CN107828655A - A microfluidic chip and its application - Google Patents
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Abstract
Description
技术领域technical field
本发明涉及一种微流控芯片及其应用，属于微流控技术领域。The invention relates to a microfluidic chip and its application, belonging to the field of microfluidic technology.
背景技术Background technique
微流控芯片(Microfluidics)又称芯片实验室(Lab-on-a-chip)，是在微米量级空间操控流体的一种科学与技术，可将生物和化学实验室的基本功能微缩到一个数平方厘米的芯片上，是21世纪最为重要的前沿技术之一，被认为是解决创新药物、化妆品和保健品研发成本过高、周期过长等关键问题，革新原有技术体系的关键技术，正面临着重大的发展机遇和挑战。Microfluidics, also known as Lab-on-a-chip, is a science and technology for manipulating fluids in micron-scale space, which can reduce the basic functions of biological and chemical laboratories to one On a chip of several square centimeters, it is one of the most important cutting-edge technologies in the 21st century. It is considered to be the key technology to solve the key problems such as high R&D costs and long cycle of innovative drugs, cosmetics and health products, and to innovate the original technology system. We are facing great development opportunities and challenges.
器官微流控芯片(Organ on a chip)是微流控芯片的一个亚类，它是在一块几平方厘米薄片内培养一种或多种功能细胞，从而模拟器官的一种仿生技术，器官芯片里的“器官”非常微小，但是具备真实器官的基本生理功能。器官芯片能仿真地模拟器官原因在于：(1)它不但同时培养器官所包含的多种细胞，而且细胞的空间排列可以模仿器官的生理结构；(2)它可以重建器官在体内的生理环境，比如流体剪切力、信号分子浓度梯度。可以说器官芯片从“组成”、“结构”和“环境”三方面对器官进行了模拟，仿真程度很高。Organ on a chip is a subclass of microfluidic chip, which is a bionic technology that simulates organs by culturing one or more functional cells in a sheet of several square centimeters. The "organs" in the game are very small, but they have the basic physiological functions of real organs. The reason why the organ chip can simulate the organ is: (1) it not only cultivates various cells contained in the organ at the same time, but also the spatial arrangement of the cells can imitate the physiological structure of the organ; (2) it can reconstruct the physiological environment of the organ in the body, Such as fluid shear force, signal molecule concentration gradient. It can be said that the organ chip simulates the organ from the three aspects of "composition", "structure" and "environment", with a high degree of simulation.
器官芯片的用途是代替真实的人体或动物的器官进行化学品的测试，常见的化学品包括药物、保健品、化妆品和环境毒物。它可以测定药物的药效、毒性和药代，可以测定保健品在肠道的吸收、肝脏里的代谢和对肠道菌群的保护作用，可以测定化妆品在皮肤内的吸收以及对皮肤的刺激性，还可以测定环境毒物对肾脏的损害作用。The purpose of organ-on-a-chip is to replace real human or animal organs for testing chemicals. Common chemicals include drugs, health products, cosmetics and environmental poisons. It can measure the efficacy, toxicity and pharmacokinetics of drugs, the absorption of health care products in the intestine, the metabolism in the liver and the protection of intestinal flora, the absorption of cosmetics in the skin and the irritation to the skin It can also determine the damage effect of environmental toxicants on the kidneys.
肾单位是肾脏的结构和功能单元，其实现血液净化的方式是血液通过肾小球的血管内皮细胞-足细胞膜结构发生选择性滤过，之后通过肾小管的管周血管内皮细胞-肾小管上皮细胞膜结构发生分泌或重吸收，从而实现血液和尿液的第二次物质交换。The nephron is the structural and functional unit of the kidney. The way blood is purified is through the selective filtration of blood through the vascular endothelial cell-podocyte membrane structure of the glomerulus, and then through the peritubular vascular endothelial cell-renal tubular epithelium of the renal tubules. The cell membrane structure undergoes secretion or reabsorption, thereby realizing the second material exchange of blood and urine.
现有的用于模拟肾脏系统物质交换膜结构的芯片主要有，三层单通道夹膜结构，例如Ingber利用三层单通道夹膜结构模拟肾小球的血管内皮细胞-足细胞形成的膜结构，在另一份工作中，Ingber利用该结构芯片模拟肾小管结构单层通道芯片，该结构通过通道中间的基质胶将两种液体隔离，细胞种植在基质胶两侧，例如Qin利用该结构模拟肾小球的血管内皮细胞-足细胞形成的膜结构，这些结构单元只能为两种流体提供一个物质交换的区域，不能有效的同时将肾小球和肾小管的结构构建在芯片上，从而使所模拟的肾单元结构单一，不能有效模拟血液净化的过程。Existing chips for simulating the material exchange membrane structure of the kidney system mainly include three-layer single-channel sandwich structures. For example, Ingber uses a three-layer single-channel sandwich structure to simulate the membrane structure formed by glomerular endothelial cells-podocytes , In another work, Ingber used this structure chip to simulate the single-layer channel chip of renal tubule structure. The membranous structure formed by the vascular endothelial cells of the glomerulus-podocytes, these structural units can only provide a material exchange area for the two fluids, and cannot effectively construct the structure of the glomeruli and renal tubules on the chip at the same time, thus The simulated nephron has a single structure and cannot effectively simulate the process of blood purification.
发明内容Contents of the invention
为了克服现有技术中存在的不足，本发明目的是提供一种微流控芯片及其应用。该微流控芯片具有三层四腔室结构，上、下层基板中的流体可通过分隔腔室的多孔膜发生两次物质交换，搭配上肾小球和肾小管的相应细胞后，可以在一定范围内有效模拟肾单位的滤过分泌和重吸收过程。In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a microfluidic chip and its application. The microfluidic chip has a three-layer four-chamber structure. The fluid in the upper and lower substrates can undergo two material exchanges through the porous membrane separating the chambers. Effectively simulate the filtration secretion and reabsorption process of the nephron within the range.
为了实现上述发明目的，解决已有技术中所存在的问题，本发明采取的技术方案是：一种微流控芯片，包括上、下层基板、第一、二多孔膜，所述第一、二多孔膜位于上、下层基板中间并通过上、下层基板压紧，所述上层基板设置有第一、二进液口，第一、二出液口，第一、二腔室及第一微通道，所述第二进液口，第一、二腔室及第二出液口通过第一微通道依次连通，所述下层基板设置有第三进液口、第三出液口，第三、四腔室及第二微通道，所述第三进液口，第三、四腔室及第三出液口通过第二微通道依次连通，所述第一进液口贯穿上层基板并与设置在下层基板上的第三进液口上、下对应，所述第一出液口贯穿上层基板并与设置在下层基板上的第三出液口上、下对应；所述第一、二、三、四腔室形状选自矩形、半圆形或半椭圆形中的一种；所述上、下层基板的材料选自石英、玻璃、PMMA、PDMS聚合物、聚碳酸酯、聚酯、琼脂糖、壳聚糖或海藻酸钠中的一种；所述第一、二多孔膜的材料选自PDMS、聚偏氟乙烯或聚碳酸酯中的一种。所述上层基板，第一、二多孔膜及下层基板之间可拆卸连接，便于维护；所述第一多孔膜上、下两表面可分别接种肾小球血管内皮细胞、肾足细胞，所述第二多孔膜上、下两表面可分别接种肾小管管周血管内皮细胞、肾小管上皮细胞，这些细胞根据需求，可以是来源于人或任何一种动物，可以是原代细胞，细胞系以及干细胞诱导分化的功能细胞。In order to achieve the purpose of the above invention and solve the problems existing in the prior art, the technical solution adopted by the present invention is: a microfluidic chip, including upper and lower substrates, first and second porous membranes, the first, The two porous membranes are located between the upper and lower substrates and are pressed tightly by the upper and lower substrates. The upper substrate is provided with first and second liquid inlets, first and second liquid outlets, first and second chambers and first The microchannel, the second liquid inlet, the first chamber, the second chamber and the second liquid outlet are sequentially connected through the first microchannel, the lower substrate is provided with a third liquid inlet and a third liquid outlet, and the first Three, four chambers and the second microchannel, the third liquid inlet, the third and fourth chambers and the third liquid outlet are connected sequentially through the second microchannel, the first liquid inlet runs through the upper substrate and Corresponding to the top and bottom of the third liquid inlet provided on the lower substrate, the first liquid outlet penetrates the upper substrate and corresponds to the top and bottom of the third liquid outlet provided on the lower substrate; the first, second, and The shape of three or four chambers is selected from one of rectangle, semicircle or semi-ellipse; the material of the upper and lower substrates is selected from quartz, glass, PMMA, PDMS polymer, polycarbonate, polyester, agar One of sugar, chitosan or sodium alginate; the material of the first and second porous membranes is selected from one of PDMS, polyvinylidene fluoride or polycarbonate. The upper substrate, the first and second porous membranes and the lower substrate are detachably connected to facilitate maintenance; the upper and lower surfaces of the first porous membrane can be inoculated with glomerular endothelial cells and renal podocytes respectively, The upper and lower surfaces of the second porous membrane can be inoculated with renal tubule perivascular endothelial cells and renal tubular epithelial cells respectively. These cells can be derived from humans or any animal according to requirements, and can be primary cells. Cell lines and stem cell-induced differentiation of functional cells.
所述一种微流控芯片，在模拟肾单位的肾小球和肾小管方面中的应用。The application of the microfluidic chip in simulating the glomeruli and renal tubules of nephrons.
本发明有益效果是：一种微流控芯片，包括上、下层基板、第一、二多孔膜，所述第一、二多孔膜位于上、下层基板中间并通过上、下层基板压紧，所述上层基板设置有第一、二进液口，第一、二出液口，第一、二腔室及第一微通道，所述第二进液口，第一、二腔室及第二出液口通过第一微通道依次连通，所述下层基板设置有第三进液口、第三出液口，第三、四腔室及第二微通道，所述第三进液口，第三、四腔室及第三出液口通过第二微通道依次连通，所述第一进液口贯穿上层基板并与设置在下层基板上的第三进液口上、下对应，所述第一出液口贯穿上层基板并与设置在下层基板上的第三出液口上、下对应；所述第一、二、三、四腔室形状选自矩形、半圆形或半椭圆形中的一种；所述上、下层基板的材料选自石英、玻璃、PMMA、PDMS聚合物、聚碳酸酯、聚酯、琼脂糖、壳聚糖或海藻酸钠中的一种；所述第一、二多孔膜的材料选自PDMS、聚偏氟乙烯或聚碳酸酯中的一种。所述上层基板，第一、二多孔膜及下层基板之间可拆卸连接，便于维护；所述第一多孔膜上、下两表面可分别接种肾小球血管内皮细胞、肾足细胞，所述第二多孔膜上、下两表面可分别接种肾小管管周血管内皮细胞、肾小管上皮细胞，这些细胞根据需求，可以是来源于人或任何一种动物，可以是原代细胞，细胞系以及干细胞诱导分化的功能细胞。与已有技术相比，本发明的一种微流控芯片形成了四个腔室，上、下流体可在分隔四个腔室的多孔膜上发生两次物质交换，从而能高仿真度的模拟肾单位的肾小球滤过肾小管分泌和重吸收的血液净化过程，进而可进行生物标记物的测试，实现对药物、化妆品、保健品、环境毒物的有效评价。另外本发明的芯片可拆卸链接，更加方便对多孔膜上的细胞进行成像检测。除此之外，上下基板经过处理，可以重复利用，节约经济。The beneficial effects of the present invention are: a microfluidic chip, comprising upper and lower substrates, first and second porous membranes, the first and second porous membranes are located between the upper and lower substrates and are compressed by the upper and lower substrates , the upper substrate is provided with the first and second liquid inlets, the first and second liquid outlets, the first and second chambers and the first microchannel, the second liquid inlet, the first and second chambers and The second liquid outlet is sequentially connected through the first microchannel, and the lower substrate is provided with a third liquid inlet, a third liquid outlet, third and fourth chambers and a second microchannel, and the third liquid inlet , the third and fourth chambers and the third liquid outlet are sequentially communicated through the second microchannel, the first liquid inlet runs through the upper substrate and corresponds to the upper and lower third liquid inlets arranged on the lower substrate, the said The first liquid outlet runs through the upper substrate and corresponds to the upper and lower sides of the third liquid outlet arranged on the lower substrate; the shapes of the first, second, third and fourth chambers are selected from rectangle, semicircle or semiellipse The material of the upper and lower substrates is selected from one of quartz, glass, PMMA, PDMS polymer, polycarbonate, polyester, agarose, chitosan or sodium alginate; the first , The material of the second porous membrane is selected from one of PDMS, polyvinylidene fluoride or polycarbonate. The upper substrate, the first and second porous membranes and the lower substrate are detachably connected to facilitate maintenance; the upper and lower surfaces of the first porous membrane can be inoculated with glomerular endothelial cells and renal podocytes respectively, The upper and lower surfaces of the second porous membrane can be inoculated with renal tubule perivascular endothelial cells and renal tubular epithelial cells respectively. These cells can be derived from humans or any animal according to requirements, and can be primary cells. Cell lines and stem cell-induced differentiation of functional cells. Compared with the prior art, a microfluidic chip of the present invention forms four chambers, and the upper and lower fluids can undergo two material exchanges on the porous membrane separating the four chambers, thereby achieving a high degree of simulation Simulate the blood purification process of renal tubular secretion and reabsorption by glomerular filtration of nephrons, and then test biomarkers to achieve effective evaluation of drugs, cosmetics, health products, and environmental toxicants. In addition, the chip of the present invention can be detachably connected, which is more convenient for imaging detection of cells on the porous membrane. In addition, the upper and lower substrates can be reused after treatment, which is economical.
附图说明Description of drawings
图1是本发明的一种微流控芯片结构示意图。Fig. 1 is a schematic structural diagram of a microfluidic chip of the present invention.
图中：1、上层基板，1a、第一进液口，1b、第二进液口，1c、第一出液口，1d、第二出液口，1e、第一微通道，1f、第一腔室，1g、第二腔室，2、第一多孔膜，2a、第二多孔膜，3、下层基板，3a、第三进液口，3b、第三出液口，3c、第二微通道，3d、第三腔室，3e、第四腔室。In the figure: 1, the upper substrate, 1a, the first liquid inlet, 1b, the second liquid inlet, 1c, the first liquid outlet, 1d, the second liquid outlet, 1e, the first microchannel, 1f, the second liquid outlet A chamber, 1g, a second chamber, 2, a first porous membrane, 2a, a second porous membrane, 3, a lower substrate, 3a, a third liquid inlet, 3b, a third liquid outlet, 3c, Second microchannel, 3d, third chamber, 3e, fourth chamber.
图2是本发明用于模拟肾单位的肾小球和肾小管的结构示意图。Fig. 2 is a schematic structural diagram of glomeruli and renal tubules used to simulate nephrons in the present invention.
图3是利用本发明模拟的肾单位测试不同浓度顺铂对四种细胞的毒性影响图。Fig. 3 is a graph showing the toxic effects of different concentrations of cisplatin on four kinds of cells tested using the simulated nephron of the present invention.
图4是利用本发明模拟的肾单位测试1.25μg/ml的阿霉素对四种细胞的毒性影响图。Fig. 4 is a graph showing the toxic effects of 1.25 μg/ml doxorubicin on four kinds of cells tested using the simulated nephron of the present invention.
具体实施方式Detailed ways
下面结合附图及实施例对本发明作进一步说明。The present invention will be further described below in conjunction with the accompanying drawings and embodiments.
如图1所示，一种微流控芯片，包括上、下层基板1、3、第一、二多孔膜2、2a，所述第一、二多孔膜2、2a位于上、下层基板1、3中间并通过上、下层基板1、3压紧，所述上层基板1设置有第一、二进液口1a、1b，第一、二出液口1c、1d，第一、二腔室1f、1g及第一微通道1e，所述第二进液口1b，第一、二腔室1f、1g及第二出液口1d通过第一微通道1e依次连通，所述下层基板3设置有第/三进液口3a、第三出液口3b，第三、四腔室3d、3e及第二微通道3c，所述第三进液口3b，第三、四腔室3d、3e及第三出液口3b通过第二微通道3c依次连通，所述第一进液口1a贯穿上层基板1并与设置在下层基板3上的第三进液口3b上、下对应，所述第一出液口1c贯穿上层基板1并与设置在下层基板3上的第三出液口3b上、下对应；所述第一、二、三、四腔室1f、1g、3d、3e的形状选自矩形、半圆形或半椭圆形中的一种；所述上、下层基板1、3的材料选自石英、玻璃、PMMA、PDMS聚合物、聚碳酸酯、聚酯、琼脂糖、壳聚糖或海藻酸钠中的一种；所述第一、二多孔膜2、2a的材料选自PDMS、聚偏氟乙烯或聚碳酸酯中的一种。所述上层基板1，第一、二多孔膜2、2a及下层基板3之间可拆卸连接，便于维护；所述第一多孔膜2的上、下两表面可分别接种肾小球血管内皮细胞、肾足细胞，所述第二多孔膜2a的上、下两表面可分别接种肾小管管周血管内皮细胞、肾小管上皮细胞，这些细胞根据需求，可以是来源于人或任何一种动物，可以是原代细胞，细胞系以及干细胞诱导分化的功能细胞。As shown in Figure 1, a microfluidic chip includes upper and lower substrates 1, 3, first and second porous membranes 2 and 2a, and the first and second porous membranes 2 and 2a are located on the upper and lower substrates 1, 3 in the middle and pressed by the upper and lower substrates 1, 3, the upper substrate 1 is provided with first and second liquid inlets 1a, 1b, first and second liquid outlets 1c, 1d, first and second chambers The chambers 1f, 1g and the first microchannel 1e, the second liquid inlet 1b, the first and second chambers 1f, 1g and the second liquid outlet 1d are sequentially connected through the first microchannel 1e, and the lower substrate 3 Provided with the third/third liquid inlet 3a, the third liquid outlet 3b, the third and fourth chambers 3d, 3e and the second microchannel 3c, the third liquid inlet 3b, the third and fourth chambers 3d, 3e and the third liquid outlet 3b are sequentially communicated through the second microchannel 3c, and the first liquid inlet 1a penetrates the upper substrate 1 and corresponds to the upper and lower third liquid inlet 3b arranged on the lower substrate 3, so The first liquid outlet 1c runs through the upper substrate 1 and corresponds to the upper and lower sides of the third liquid outlet 3b provided on the lower substrate 3; the first, second, third, and fourth chambers 1f, 1g, 3d, 3e The shape is selected from one of rectangle, semicircle or semi-ellipse; the material of the upper and lower substrates 1, 3 is selected from quartz, glass, PMMA, PDMS polymer, polycarbonate, polyester, agarose , chitosan or sodium alginate; the material of the first and second porous membranes 2 and 2a is selected from one of PDMS, polyvinylidene fluoride or polycarbonate. The upper substrate 1, the first and second porous membranes 2, 2a and the lower substrate 3 are detachably connected to facilitate maintenance; the upper and lower surfaces of the first porous membrane 2 can be respectively inoculated with glomerular blood vessels Endothelial cells, renal podocytes, the upper and lower surfaces of the second porous membrane 2a can be inoculated with renal tubule perivascular endothelial cells and renal tubular epithelial cells respectively. These cells can be derived from human or any Animals can be primary cells, cell lines and functional cells induced and differentiated from stem cells.
如图2所示，将肾小球血管内皮细胞、肾足细胞、肾小管管周血管内皮细胞和肾小管上皮细胞种在多孔膜上，仿血液流体培养液通过上层基板的进液口进入上层基板通道，流经肾小球血管内皮细胞和肾小管管周血管内皮细胞侧的腔室，通过上层基板的出液口离开芯片，仿尿液流体培养液通过贯穿上层基板的下层基板进液口进入下层基板通道，流经肾足细胞和肾小管上皮细胞侧的腔室，通过贯穿上层基板的下层基板出液口离开通道。仿血液和仿尿液流体在正面种有肾小球血管内皮细胞，背面种有足细胞的多孔膜处发生第一次物质交换，模拟肾单位血液净化中的肾小球滤过过程，在正面种有肾小管管周血管内皮细胞，背面种有肾小管上皮细胞的多孔膜处发生第二次物质交换，模拟肾单位血液净化中的肾小管分泌和重吸收过程。根据具体需要，仿血液流体和选择循环流动或者单向流动，仿尿液流体可选择单向流动。As shown in Figure 2, the glomerular vascular endothelial cells, renal podocytes, renal tubular peritubular vascular endothelial cells and renal tubular epithelial cells were planted on the porous membrane, and the imitation blood fluid culture medium entered the upper layer through the liquid inlet of the upper substrate The substrate channel flows through the chambers of the glomerular endothelial cells and the perivascular endothelial cells of the renal tubules, and leaves the chip through the liquid outlet of the upper substrate, and the urine-like fluid culture solution passes through the lower substrate liquid inlet that runs through the upper substrate Enters the channel of the lower substrate, flows through the chamber on the side of the renal podocytes and tubular epithelial cells, and exits the channel through the outlet of the lower substrate through the upper substrate. The imitation blood and urine imitation fluids are planted with glomerular endothelial cells on the front side, and podocytes are planted on the back side where the first material exchange occurs, simulating the glomerular filtration process in nephron blood purification, and on the front side There are vascular endothelial cells around the renal tubules, and the second material exchange occurs at the porous membrane with renal tubular epithelial cells on the back, simulating the process of renal tubular secretion and reabsorption in nephron blood purification. According to specific needs, the imitation blood fluid can choose to circulate or one-way flow, and the imitation urine fluid can choose one-way flow.
实施例1Example 1
以用芯片肾单位检测不同浓度的顺铂对肾单位细胞的极性毒性反应为例，如附图3所示，在血液极入口加入0μg/ml,3μg/ml,10μg/ml,30μg/ml顺铂，24小时后，可以观察到当系统内加入30μg/ml顺铂时，肾小球血管内皮细胞，肾足细胞，肾小管上皮细胞活性有显著性下降，说明30μg/ml顺铂对肾小球血管内皮细胞，肾足细胞，肾小管上皮细胞有明显毒性。Take the nephron chip to detect the polar toxicity of different concentrations of cisplatin on nephron cells as an example, as shown in Figure 3, add 0μg/ml, 3μg/ml, 10μg/ml, 30μg/ml to the blood pole inlet Cisplatin, after 24 hours, it can be observed that when 30 μg/ml cisplatin is added to the system, the activity of glomerular vascular endothelial cells, renal podocytes, and renal tubular epithelial cells is significantly reduced, indicating that 30 μg/ml cisplatin has a positive effect on kidney function. Glomerular vascular endothelial cells, renal podocytes, and renal tubular epithelial cells were significantly toxic.
实施例2Example 2
以用芯片肾单位检测1.25μg/ml的阿霉素对肾单位细胞的极性毒性反应为例，如附图4所示，在血液极入口加入1.25μg/ml的阿霉素，48小时后，可以观察到，肾小球血管内皮细胞、肾足细胞，管周血管内皮细胞活性有明显下降，说明在48小时，1.25μg/ml的阿霉素对肾小球血管内皮细胞、肾足细胞，管周血管内皮细胞有明显毒性，对肾小管上皮细胞影响较小。Take the nephron chip to detect the polar toxic reaction of 1.25 μg/ml doxorubicin to nephron cells as an example, as shown in Figure 4, add 1.25 μg/ml doxorubicin to the blood pole inlet, after 48 hours , it can be observed that the activity of glomerular vascular endothelial cells, renal podocytes, and peritube vascular endothelial cells has decreased significantly, indicating that in 48 hours, 1.25 μg/ml of doxorubicin has an effect on glomerular vascular endothelial cells, renal podocytes , Peritubular vascular endothelial cells have obvious toxicity, and have little effect on renal tubular epithelial cells.
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| EP3830244A4 (en) * | 2018-07-27 | 2022-04-27 | The Trustees of Columbia University in the City of New York | ORGAN-ON-CHIP MODELS FOR PREDICTIVE SCREENING |
| CN109456934A (en) * | 2018-10-31 | 2019-03-12 | 清华大学 | A kind of preparation method of three-dimensional Glomerulus model |
| CN109456934B (en) * | 2018-10-31 | 2021-05-11 | 清华大学 | A kind of preparation method of three-dimensional glomerular model |
| WO2022134294A1 (en) * | 2020-12-25 | 2022-06-30 | 苏州大学 | Detachable and reusable hydrophobic or super-hydrophobic microfluidic organ-on-a-chip |
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