluidic chip was placed in a 37°C incubator containing 5% CO 2 and incubated for 6 to 8 hours. After various MSCs adhered to the wall, the microfluidic chip was taken out, and the growth status of SiHa cells, ECT1/E6E7 cells and MSCs from various tissues was observed and recorded under an inverted fluorescence microscope, and the migration behavior of MSCs from different tissues in each matrix gel perfusion channel was observed and recorded, which was recorded as 0h. Each microfluidic chip was placed in a 37°C incubator containing 5% CO 2 and continued to be cultured. After incubation for 24 hours, the microfluidic chip was taken out, and the growth status of SiHa cells, ECT1/E6E7 cells and MSCs from various tissues was observed and recorded under an inverted fluorescence microscope, and the migration behavior of MSCs from different tissues in each matrix gel perfusion channel was observed and recorded, which was recorded as 24h. Image-Pro Plus software was used to measure and compare the migration area and maximum migration distance of different MSCs in the matrix gel perfusion channel toward SiHa cells, ECT1/E6E7 cells and cell-free DMEM medium within 24 hours.
本实施例选取了四种组织来源的MSCs，包括脂肪间充质干细胞(Adipose tissue-derived MSCs,AT-MSCs)、脐带间充质干细胞(Umbilical cord-derived MSCs,UC-MSCs)、羊膜间充质干细胞(Amniotic membrane-derived MSCs,AM-MSCs)和绒毛膜间充质干细胞(Chorionic plate-derived MSCs,CP-MSCs)。结果如图4所示，首先，通过比较实验用微流控芯片与正常细胞对照用微流控芯片和空白对照用微流控芯片的结果，可以看出各MSCs对SiHa细胞具有趋化能力；其次，通过比较实验用微流控芯片各细胞迁移单元的结果，可以看出CP-MSCs对SiHa细胞趋化能力最强，因此可作为后续宫颈癌靶向治疗研究的潜在药物载体。在本实施例中，微流控芯片的观察时间点可选择培养过程中的任一时刻，本例以培养24h为例。In this embodiment, four tissue-derived MSCs were selected, including adipose tissue-derived MSCs (AT-MSCs), umbilical cord-derived MSCs (UC-MSCs), amniotic membrane-derived MSCs (AM-MSCs) and chorionic plate-derived MSCs (CP-MSCs). The results are shown in Figure 4. First, by comparing the results of the experimental microfluidic chip with the normal cell control microfluidic chip and the blank control microfluidic chip, it can be seen that each MSC has chemotaxis to SiHa cells; secondly, by comparing the results of each cell migration unit of the experimental microfluidic chip, it can be seen that CP-MSCs has the strongest chemotaxis to SiHa cells, so it can be used as a potential drug carrier for subsequent targeted treatment of cervical cancer. In this embodiment, the observation time point of the microfluidic chip can be selected at any time during the culture process. This example takes 24h of culture as an example.
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 embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.
Claims (10)
- 一种用于间充质干细胞迁移筛选的肿瘤微流控芯片，其特征在于：A tumor microfluidic chip for mesenchymal stem cell migration screening, characterized in that:包括表面加工出微结构通道的芯片主体和紧密贴合在芯片主体上的基板；It includes a chip body with a microstructure channel processed on the surface and a substrate closely attached to the chip body;芯片主体上的微结构通道包括一条中央细胞培养通道和若干个对称排列在中央细胞培养通道两侧的相互独立的细胞迁移单元；所述细胞迁移单元包括一条内侧的基质胶灌注通道和一条外侧的迁移细胞培养通道；The microstructure channel on the chip body includes a central cell culture channel and a plurality of independent cell migration units symmetrically arranged on both sides of the central cell culture channel; the cell migration unit includes an inner matrix gel perfusion channel and an outer migration cell culture channel;中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道的两端均具有进样口和出样口；Both ends of the central cell culture channel and each matrix gel perfusion channel and migration cell culture channel are provided with an inlet and an outlet;所述中央细胞培养通道用于肿瘤细胞的培养；基质胶灌注通道用于基质胶的填充；若干迁移细胞培养通道用于不同组织来源间充质干细胞的培养；The central cell culture channel is used for culturing tumor cells; the matrix gel perfusion channel is used for filling matrix gel; and the plurality of migration cell culture channels are used for culturing mesenchymal stem cells from different tissue sources;所述中央细胞培养通道与各细胞迁移单元的基质胶灌注通道之间、以及同一细胞迁移单元的基质胶灌注通道与迁移细胞培养通道之间均设有截流结构，截流结构由若干间隔设置的柱状结构构成，中央细胞培养通道与各细胞迁移单元的基质胶灌注通道、以及同一细胞迁移单元的基质胶灌注通道与迁移细胞培养通道通过截流结构进行细胞之间的信号传输和相互作用以及细胞迁移。An interception structure is provided between the central cell culture channel and the matrix gel perfusion channel of each cell migration unit, and between the matrix gel perfusion channel of the same cell migration unit and the migration cell culture channel. The interception structure is composed of a plurality of columnar structures arranged at intervals. The central cell culture channel and the matrix gel perfusion channel of each cell migration unit, and between the matrix gel perfusion channel of the same cell migration unit and the migration cell culture channel, perform signal transmission and interaction between cells and cell migration through the interception structure.
- 根据权利要求1所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片，其特征在于：The tumor microfluidic chip for mesenchymal stem cell migration screening according to claim 1, characterized in that:其中，所述细胞迁移单元中，基质胶灌注通道和迁移细胞培养通道的两端均向外侧方向弯折。Wherein, in the cell migration unit, both ends of the matrix gel perfusion channel and the migration cell culture channel are bent outward.
- 根据权利要求1所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片，其特征在于：The tumor microfluidic chip for mesenchymal stem cell migration screening according to claim 1, characterized in that:其中，所述芯片主体的材料为聚二甲基硅氧烷、聚甲基丙烯酸甲酯或聚碳酸酯；Wherein, the material of the chip body is polydimethylsiloxane, polymethyl methacrylate or polycarbonate;所述基板的材料为玻璃或硅片。The substrate is made of glass or silicon wafer.
- 根据权利要求1所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片，其特征在于：The tumor microfluidic chip for mesenchymal stem cell migration screening according to claim 1, characterized in that:其中，所述芯片主体与基板通过等离子体键合工艺实现两者之间的紧密贴合。Wherein, the chip body and the substrate are tightly fitted together by a plasma bonding process.
- 根据权利要求1所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片，其特征在于：The tumor microfluidic chip for mesenchymal stem cell migration screening according to claim 1, characterized in that:其中，所述中央细胞培养通道和迁移细胞培养通道培养肿瘤细胞和间充质干细胞前涂覆有培养基质，培养基质为明胶、壳聚糖、丝素蛋白、鼠尾胶原蛋白、纤维蛋白胶、基质胶中的一种或几种。Wherein, the central cell culture channel and the migration cell culture channel are coated with a culture matrix before culturing tumor cells and mesenchymal stem cells, and the culture matrix is one or more of gelatin, chitosan, silk fibroin, rat tail collagen, fibrin glue, and matrix glue.
- 根据权利要求1所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片，其特征在于：The tumor microfluidic chip for mesenchymal stem cell migration screening according to claim 1, characterized in that:其中，所述芯片主体的中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道均位于同一平面，且宽度和高度均相同，分别为1000μm和100μm；基质胶灌注通道的长度为1.0cm，迁移细胞培养通的长度为0.8cm；The central cell culture channel of the chip body and each matrix gel perfusion channel and migration cell culture channel are located in the same plane, and have the same width and height, which are 1000 μm and 100 μm respectively; the length of the matrix gel perfusion channel is 1.0 cm, and the length of the migration cell culture channel is 0.8 cm;所述截流结构由5个六棱柱结构等间距排列而成，各六棱柱结构的边长为100μm，六棱柱结构之间的间距为50μm。The intercepting structure is composed of five hexagonal prism structures arranged at equal intervals, the side length of each hexagonal prism structure is 100 μm, and the interval between the hexagonal prism structures is 50 μm.
- 如权利要求1-6任意一项所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片的制备方法，其特征在于：The method for preparing a tumor microfluidic chip for mesenchymal stem cell migration screening according to any one of claims 1 to 6, characterized in that:包括以下步骤：The following steps are involved:步骤一、用计算机辅助软件设计和绘制所述芯片主体的微结构通道图形；Step 1: designing and drawing the microstructure channel pattern of the chip body using computer-aided software;步骤二、通过微加工技术制备具有微结构通道的芯片主体，通过手动打孔器制备中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道的进样口及出样口；Step 2: Prepare a chip body with microstructure channels by micromachining technology, and prepare a central cell culture channel and each matrix gel perfusion channel and an inlet and outlet of a migration cell culture channel by a manual puncher;步骤三、将芯片主体和基板用无水乙醇清洗后，进行高压灭菌处理，最后进行等离子体键合，得到微流控芯片。Step 3: Clean the chip body and substrate with anhydrous ethanol, sterilize them under high pressure, and finally perform plasma bonding to obtain a microfluidic chip.
- 如权利要求1-6任意一项所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片的应用方法，其特征在于：The application method of the tumor microfluidic chip for mesenchymal stem cell migration screening according to any one of claims 1 to 6, characterized in that:包括以下步骤：The following steps are involved:步骤一、微流控芯片的预处理：取三个微流控芯片，分别为实验用微流控芯片、正常细胞对照用微流控芯片和空白对照用微流控芯片；Step 1: Pretreatment of microfluidic chips: Take three microfluidic chips, namely, a microfluidic chip for experiment, a microfluidic chip for normal cell control and a microfluidic chip for blank control;涂覆培养基质：对于各微流控芯片，向所述中央细胞培养通道和各迁移细胞培养通道填充培养基质溶液，然后将微流控芯片置于37℃恒温箱中1小时，使中央细胞培养通道和各迁移细胞培养通道的表面涂覆上培养基质；Coating the culture matrix: for each microfluidic chip, the central cell culture channel and each migration cell culture channel are filled with a culture matrix solution, and then the microfluidic chip is placed in a 37° C. incubator for 1 hour to coat the surface of the central cell culture channel and each migration cell culture channel with the culture matrix;恢复疏水性：对于各微流控芯片，用无菌水冲洗中央细胞培养通道和各迁移细胞培养通道中多余的培养基质溶液，将微流控芯片置于80℃的烘箱中1-2小时，使微流控芯片干燥并恢复疏水性；Restoring hydrophobicity: For each microfluidic chip, rinse the excess culture matrix solution in the central cell culture channel and each migration cell culture channel with sterile water, and place the microfluidic chip in an oven at 80°C for 1-2 hours to dry the microfluidic chip and restore its hydrophobicity;步骤二、荧光标记细胞：取生长状态良好的肿瘤细胞株、与肿瘤细胞同器官来源的正常细胞株和不同组织来源间充质干细胞，用活细胞示踪剂分别标记肿瘤细胞、与肿瘤细胞同器官来源的正常细胞和各间充质干细胞，其中，标记肿瘤细胞、与肿瘤细胞同器官来源的正常细胞的活细胞示踪剂的颜色与标记间充质干细胞的活细胞示踪剂的颜色不同；Step 2, fluorescently labeling cells: taking tumor cell lines with good growth status, normal cell lines derived from the same organ as the tumor cells, and mesenchymal stem cells derived from different tissues, and labeling the tumor cells, normal cells derived from the same organ as the tumor cells, and mesenchymal stem cells with live cell tracers, respectively, wherein the color of the live cell tracer labeling the tumor cells and the normal cells derived from the same organ as the tumor cells is different from the color of the live cell tracer labeling the mesenchymal stem cells;步骤三、接种及培养细胞：对于各微流控芯片，将基质胶注入基质胶灌注通道，将微流控芯片放入37℃培养箱30分钟，促进基质胶固化；Step 3: Inoculating and culturing cells: For each microfluidic chip, inject Matrigel into the Matrigel perfusion channel, and place the microfluidic chip in a 37°C incubator for 30 minutes to promote the solidification of Matrigel;将活细胞示踪剂标记的肿瘤细胞和与肿瘤细胞同器官来源的正常细胞分别加入培养基配制成细胞悬液，然后分别种植于实验用微流控芯片和正常细胞对照用微流控芯片的中央细胞培养通道；将培养基灌入空白对照用微流控芯片的中央细胞培养通道；Tumor cells labeled with a live cell tracer and normal cells from the same organ as the tumor cells are added to a culture medium to prepare a cell suspension, and then planted in the central cell culture channel of an experimental microfluidic chip and a normal cell control microfluidic chip respectively; the culture medium is poured into the central cell culture channel of a blank control microfluidic chip;将各活细胞示踪剂标记的间充质干细胞分别加入培养基配制成细胞悬液，然后分别种植于各微流控芯片的不同细胞迁移单元的移细胞培养通道；The mesenchymal stem cells labeled with each living cell tracer are added to the culture medium to prepare a cell suspension, and then are respectively planted in the cell culture channels of different cell migration units of each microfluidic chip;将三个微流控芯片放入培养箱孵育；Place three microfluidic chips in an incubator for incubation;步骤四、观察和监测细胞的迁移：待间充质干细胞贴壁后，取出微流控芯片观察并记录不同间充质干细胞在各基质胶灌注通道的迁移情况，记为0小时；继续培养n小时后，取出微流控芯片观察并记录不同间充质干细胞在相应基质胶灌注通道的迁移情况，记为n小时；测量并比较n小时内不同间充质干细胞向肿瘤细胞、与肿瘤细胞同器官来源的正常细胞及培养基的趋化迁移的迁移面积和最大迁移距离。Step 4, observe and monitor cell migration: after the mesenchymal stem cells adhere to the wall, take out the microfluidic chip to observe and record the migration of different mesenchymal stem cells in each matrix gel perfusion channel, which is recorded as 0 hour; after continuing to culture for n hours, take out the microfluidic chip to observe and record the migration of different mesenchymal stem cells in the corresponding matrix gel perfusion channel, which is recorded as n hours; measure and compare the migration area and maximum migration distance of the chemotactic migration of different mesenchymal stem cells to tumor cells, normal cells from the same organ as the tumor cells, and culture medium within n hours.
- 根据权利要求8所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片的 应用方法，其特征在于：The application method of the tumor microfluidic chip for mesenchymal stem cell migration screening according to claim 8 is characterized in that:其中，涂覆培养基质前，对微流控芯片进行无菌处理；Among them, before coating the culture matrix, the microfluidic chip is sterilized;无菌处理方法为：用75％酒精润洗中央细胞培养通道和各基质胶灌注通道、迁移细胞培养通道，然后将微流控芯片置于紫外光照射下灭菌过夜，待75％酒精完全挥发后再涂覆培养基质。The aseptic treatment method is: rinse the central cell culture channel and each matrix gel perfusion channel and migration cell culture channel with 75% alcohol, then place the microfluidic chip under ultraviolet light for sterilization overnight, and then coat the culture matrix after the 75% alcohol is completely evaporated.
- 根据权利要求8所述的用于间充质干细胞迁移筛选的肿瘤微流控芯片的应用方法，其特征在于：The application method of the tumor microfluidic chip for mesenchymal stem cell migration screening according to claim 8 is characterized in that:其中，步骤三中，种植于中央细胞培养通道的肿瘤细胞和与肿瘤细胞同器官来源的正常细胞的细胞悬液的细胞密度为2×10 6/ml； Wherein, in step 3, the cell density of the cell suspension of the tumor cells and the normal cells from the same organ as the tumor cells planted in the central cell culture channel is 2×10 6 /ml;种植于移细胞培养通道的间充质干细胞的细胞悬液的细胞密度为1×10 6/ml。 The cell density of the cell suspension of mesenchymal stem cells seeded in the cell culture channel was 1×10 6 /ml.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211270154.3A CN117264763A (en) | 2022-10-18 | 2022-10-18 | Tumor micro-fluidic chip for mesenchymal stem cell migration screening and preparation and application methods thereof |
| CN202211270154.3 | 2022-10-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024082430A1 true WO2024082430A1 (en) | 2024-04-25 |
Family
ID=89203252
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/140871 WO2024082430A1 (en) | 2022-10-18 | 2022-12-22 | Tumor microfluidic chip for screening mesenchymal stem cell migration and preparation and use method therefor |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN117264763A (en) |
| LU (1) | LU505383B1 (en) |
| WO (1) | WO2024082430A1 (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110159522A1 (en) * | 2008-04-08 | 2011-06-30 | Kamm Roger D | Three-Dimensional Microfluidic Platforms and Methods of Use Thereof |
| CN103627635A (en) * | 2013-11-18 | 2014-03-12 | 辽宁中医药大学 | Multifunctional micro-fluidic chip for cell migration and invasion assay |
| CN105713835A (en) * | 2014-12-05 | 2016-06-29 | 中国科学院大连化学物理研究所 | Multi-functional-region cell three-dimensional co-culture method based on micro-fluidic chip |
| CN106544271A (en) * | 2016-12-07 | 2017-03-29 | 清华大学深圳研究生院 | A kind of many cells 3D co-culture devices and method of research tumor invasion blood vessel |
| CN106566863A (en) * | 2015-10-10 | 2017-04-19 | 中国科学院大连化学物理研究所 | Cell bidirectional invasion monitoring method based on micro-fluidic chip |
| CN107955783A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of construction method based on the external diabetes Glomerulus model of micro-fluidic chip |
| CN107955784A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of three-dimensional cell ball migration monitoring method based on microfluidic chip technology |
| CN114480122A (en) * | 2022-01-24 | 2022-05-13 | 中国人民解放军海军军医大学 | Establishment method and application of blood brain barrier and brain glioma co-culture model based on micro-fluidic chip |
-
2022
- 2022-10-18 CN CN202211270154.3A patent/CN117264763A/en active Pending
- 2022-12-22 WO PCT/CN2022/140871 patent/WO2024082430A1/en unknown
- 2022-12-22 LU LU505383A patent/LU505383B1/en active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110159522A1 (en) * | 2008-04-08 | 2011-06-30 | Kamm Roger D | Three-Dimensional Microfluidic Platforms and Methods of Use Thereof |
| CN103627635A (en) * | 2013-11-18 | 2014-03-12 | 辽宁中医药大学 | Multifunctional micro-fluidic chip for cell migration and invasion assay |
| CN105713835A (en) * | 2014-12-05 | 2016-06-29 | 中国科学院大连化学物理研究所 | Multi-functional-region cell three-dimensional co-culture method based on micro-fluidic chip |
| CN106566863A (en) * | 2015-10-10 | 2017-04-19 | 中国科学院大连化学物理研究所 | Cell bidirectional invasion monitoring method based on micro-fluidic chip |
| CN107955783A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of construction method based on the external diabetes Glomerulus model of micro-fluidic chip |
| CN107955784A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of three-dimensional cell ball migration monitoring method based on microfluidic chip technology |
| CN106544271A (en) * | 2016-12-07 | 2017-03-29 | 清华大学深圳研究生院 | A kind of many cells 3D co-culture devices and method of research tumor invasion blood vessel |
| CN114480122A (en) * | 2022-01-24 | 2022-05-13 | 中国人民解放军海军军医大学 | Establishment method and application of blood brain barrier and brain glioma co-culture model based on micro-fluidic chip |
Also Published As
| Publication number | Publication date |
|---|---|
| LU505383B1 (en) | 2024-04-26 |
| CN117264763A (en) | 2023-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111411083B (en) | Culture medium and culture method for stomach cancer organoid | |
| CN111394314A (en) | Culture medium and culture method for intestinal cancer organoid | |
| CN104312975B (en) | A kind of external dimensional culture model of glioma stem cells and application | |
| CN109182257A (en) | A kind of mechanical environment cultural method for improving chondrocyte proliferation activity, maintaining cartilage phenotype | |
| JPWO2005014774A1 (en) | Animal cell culture carrier, animal cell culture method and transplantation method using the culture carrier | |
| CN103230623A (en) | Method for in-vitro construction of tissue engineered nerves | |
| CN112481212A (en) | Method for generating brain organoid by using pluripotent stem cells | |
| CN111534483B (en) | Application of insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells | |
| CN113846016B (en) | High-flux porous array chip, device, preparation method and application | |
| CN111718895A (en) | Artificial myocardial tissue fibrosis model, preparation method, preparation device and application thereof | |
| CN101993852A (en) | Culture medium and culture method of breast stem cells and breast stem cell-rich mixture | |
| CN112852709B (en) | Method for culturing mouse lung organoid | |
| CN113957036A (en) | Culture medium and culture method for endometrioid organs | |
| WO2024082430A1 (en) | Tumor microfluidic chip for screening mesenchymal stem cell migration and preparation and use method therefor | |
| CN106244530A (en) | A kind of method that cell culture fluid and application and induction skeletal muscle stem Cells Cardiocytes thereof break up | |
| US20220145247A1 (en) | Generation of human pluripotent stem cell derived artificial tissue structures without three dimensional matrices | |
| CN103877613B (en) | The system and method for injection-type three-dimensional cell microenvironment is built based on micro-ice glue | |
| CN107185047A (en) | Organization engineered cartilage graft and preparation method thereof | |
| CN114214282B (en) | Method for culturing lung tumor organoids | |
| CN115449504A (en) | Method for establishing 2D culture model of porcine small intestine organs | |
| Hsieh et al. | Biological properties of human periodontal ligament cell spheroids cultivated on chitosan and polyvinyl alcohol membranes | |
| CN110499279A (en) | A method of induce human urine derived stem cells to hepatocyte differentiation | |
| Sundaram et al. | 3D decellularized native extracellular matrix scaffold for in vitro culture expansion of human Wharton’s jelly-derived mesenchymal stem cells (hWJ MSCs) | |
| CN103305460B (en) | Method for separating and culturing human epidermal stem cells | |
| CN112813029A (en) | 3D culture method of medulloblastoma cells and application of medulloblastoma cells in drug screening |