A barrier-stem cell homing bionic microfluidic chip and its application

Abstract

The present invention proposes a barrier-stem cell homing biomimetic microfluidic chip, comprising a barrier chip and a stem cell homing chip. The barrier chip is layered and cultured from lower to upper layers according to the cell composition order of the barrier structure of the target organ. The upper chamber of the stem cell homing chip cultured stem cells, the middle chamber embedded with a 3D "nest" of mammalian decellularized matrix corresponding to the target organ, and the lower chamber is a perfusion chamber, which is perfused with cell culture fluid flowing from the upper chamber of the barrier chip. The microfluidic biomimetic chip of the present invention can achieve the complex and dynamic microenvironment required to simulate organs. For example, it can be designed with fluid transport, cell culture, and detection units, etc., to achieve a continuously controllable flow environment and interconnected culture units, which are used to highly restore the physiological characteristics of the target organ and simulate the barrier function of the target organ and the biological process of stem cell homing.

Description

Technical Field

[0001] The present invention belongs to the field of organ chips, and specifically relates to a barrier-stem cell homing bionic microfluidic chip and its application. Background Art

[0002] Conventional life science research is primarily based on animals and two-dimensional (2D) cells. While animal models have contributed to a rich understanding of physiology and disease, and to the development of new drugs, they also present numerous limitations as vehicles for human research. For example, drug candidates may be discontinued due to lack of efficacy in animals or the discovery of hazards or toxicities in animals that may not be relevant to humans. Despite significant advances in in vitro biology and toxicology over the past 20 years, over 80% of investigational drugs currently fail in clinical trials, 60% due to lack of efficacy and another 30% due to toxicity. 2D cell culture has been used in life science research for over a century. However, this culture format struggles to support tissue-specific differentiation of multiple cell types and to provide insights into the complexity of living systems. Consequently, there is an urgent need for in vitro modeling and testing platforms that more realistically reflect the complexities of the human body. Rapid advances in microfabrication technologies have made it possible to conduct benchtop experiments in small systems, known as microfluidic lab-on-a-chip (LOC) systems. Organ-on-a-chips are 3D microfluidic cell culture devices that combine microfluidic chip technology, stem cell differentiation, and tissue engineering techniques to construct physiological organ microsystems. They can simulate the primary structures and functions of different human tissues and organs, as well as the complex inter-organ connections, in vitro. Human organ-on-a-chip technology is gaining traction among researchers and may replace animal experiments in the future, becoming a promising research tool.

[0003] Stem cells have unique advantages in regenerative medicine. Due to the continuous progress of stem cell research at this stage, they are available, have the characteristics of induced differentiation in multiple organ directions, and have the unique ability to self-renew and produce different cell types. They are more consistent with human genetic characteristics, more suitable for in vitro modeling, and have been applied to human disease research, drug development, and personalized medicine. Based on stem cell technology, accurate in vitro modeling of donor characteristics can also be achieved. By simulating the homing process of autologous or exogenous stem cells under the influence of various factors, they tend to migrate to the target tissue and colonize and survive on the chip. In addition, the in vitro modeling of this biological process can be achieved through organ chip technology, which also allows the monitoring of cell status during the culture process, as well as more in-depth scientific research and disease diagnosis and prognosis.

[0004] Currently, most organ-on-a-chip research is limited to in vitro modeling of individual organ functions (or functional interfaces), which ignores the interrelationships between organs and tissue-organ barriers. In fact, the normal physiological functioning of organs requires the metabolism of substances achieved through the circulation of body fluids and blood, as well as the digestion, absorption, transportation, decomposition, and metabolism of substances in the body, and other physiologically relevant chemical processes. Summary of the Invention

[0005] To overcome the problems of the prior art, the present invention provides a barrier-homing biomimetic microfluidic chip and its application. Through structural design, the present invention achieves in vitro modeling of the target organ-organ barrier and simulates specific elements of the target organ's cell-extracellular matrix-microenvironment. This provides feasible technical support for applying this in vitro modeling design to various scientific research areas, such as disease modeling based on stem cell technology, drug development, and personalized medicine, as well as life science research in special environments such as the deep sea, space environments, high temperature and high pressure, and strong light and radiation environments.

[0006] The present invention provides the following technical solutions:

[0007] A barrier-stem cell homing bionic microfluidic chip comprises a barrier chip and a stem cell homing chip. The barrier chip is layered and cultured from lower to upper layers according to the cell composition order of the barrier structure of the target organ. The upper chamber of the stem cell homing chip cultures stem cells, the middle chamber embeds a 3D "nest" formed by a mammalian decellularized matrix corresponding to the target organ, and the lower chamber is a perfusion chamber that is perfused with the cell culture fluid flowing out of the upper chamber of the barrier chip.

[0008] Furthermore, the barrier chip is separated into an upper chamber and a lower chamber by a porous membrane, and a first inlet and a first outlet are provided in the barrier chip interface layer, communicating with the upper chamber; a second inlet and a second outlet are provided in the barrier chip interface layer, communicating with the lower chamber.

[0009] Furthermore, the barrier chip perfuses the lower chamber with a culture medium for culturing lower cells, and the upper chamber with a mixed culture medium of the culture medium for culturing upper cells and the culture medium for culturing upper stem cells in the stem cell homing chip.

[0010] Furthermore, the porous membrane is adapted to the size of key functional interface cells of the target organ, and has a pore size of 3-10 μm.

[0011] Furthermore, the barrier chip has electrodes inserted into the upper chamber and the lower chamber respectively for measuring the transmembrane resistance value.

[0012] Furthermore, the stem cell homing chip is separated from the lower chamber and the middle chamber by a first porous membrane, and is separated from the middle chamber and the upper chamber by a second porous membrane. A third inlet and a third outlet are provided on the interface layer of the stem cell homing chip, communicating with the lower chamber. A fourth inlet and a fourth outlet are provided on the interface layer of the stem cell homing chip, communicating with the middle chamber. A fifth inlet and a fifth outlet are provided on the interface layer of the stem cell homing chip, communicating with the upper chamber.

[0013] Furthermore, the middle chamber of the stem cell homing chip is embedded with a 3D nest structure mixed with a bioactive hydrogel and a cell-free matrix of a mammalian organ corresponding to the target organ, and the upper chamber cultures pluripotent stem cells, embryonic stem cells, mesenchymal stem cells or other stem cells with differentiation potential that can be induced to differentiate into the target organ.

[0014] Furthermore, the pore size of the first porous membrane of the stem cell homing chip is 100-500 μm; the pore size of the second porous membrane is 10-50 μm.

[0015] Furthermore, the stem cell homing chip is connected to gold / platinum electrodes in the upper chamber and the middle chamber respectively, the upper chamber is connected to negative electricity, and the middle chamber is connected to positive electricity, with a voltage of 0-2.0V.

[0016] Furthermore, the stem cell homing chip is provided with an upper chamber observation window and a middle chamber observation window.

[0017] Furthermore, a multifunctional liquid supply unit is provided upstream of the barrier chip and the stem cell homing chip liquid path, and the liquid path is connected to a fluid drive system; a multifunctional liquid collection unit is connected downstream of the barrier chip and the stem cell homing chip liquid path.

[0018] Furthermore, the stem cell homing chip is a high-throughput biomimetic microfluidic chip, and a plurality of the barrier chips are simultaneously connected to the same stem cell homing chip for perfusion.

[0019] A barrier-stem cell homing bionic microfluidic chip is used for the application. The upper chamber of the barrier chip cultures astrocytes, and the lower chamber of the barrier chip cultures microvascular endothelial cells and pericytes. The upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with brain organ differentiation potential, and the middle chamber is formed by the mammalian brain decellularized matrix corresponding to the target organ to form a "nest" environment, which is used for a blood-brain barrier-brain organoid bionic microfluidic chip.

[0020] A barrier-stem cell homing bionic microfluidic chip is used for application. The upper chamber of the barrier chip cultures alveolar epithelial cells, and the lower chamber of the barrier chip cultures microvascular endothelial cells and pericytes. The upper cells of the stem cell homing chip culture pluripotent stem cells or stem cells with lung organoid differentiation potential, and the middle chamber is formed by a 3D "nest" of mammalian lung decellularized matrix corresponding to the target organ, which is used for a blood-gas barrier-lung organoid bionic microfluidic chip.

[0021] A barrier-stem cell homing bionic microfluidic chip is used for the application. The upper chamber of the barrier chip cultures renal capsule epithelial cells, and the lower chamber of the barrier chip cultures microvascular endothelial cells. The upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with kidney organ differentiation potential, and the middle chamber is formed by a 3D "nest" of mammalian kidney decellularized matrix corresponding to the target organ, which is used for a blood-urine barrier-kidney organ bionic microfluidic chip.

[0022] A barrier-stem cell homing bionic microfluidic chip is used for the application. The upper chamber of the barrier chip cultures intestinal epithelial cells, and the lower chamber of the barrier chip cultures microvascular endothelial cells. The upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with intestinal organoid differentiation potential, and the middle chamber is formed by the mammalian intestinal decellularized matrix corresponding to the target organ to form a 3D "nest", which is used for the intestinal vascular barrier-intestinal organoid bionic microfluidic chip.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects:

[0024] (1) The microfluidic bionic chip of the present invention can realize the complex and dynamic microenvironment required for simulating organs, realize a continuously controllable flow environment, and interconnected culture units, which are used to simulate the barrier function of target organs and the biological process of stem cell homing.

[0025] (2) Organ chip technology, combining microfluidic chip technology and tissue engineering technology, can realize the construction of organ physiological microsystems on the chip by using microchannels, microchambers and other designs, and can simulate the main structural functions of different tissues and organs of the human body in vitro. It can not only cultivate cells with stable phenotypic and genetic characteristics that can be cultured for a long time in vitro, but also model the development, homeostasis and injury (disease) processes of various human organs. The technology platform based on organ chips can replace animals to a large extent. The chip system can be added with more internal or external environment operations, such as mechanical, chemical, electromagnetic or optical additional components, to build an integrated and automated system, which can conduct single-chip (or multi-chip) multi-task and single-chip (or multi-chip) multi-physiological process research. It can achieve long-term organoid culture, precise regulation of the local (micro) environment of organoids, controlled application of special (environmental) conditions, real-time monitoring, timely detection, controlled drug delivery, etc.

[0026] (3) The high-throughput chip design of the present invention allows multiple biological experiments to be conducted in different chip chambers under the same experimental conditions, and each chamber allows independent experimental operations, reagent addition, and design of a separate observation chamber without interference. The barrier-organ chip is more versatile and can model multiple organs in vitro, which is more consistent with the actual situation in vivo. The barrier-stem cell homing chip of the present invention can be used either connected or separately.

[0027] (4) Electrophysiology plays an important role in the physiological and pathological processes of biological organisms. The electrode design of the present invention allows for interpretation of biological signals by interpreting electrical signals. Furthermore, it is possible to achieve an electrical drive that can enhance the homing efficiency and differentiation rate of stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a flow diagram of the barrier-stem cell homing biomimetic microfluidic chip of the present invention;

[0029] Figure 2 Schematic diagram of the connection of the barrier-stem cell homing bionic microfluidic chip of the present invention;

[0030] Figure 3 It is a schematic structural diagram of the barrier chip of the present invention;

[0031] Figure 4 This is a schematic diagram of the structure of the stem cell homing chip of the present invention;

[0032] Figure 5 Schematic diagram of the fluid pathway of the stem cell homing chip of the present invention.

[0033] Description of Reference Numerals

[0034] 1. Interface layer, 2. Porous membrane, 3. Slot layer, 4. Base layer. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] Example 1

[0037] This invention provides a barrier-stem cell homing biomimetic microfluidic chip, comprising a barrier chip and a stem cell homing chip. The barrier chip is layered and cultured from bottom to top, following the cell composition order of the target organ's barrier structure. The barrier chip is used to construct the key functional interface of the target organ, realizing the complex organ barrier function between cells, tissues, organs, and microenvironments.

[0038] The upper chamber of the Stem Cell Homing Chip houses stem cell culture, while the middle chamber contains a 3D "nest" made of a mammalian acellular matrix corresponding to the target organ. The lower chamber serves as a perfusion chamber, receiving cell culture fluid from the upper chamber of the barrier chip. The Stem Cell Homing Chip simulates stem cell homing. Under the influence of various factors, pluripotent stem cells within the chip can be directed to migrate to the targeted region and, through directed differentiation induction, establish and survive.

[0039] Decellularized matrices are derived from natural ingredients and possess excellent cytocompatibility and bioactivity. Decellularized matrices derived from biological tissues and organs can preserve the unique native microenvironment of the target tissue, including the complex extracellular matrix components and microstructure. These matrices provide specific guidance signals for cell adhesion, proliferation, migration, differentiation, and other behaviors, offering advantages in regulating cell fate that are unmatched by other natural materials. By combining with hydrogels, they can form 3D structures, which are more conducive to cell survival and more realistically simulate the physiological structure of the body.

[0040] like Figure 1 、 2 As shown, a multifunctional liquid delivery unit is installed before the inlet of the barrier chip. A liquid-driven device, such as a peristaltic pump or syringe pump, is connected to the liquid path. Culture fluid is perfused through the upper and lower chambers of the barrier chip. A multifunctional liquid collection unit is connected to the liquid path at the outlet of the lower chamber of the barrier chip to collect the residual liquid. The outlet of the upper chamber is connected to the liquid path inlet of the lower chamber of the stem cell homing chip, enabling perfusion of the stem cell homing chip. The outlet of each chamber of the stem cell homing chip is connected to the multifunctional liquid collection unit. The stem cell homing chip is a high-throughput biomimetic microfluidic chip, in which multiple barrier chips are simultaneously connected to the same stem cell homing chip for perfusion.

[0041] In this embodiment, the bionic microfluidic chip and system can construct an in vitro organ modeling device with a continuously controllable flow environment and interconnected cell culture units through units such as fluid delivery, cell culture and functional testing, simulate the complex and dynamic microenvironment required for organ development, and realize the biological process of simulating stem cell homing.

[0042] The above-mentioned system can be used for different scientific research such as disease modeling, drug development, personalized medicine based on stem cell technology, as well as life science research in special environments such as deep sea, space environment, high temperature and high pressure, strong light and strong radiation environment, and provides feasible technical support. It has low cost, small size, high integration, strong design and adjustability, and simple operation. It is very suitable for special environments, and can carry out single-chip (or multi-chip) multi-task and single-chip (or multi-chip) multi-physiological process research, realizing the advantages of long-term organoid culture, precise regulation of the local (micro) environment of organoids, controllable application of special (environmental) conditions, real-time monitoring, timely detection, and controllable drug delivery.

[0043] Example 2

[0044] like Figure 3 As shown, the barrier chip is separated by a porous membrane 2 into an upper chamber and a lower chamber. In this embodiment, the upper chamber is arranged on the back of the interface layer 1, the interface is arranged on the top surface of the interface layer, and the lower chamber is arranged on the inner side of the base layer 4.

[0045] Barrier chips are constructed by culturing cells in layers from bottom to top, following the order of the target organ's barrier structure. For example, in a blood-brain barrier chip, the lower chamber is used to culture brain microvascular endothelial cells and pericytes, while the upper chamber is used to culture astrocytes.

[0046] The interface layer is provided with a first inlet and a first outlet, which are connected to the upper chamber. The barrier chip interface layer is provided with a second inlet and a second outlet, which are connected to the lower chamber. The cells in the lower chamber are perfused and cultured, which can realize the entry and exit of liquids in the upper and lower chambers. Fresh culture fluid for perfusion culture of target organ cells can also be perfused. Biochemical reagents used for processing and detecting the cells can also be perfused, such as staining solution, fixative, detection reagent, cell digestion solution, etc.

[0047] The porous membrane is arranged in the card slot layer 3, and its position corresponds to the upper chamber and the lower chamber. The porous membrane is adapted to the size of the key functional interface cells of the target organ. The pore size of the porous membrane is 3-10 μm, and the material is PET, PC or PDMS. The porous membrane serves as a separator and support site for the multilayer structure of cell culture. Due to its porous and small pore size characteristics, it allows chemical signals secreted by the upper and lower cells to pass through, realizing information exchange between the upper and lower layers, simulating the interaction between cells in the complex microenvironment in the body, and constructing an in vitro model that is closer to the physiological state in the body. The chip material or porous membrane, as well as other materials that will contact cells on the chip, are selected from materials with good biocompatibility and good light transmittance.

[0048] Electrodes are inserted into the upper and lower chambers to measure transmembrane resistance. Recommended electrode materials are gold, platinum, or silver, which are biocompatible and stable. The specific electrode material selected depends on the cell growth characteristics. After the electrodes are inserted, the holes around the electrodes can be sealed with temperature-sensitive glue or light-curing glue to prevent leakage.

[0049] Example 3

[0050] like Figure 4 、 Figure 5 As shown, the stem cell homing chip is separated from the lower chamber and the middle chamber by a first porous membrane, and from the middle chamber and the upper chamber by a second porous membrane. A third inlet and a third outlet are provided on the interface layer of the stem cell homing chip, which are connected to the lower chamber and can be perfused separately. The perfusion fluid is the cell culture fluid or biochemical reagent flowing out of the upper chamber of the barrier chip. A fourth inlet and a fourth outlet are provided on the interface layer of the stem cell homing chip, which are connected to the middle chamber and can be perfused separately. The perfusion fluid can be culture medium or other liquid biochemical reagents. A fifth inlet and a fifth outlet are provided on the interface layer of the stem cell homing chip, which are connected to the upper chamber and can be perfused separately. The perfusion fluid can be culture medium or other liquid biochemical reagents.

[0051] The pore size of the first porous membrane in the stem cell homing chip is 100-500μm, which is used to support the hydrogel "nest" layer and separate the hydrogel "nest" layer and the perfusion layer, allowing nutrients and other small molecules in the perfusion process to pass through the porous membrane to reach the middle chamber; the pore size of the second porous membrane is 10-50μm, which is used to separate the stem cell culture layer and the hydrogel "nest" layer and form a microchannel that allows stem cells to migrate.

[0052] Gold / platinum electrodes were connected to the upper and middle chambers, respectively. The upper chamber was negatively charged, while the middle chamber was positively charged, with a voltage of 0 to 2.0V, to simulate electrical stimulation and promote stem cell homing. The middle chamber contained a 3D nest structure composed of a mixture of bioactive hydrogel and acellular matrix from a mammalian organ corresponding to the target organ. This creates the biological, physical, and chemical factors that determine the microenvironment for stem cell differentiation induction, thereby determining the differentiation, proliferation, migration, and other behaviors of the stem cells in the upper chamber, as well as their phenotype. The middle chamber was embedded with a bioactive, porous, three-dimensional composite hydrogel structure to create a specific microenvironment and form a stem cell "nest."

[0053] The upper chamber houses pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or other stem cells with differentiation potential that can be induced to differentiate into target organs. Through the porous membrane with a defined pore size, cells can migrate to the targeted area and, after undergoing the directed differentiation process, settle and survive in the hydrogel "nest" in the middle chamber.

[0054] Example 4

[0055] This embodiment provides a blood-brain barrier + brain-like organ homing bionic microfluidic chip.

[0056] The barrier chip consists of a four-layer structure: from bottom to top, a base layer, a slot layer, and a porous membrane. The interface layer is made of glass, with pathways and chambers etched using chemical etching. The slot layer is made of PDMS, with a central circular slot formed using a 3D-printed mold. The porous membrane is made of PET with a pore size of 3μm. The interface layer is injection-molded from PC, with a spiral interface designed to accommodate a Luer external female connector. A gold electrode rod is embedded in the center top of the interface layer chamber, and the electrode interface is secured and sealed with hot-melt adhesive.

[0057] Preparation of stem cell homing chip (operated in a clean room): The multiple stem cell homing unit structures are, from bottom to top, the lower perfusion layer, the first porous membrane, the middle hydrogel "nest" layer, the second porous membrane, the upper stem cell culture layer, and the interface layer. The lower perfusion layer uses glass material for the passage, and the passage is etched using chemical etching. The first porous membrane is made of PDMS with a pore size of 100μm, and the middle hydrogel "nest" layer is made of PDMS. The chamber is formed by pouring a 3D printed mold. The second porous membrane is made of PDMS with a pore size of 20μm. The upper stem cell culture layer chamber and observation window are poured into a 3D printed mold. The interface layer is made of PC. The electrode wires of the stem cell culture layer and the hydrogel nest layer are pressed onto the surface before the PDMS is fully formed, and then continue to solidify and form. According to the structural design, each layer of the chamber corresponds to an inlet and an outlet, allowing independent liquid inlet and outlet operations.

[0058] Chip Sterilization: All components of the chip must be sterilized before use. High-temperature-resistant materials must be sterilized using moist heat, while other components must be sterilized by soaking in 75% alcohol or using a gas sterilizer. Chip assembly, cell culture, and subsequent fluid injection and extraction experiments must be performed under a sterile operating table.

[0059] Chip Embedding and Fluidic Pathway Connection: The upper chamber of the stem cell homing chip was coated with a 2:1 mixture of 0.1 mg / ml Matrigel and 0.2 mg / ml type I collagen. The cells were incubated in a 37°C, 5% CO2 incubator for 2 hours before subsequent cell culture. The middle chamber was embedded with a mixture of porcine brain matrix and GELMA with a pore size of 200 μm.

[0060] The steps for preparing the porcine brain decellularized matrix are as follows: Cut the porcine brain tissue into 5mm x 5mm x 5mm pellets and wash them in a DW solution containing 1% penicillin / streptomycin (P / S) for 48 hours to remove blood and impurities. Wash the tissue in a solution containing 0.5% sodium dodecyl sulfate (SDS) and 1% P / S for 24 hours, changing the solution every 12 hours, to remove cell membranes and some intracellular matrix. Wash the tissue in a solution containing 50U / ml DNase and 1M NaCl for 12 hours to remove cellular genetic material through enzymatic digestion and hypertonic salt treatment. Wash the tissue in a solution containing 0.5% Triton X-100 for 24-96 hours until the porcine brain tissue turns grayish white and most cellular components are removed. Wash the tissue in a solution containing 0.1% peracetic acid and 4% ethanol for 2 hours to sterilize the tissue. Wash the tissue in 1X PBS for 1 hour to remove any residual decellularization reagent. The pig brain tissue after the above treatment was frozen at -80°C overnight and freeze-dried for more than 72 hours to obtain the pig brain decellularized matrix.

[0061] The first inlet of the barrier chip is connected to the multifunctional liquid feeding unit 1, the second inlet is connected to the multifunctional liquid feeding unit 2, the first outlet is connected to the multifunctional liquid collection unit, the second outlet is connected to the two-way valve, and the two separate pathways are connected. One pathway is connected to the multifunctional liquid collection unit, and the other is connected to the third inlet of the stem cell chip.

[0062] The third inlet of the stem cell homing chip (using one unit as an example) is connected to the second outlet of the barrier chip. The fourth and fifth inlets are connected to multifunctional liquid delivery unit 3, respectively. All outlets are connected to the multifunctional liquid collection unit. Check valves are installed in the pathways connecting all outlets of the barrier-stem cell homing biomimetic microfluidic chip and the multifunctional liquid collection units to prevent fluid backflow. Each multifunctional liquid collection unit has a knob to allow for timely removal of liquid for connection to downstream analysis systems, further cell experiments, or storage.

[0063] Chip cell culture: Human brain microvascular endothelial cells hBMEC and human brain vascular pericytes HBVP were mixed in a ratio of 1:1 and seeded at a concentration of 1×10 6 / mL, human astrocytes, seeding concentration was 1×10 6 Porcine brain de-matrix was mixed with 5 mg / mL Matrigel hydrogel and injected into the lower and upper chambers of the barrier chip and the middle chamber of the stem cell chip, respectively. The mixture was placed in a 37°C 5% CO2 incubator for 30 minutes. Stem cells were seeded in the upper chamber of the stem cell homing chip and cultured using the mTeSR Plus Kit. After overnight stasis, perfusion was performed.

[0064] The lower layer of the barrier chip was perfused at a rate of 10 μL / min using EBM-2 medium supplemented with Single-Quots (hydrocortisone, ascorbic acid, and heparin), antibiotic-antimycotic solution, and 20% fetal bovine serum. The upper layer of the barrier chip was perfused at a rate of 0.15 μL / min using mTeSR Plus Kit medium and a 10:1 mixture of RPMI 1640 medium supplemented with 5% human serum, 1% glutamine, and 1% penicillin / streptomycin.

[0065] On-chip cell online monitoring and multifunctional fluid delivery: Cell growth status is observed every 12 hours. The barrier chip allows real-time observation under an optical microscope. The stem cell homing chip features observation windows in both the upper and middle chambers, allowing real-time observation under an optical microscope.

[0066] The barrier function of the chip was measured every 12 hours by measuring transmembrane electrical resistance and the presence of fluorescein isothiocyanate (FITC)-dextran (70 kDa, 20 kDa, and 4 kDa, 0.5 mg / ml). The stem cell homing chip was also intermittently applied with 0-2 V alternating current to different cells daily. The differentiation of stem cells was measured in different cells at 3, 7, 12, and 18 days of culture, and immunostaining was used to determine the differentiation.

[0067] Furthermore, the biomimetic microfluidic chip of the present invention can be used to test drugs that promote stem cell differentiation. Two drugs with different permeabilities through the barrier can be administered through the lower channel of the barrier chip, while another drug administered directly within the barrier can be administered through the upper channel of the stem cell homing chip. Preliminary experiments can be conducted to determine the drug concentration and administration time. This allows for verification of the drug's barrier permeability, its effect on the blood-brain barrier, and the pharmacokinetics of these different drugs, enabling drug screening.

[0068] Example 5

[0069] Similar to the above-mentioned preparation method and cell culture method, the present invention provides an application of a barrier-stem cell homing bionic microfluidic chip. The upper chamber of the barrier chip cultures astrocytes, the lower chamber of the barrier chip cultures microvascular endothelial cells and pericytes, the upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with brain organ differentiation potential, and the middle chamber is composed of a mammalian brain decellularized matrix corresponding to the target organ to form a "nest" environment, which is used for a blood-brain barrier-brain organoid bionic microfluidic chip.

[0070] An application of a barrier-stem cell homing bionic microfluidic chip is characterized in that the upper chamber of the barrier chip cultures alveolar epithelial cells, the lower chamber of the barrier chip cultures microvascular endothelial cells and pericytes, the upper cells of the stem cell homing chip culture pluripotent stem cells and stem cells with lung organoid differentiation potential, and the middle chamber forms a 3D "nest" with mammalian lung decellularized matrix corresponding to the target organ, which is used for a blood-gas barrier-lung organoid bionic microfluidic chip.

[0071] A barrier-stem cell homing bionic microfluidic chip is used. The upper chamber of the barrier chip cultures renal capsule epithelial cells, the lower chamber of the barrier chip cultures microvascular endothelial cells, the upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with kidney organ differentiation potential, and the middle chamber forms a 3D "nest" with mammalian kidney decellularized matrix corresponding to the target organ, which is used for a blood-urine barrier-kidney-like bionic microfluidic chip.

[0072] An application of a barrier-stem cell homing bionic microfluidic chip, in which the upper chamber of the barrier chip cultures intestinal epithelial cells, and the lower chamber of the barrier chip cultures microvascular endothelial cells. The upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with intestinal organoid differentiation potential, and the middle chamber forms a 3D "nest" with mammalian intestinal decellularized matrix corresponding to the target organ, which is used for the intestinal vascular barrier-intestinal organoid bionic microfluidic chip.

[0073] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A barrier-stem cell homing bionic microfluidic chip, comprising a barrier chip and a stem cell homing chip, characterized in that: The barrier chip is cultured in layers from lower to upper layers according to the cell composition order of the barrier structure of the target organ; the upper chamber of the stem cell homing chip cultures stem cells, the middle chamber embeds a 3D "nest" formed by the decellularized matrix of the mammal corresponding to the target organ, and the lower chamber is a perfusion chamber, which is perfused with the cell culture fluid flowing out of the upper chamber of the barrier chip; the stem cell homing chip is separated from the lower chamber and the middle chamber by a first porous membrane, and from the middle chamber and the upper chamber by a second porous membrane; a third inlet and a third outlet are provided on the interface layer of the stem cell homing chip, which are connected to the lower chamber; a fourth inlet and a fourth outlet are provided on the interface layer of the stem cell homing chip, which are connected to the middle chamber; and a fifth inlet and a fifth outlet are provided on the interface layer of the stem cell homing chip, which are connected to the upper chamber.

2. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The middle chamber of the stem cell homing chip embeds a 3D nest structure mixed with a bioactive hydrogel and a cell-free matrix of a mammalian organ corresponding to the target organ, and the upper chamber cultures pluripotent stem cells, embryonic stem cells, mesenchymal stem cells or stem cells with differentiation potential that can be induced to differentiate into the target organ.

3. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The pore size of the first porous membrane of the stem cell homing chip is 100-500 μm; the pore size of the second porous membrane is 10-50 μm.

4. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The stem cell homing chip is connected to gold / platinum electrodes in the upper chamber and the middle chamber respectively. The upper chamber is connected to negative electricity, and the middle chamber is connected to positive electricity, with a voltage of 0-2.0V.

5. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The stem cell homing chip is provided with an upper chamber observation window and a middle chamber observation window.

6. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: A multifunctional liquid supply unit is provided upstream of the liquid path of the barrier chip and the stem cell homing chip, and the liquid path is connected to a fluid drive system; a multifunctional liquid collection unit is connected downstream of the liquid path of the barrier chip and the stem cell homing chip.

7. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The stem cell homing chip is a high-throughput microfluidic chip, and a plurality of barrier chips are simultaneously connected to the same stem cell homing chip for perfusion.

8. An application of the barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The upper chamber of the barrier chip cultures astrocytes, the lower chamber of the barrier chip cultures microvascular endothelial cells and pericytes, the upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with brain organ differentiation potential, and the middle chamber forms a "nest" environment with a decellularized mammalian brain matrix corresponding to the target organ, which is used for a blood-brain barrier-brain organoid bionic microfluidic chip.

9. An application of the barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The upper chamber of the barrier chip cultures alveolar epithelial cells, the lower chamber of the barrier chip cultures microvascular endothelial cells and pericytes, the upper cells of the stem cell homing chip culture pluripotent stem cells or stem cells with lung organoid differentiation potential, and the middle chamber forms a 3D "nest" with mammalian lung decellularized matrix corresponding to the target organ, which is used for a blood-gas barrier-lung organoid bionic microfluidic chip.

10. An application of the barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The upper chamber of the barrier chip cultures renal capsule epithelial cells, the lower chamber of the barrier chip cultures microvascular endothelial cells, the upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with kidney organ differentiation potential, and the middle chamber forms a 3D "nest" with mammalian kidney decellularized matrix corresponding to the target organ, which is used for the blood-urine barrier-kidney organ bionic microfluidic chip.

11. An application of the barrier-stem cell homing biomimetic microfluidic chip according to claim 1, characterized in that: The upper chamber of the barrier chip cultures intestinal epithelial cells, the lower chamber of the barrier chip cultures microvascular endothelial cells, the upper chamber of the stem cell homing chip cultures pluripotent stem cells or stem cells with intestinal organ differentiation potential, and the middle chamber forms a 3D "nest" with mammalian intestinal decellularized matrix corresponding to the target organ, which is used for the intestinal vascular barrier-intestinal organoid bionic microfluidic chip.

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