CN114854588A - Barrier-stem cell homing bionic micro-fluidic chip and application thereof - Google Patents

Abstract

The invention provides a barrier-stem cell homing bionic microfluidic chip which comprises a barrier chip and a stem cell homing chip, wherein the barrier chip is cultured in a layering way from the lower layer to the upper layer according to the cell composition sequence of a barrier structure of a target organ; the upper layer cavity of the stem cell homing chip is used for culturing stem cells, the middle layer cavity is embedded with a 3D 'nest' formed by mammal acellular matrixes corresponding to target organs, and the lower layer cavity is a perfusion cavity which is perfused by cell culture solution flowing out from the upper layer cavity of the barrier chip. The microfluidic bionic chip can realize complex and dynamic microenvironment required by simulated organs, for example, fluid conveying, cell culture and detection units and the like can be designed, continuous and controllable flow environment and mutually communicated culture units can be realized, and the microfluidic bionic chip is used for highly reducing the physiological characteristics of target organs and simulating the barrier function of the target organs and the biological process of stem cell homing.

Description

Barrier-stem cell homing bionic micro-fluidic chip and application thereof

Technical Field

The invention belongs to the field of organ chips, and particularly relates to a barrier-stem cell homing bionic microfluidic chip and application thereof.

Background

The vector for routine life science research, is essentially based on animal and two-dimensional (2D) cells. Although animal models have become established for human understanding of the abundance of physiology and disease and the development of new drugs, animal models have been used as carriers for human research and have a number of drawbacks, such as candidate drugs that may be terminated by lack of efficacy in animals or by the discovery of risks or toxicities in animals that may not be relevant to humans. Despite major advances in vitro biology and toxicology over the last 20 years, over 80% of research drugs currently fail clinical trials, 60% of which are due to lack of efficacy and 30% to toxicity. 2D cell culture has been used for life science research for over a century. However, this culture approach has difficulty supporting tissue-specific differentiation functions of multiple cell types and providing information about the complexity of living systems. There is therefore an urgent need for in vitro modeling and testing platforms that are more realistic in responding to complex human features. Indeed, with the rapid development of microfabrication technology, bench-top experiments can be performed in small systems, which are called microfluidic lab-on-a-chip (LOC) systems. The organ chip is a 3D micro-fluidic cell culture device, and can be combined with a micro-fluidic chip technology, a stem cell induced differentiation technology, a tissue engineering technology and the like to construct an organ physiological microsystem, and can simulate the main structural functions of different tissues and organs of a human body and the connection between complex organs in vitro. The human organ chip technology is gaining favor of researchers, and is a promising research means for replacing animal experiments in the future.

Stem cells have unique advantages in regenerative medicine. Due to the continuous progress of stem cell research at present, the stem cells have the characteristics of availability, induced differentiation in various organ directions and unique capabilities of self-renewal and generation of different cell types, are more consistent with human genetic characteristics, are more suitable for in vitro modeling, and are applied to human disease research, drug development, personalized medicine and the like. Based on stem cell technology, accurate in vitro modeling of donor characteristics can also be achieved. The homing process of the stem cells which survive is directionally tended to migrate to the target tissue and colonized by simulating the autologous or exogenous stem cells on the chip under the action of various factors. In addition, the in vitro modeling of the biological process can be realized by an organ chip technology, and the monitoring of the cell state in the culture process, the deeper scientific research, the diagnosis and treatment prognosis of diseases and the like are allowed.

At this stage, most organ chip studies are limited to in vitro modeling of individual organ functions (or functional interfaces), which neglects the organ/tissue-organ barrier interrelationships, in fact. The normal physiological operation of organs requires a chemical process related to physiology, such as metabolism of substances by the circulation of body fluids and blood, and digestion, absorption, transportation, decomposition, and metabolism of substances in vivo.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a barrier-homing bionic micro-fluidic chip and application thereof. The invention realizes the in vitro modeling of the target organ-organ barrier through the structural design, simulates the specific elements of the cell-extracellular matrix-microenvironment of the target organ, and provides a feasible technical support for applying the in vitro modeling design to different scientific researches such as disease modeling, drug research and development, personalized medical treatment and the like based on stem cell technology, and to life science researches in special environments such as deep sea, space environment, high temperature and high pressure and strong light and strong radiation environment.

The invention provides the following technical scheme:

a barrier-stem cell homing bionic microfluidic chip comprises a barrier chip and a stem cell homing chip, wherein the barrier chip is cultured in a layered manner from a lower layer to an upper layer according to the sequence of cell composition of a barrier structure of a target organ; the upper layer cavity of the stem cell homing chip is used for culturing stem cells, the middle layer cavity is embedded with a 3D 'nest' formed by mammal acellular matrixes corresponding to target organs, and the lower layer cavity is a perfusion cavity which is perfused by cell culture solution flowing out from the upper layer cavity of the barrier chip.

Furthermore, the barrier chip is divided into an upper layer chamber and a lower layer chamber by a porous membrane, and a first inlet and a first outlet which are communicated with the upper layer chamber are arranged on an interface layer of the barrier chip; and the barrier chip interface layer is provided with a second inlet and a second outlet which are communicated with the lower-layer cavity.

Further, the barrier chip perfuses the lower chamber with a culture solution for culturing the lower cells, and the upper chamber perfuses the upper chamber with a mixed culture solution for culturing the upper stem cells in the stem cell homing chip.

Furthermore, the porous membrane is adaptive to the size of key functional interface cells of a target organ, and the pore diameter is 3-10 mu m.

Further, the barrier chip inserts electrodes into the upper chamber and the lower chamber respectively for measuring the transmembrane resistance value.

Furthermore, the stem cell homing chip is provided with a lower layer cavity and a middle layer cavity which are separated by a first porous membrane, a middle layer cavity and an upper layer cavity which are separated by a second porous membrane, a third inlet and a third outlet which are communicated with the lower layer cavity are arranged on an interface layer of the stem cell homing chip, a fourth inlet and a fourth outlet which are communicated with the middle layer cavity are arranged on the interface layer of the stem cell homing chip, and a fifth inlet and a fifth outlet which are communicated with the upper layer cavity are arranged on the interface layer of the stem cell homing chip.

Furthermore, a 3D nest structure formed by mixing bioactive hydrogel and acellular matrixes of mammal organs corresponding to target organs is embedded in a middle-layer cavity of the stem cell homing chip, and pluripotent stem cells, embryonic stem cells, mesenchymal stem cells or other stem cells with differentiation potential capable of being induced and differentiated to the target organs are cultured in an upper-layer cavity.

Further, the pore diameter of the first porous membrane of the stem cell homing chip is 100-500 μm; the pore diameter of the second porous membrane is 10 to 50 μm.

Furthermore, the stem cell homing chip is respectively connected with gold/platinum electrodes in the upper layer cavity and the middle layer cavity, the upper layer cavity is connected with negative electricity, the middle layer cavity is connected with positive electricity, and the voltage is 0-2.0 v.

Furthermore, the stem cell homing chip is provided with an upper-layer cavity observation window and a middle-layer cavity observation window.

Furthermore, a multifunctional liquid supply unit is arranged at the upstream of a liquid path of the barrier chip and the stem cell homing chip, and a fluid driving system is connected to the liquid path; and a multifunctional liquid collection unit is connected with the downstream of the liquid paths of the barrier chip and the stem cell homing chip.

Furthermore, the stem cell homing chip is a high-flux bionic micro-fluidic chip, and a plurality of barrier chips and the same stem cell homing chip are connected and perfused at the same time.

The barrier-stem cell homing bionic microfluidic chip is applied to a blood brain barrier-brain organoid bionic microfluidic chip, wherein an upper layer cavity of the barrier chip is used for culturing astrocytes, a lower layer cavity of the barrier chip is used for culturing microvascular endothelial cells and pericytes, an upper layer cavity of the stem cell homing chip is used for culturing multipotent stem cells or stem cells with brain organoid differentiation potential, and a middle layer cavity is used for forming a 'nest' environment by mammal brain acellular matrixes corresponding to target organs.

The barrier-stem cell homing bionic microfluidic chip is applied to culturing alveolar epithelial cells in an upper layer chamber of the barrier chip, culturing microvascular endothelial cells and pericytes in a lower layer chamber of the barrier chip, culturing multipotential stem cells or stem cells with lung-like organ differentiation potential in the upper layer cells of the stem cell homing chip, and forming 3D 'nest' by mammal lung acellular matrixes corresponding to target organs in a middle layer chamber, and is used for the blood-gas barrier-lung-like organ bionic microfluidic chip.

The barrier-stem cell homing bionic microfluidic chip is applied to a hematuria barrier-kidney organ bionic microfluidic chip, wherein an upper layer cavity of the barrier chip is used for culturing renal vesicle epithelial cells, a lower layer cavity of the barrier chip is used for culturing microvascular endothelial cells, an upper layer cavity of the stem cell homing chip is used for culturing multipotential stem cells or stem cells with kidney organ differentiation potential, and a middle layer cavity is used for forming a 3D 'nest' by a kidney acellular matrix of a mammal corresponding to a target organ.

The upper layer chamber of the stem cell homing chip is used for culturing multipotential stem cells or stem cells with intestinal organoid differentiation potential, and the middle layer chamber is used for forming a 3D nest by an intestinal acellular matrix of mammals corresponding to a target organ and is used for the intestinal vascular barrier-intestinal organoid bionic microfluidic chip.

By adopting the technical scheme, the invention has the following beneficial effects:

(1) the microfluidic bionic chip can realize complex and dynamic microenvironment required by simulated organs, realize continuous controllable flow environment and interconnected culture units, and is used for simulating the barrier function of target organs and the biological process of stem cell homing.

(2) The organ chip technology combines the micro-fluidic chip technology and the tissue engineering technology, utilizes the design of micro-channels, micro-chambers and the like, can realize the construction of an organ physiological microsystem on a chip, can simulate the main structural functions of different tissues and organs of a human body in vitro, can culture cells with stable phenotype and genetic characteristics which can be cultured in vitro for a long time, and can also model the development, the steady state and the damage (disease) processes of various human organs. The organ chip-based technical platform can replace animals to a great extent, and the chip system can be added with more operations of internal or external environment, for example, the chip system can be combined with additional components such as machinery, chemistry, electromagnetism or optics and the like to construct an integrated and automatic system, and can carry out single-chip (or multi-chip) multitask and single-chip (or multi-chip) multi-physiological process research. Realizes long-period organoid culture, precise regulation and control of local (micro) environment of organoids, controllable application of special (environmental) conditions, real-time monitoring, timely detection, controllable drug delivery and the like.

(3) The high throughput chip design of the present invention allows for multiple biological experiments to be performed in different chip chambers under the same experimental conditions, and each chamber allows for separate experimental operations, adding reagents and designing separate observation chambers without interfering with each other. The barrier-organ chip has universality, can be used for in-vitro modeling of various organs, and is more suitable for the real situation in vivo. The barrier-stem cell homing chip of the present invention can be used either in a connected manner or in a separated manner.

(4) Electrophysiology plays an important role in the physiological and pathological processes of biological organisms. The electrode design of the invention allows the interpretation of the biological signal by interpreting the electrical signal. In addition, it is also possible to achieve that the "homing" efficiency and differentiation rate of stem cells can be increased in an electrically driven manner.

Drawings

Fig. 1 is a flow path diagram of a barrier-stem cell homing biomimetic microfluidic chip of the present invention;

FIG. 2 is a schematic connection diagram of the barrier-stem cell homing bionic microfluidic chip of the present invention;

FIG. 3 is a schematic structural diagram of a barrier chip according to the present invention;

FIG. 4 is a schematic structural diagram of a stem cell homing chip of the present invention;

fig. 5 is a schematic view of a flow path of the stem cell homing chip of the present invention.

Description of the reference numerals

1. Interface layer, 2, porous membrane, 3, card groove layer, 4, basal layer.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the block diagrams and specific examples are set forth only for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Example 1

The invention provides a barrier-stem cell homing bionic microfluidic chip which comprises a barrier chip and a stem cell homing chip, wherein the barrier chip is cultured in a layered manner from a lower layer to an upper layer according to the cell composition sequence of a barrier structure of a target organ. The barrier chip is used for constructing a key functional interface of a target organ and realizing a complex organ barrier function between a cell-cell/tissue/organ-microenvironment.

The upper layer cavity of the stem cell homing chip is used for culturing stem cells, the middle layer cavity is embedded with a 3D nest formed by mammal acellular matrixes corresponding to a target organ, the lower layer cavity is a perfusion cavity, and cell culture solution flowing out of the upper layer cavity of the barrier chip is perfused. The stem cell homing chip is used for simulating stem cell homing, and under the action of various factors, the pluripotent stem cells in the chip can directionally tend to migrate to a target area and can be planted and survive after being directionally induced and differentiated.

The acellular matrix is derived from natural components and has excellent cell compatibility and bioactivity; the acellular matrix obtained from organism tissue organs can retain the unique native microenvironment of a target tissue, comprises complex extracellular matrix components and microstructures, provides specific guide signals for the behaviors of adhesion, proliferation, migration, differentiation and the like of cells, and has incomparable advantages compared with other natural materials in the aspect of regulating and controlling cell fate. By combining with the hydrogel, a 3D structure can be formed, which is more beneficial to cell survival and more truly simulates the physiological structure in vivo.

As shown in fig. 1 and 2, a multifunctional liquid supply unit is arranged in front of an inlet of the barrier chip, a liquid path is connected with a liquid driving device such as a peristaltic pump or an injection pump, culture liquid is perfused and cultured through an upper layer chamber and a lower layer chamber of the barrier chip respectively, and a multifunctional liquid collection unit is connected on a liquid path at an outlet of the lower layer chamber of the barrier chip to receive a residual liquid. The outlet of the upper-layer cavity is communicated with the inlet of the lower-layer cavity of the stem cell homing chip, so that the stem cell homing chip is perfused, and the outlets of the cavities of the stem cell homing chip are connected with the multifunctional liquid collecting unit. The stem cell homing chip is a high-flux bionic micro-fluidic chip, and a plurality of barrier chips are connected with the same stem cell homing chip for perfusion at the same time.

In the embodiment, the bionic microfluidic chip and the system can construct an organ in-vitro modeling device with continuously controllable flowing environment and mutually communicated cell culture units through units such as fluid conveying, cell culture, function detection and the like, simulate a complex and dynamic microenvironment required by organ development, and realize the biological process of simulating stem cell homing.

The system can be used for different scientific researches such as disease modeling, drug research and development, personalized medical treatment and the like based on stem cell technology, is applied to life science research in special environments such as deep sea, space environment, high temperature, high pressure and strong light intensity radiation environment, provides feasible technical support, has the advantages of low cost, small volume, high integration level, strong design adjustability and simplicity in operation, is very suitable for the special environments, can perform single-chip (or multi-chip) multi-task and single-chip (or multi-chip) multi-physiological process research, and realizes the advantages of long-period organoid culture, precise regulation and control of local (micro) environment of organoid, controllable application of special (environmental) conditions, real-time monitoring, timely detection, controllable drug delivery and the like.

Example 2

As shown in fig. 3, 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 disposed on the back side of an interface layer 1, the interface is disposed on the top side of the interface layer, and the lower chamber is disposed inside a substrate layer 4.

The barrier chip is cultured in layers from the lower layer to the upper layer according to the cell composition sequence of the barrier structure of the target organ. For example, a blood brain barrier chip is prepared, the lower cavity is used for culturing brain microvascular endothelial cells and brain perivascular cells, and the upper cavity is used for culturing astrocytes.

Interface layer is equipped with first import and first export, with upper chamber intercommunication, is equipped with second import and second export at barrier chip interface layer, with lower floor's chamber intercommunication, lower floor's chamber cell is cultivateed in the perfusion, can realize the business turn over of upper and lower floor's chamber liquid, and the fresh culture solution of target organ cell is cultivateed in the perfusion, also can the perfusion handle simultaneously, detect the biochemical reagents that this cell was used, for example dyeing liquor, stationary liquid, detection reagent, cell digest etc..

The porous membrane is arranged in the clamping groove layer 3, the positions of the porous membrane correspond to the upper layer cavity and the lower layer cavity, the size of the porous membrane is adapted to the size of key function interface cells of a target organ, the pore diameter of the porous membrane is 3-10 mu m, and the porous membrane is made of PET, PC or PDMS. The porous membrane is used as a separation and support site of a multilayer structure of cell culture, and chemical signals secreted by upper and lower layers of cells are allowed to permeate due to the characteristics of porosity and tiny pore diameter, so that information interaction between the upper and lower layers is realized, intercellular interaction in a complex microenvironment in vivo is simulated, and an in vitro model closer to the physiological state in vivo is constructed. The chip material or the porous membrane and other materials which can contact with cells on the chip are all selected from materials with good biocompatibility and good light transmittance.

Electrodes are inserted into the upper chamber and the lower chamber, respectively, for measuring the transmembrane resistance value. The electrode material is gold or platinum or silver, the biocompatibility of the electrode is good and stable, and the specific electrode material is selected according to the growth characteristics of cells. After the electrode is inserted, holes around the electrode can be blocked by temperature-sensitive glue or photocuring glue to prevent liquid leakage.

Example 3

As shown in fig. 4 and 5, the stem cell homing chip has a lower chamber and a middle chamber separated by a first porous membrane, and a middle chamber and an upper chamber separated by a second porous membrane, and the interface layer of the stem cell homing chip has a third inlet and a third outlet, which are communicated with the lower chamber and can perform separate perfusion on the lower chamber, and the perfusion liquid is a cell culture liquid or a biochemical reagent flowing out from the upper chamber of the barrier chip. The interface layer of the stem cell homing chip is provided with a fourth inlet and a fourth outlet which are communicated with the middle layer cavity and can independently perfuse the middle layer cavity, and the perfusion liquid can be a culture medium or other liquid biochemical reagents. The interface layer of the stem cell homing chip is provided with a fifth inlet and a fifth outlet which are communicated with the upper-layer cavity to realize independent perfusion to the upper-layer cavity, and the perfusion liquid can be a culture medium or other liquid biochemical reagents.

The first porous membrane in the stem cell homing chip has the pore size of 100-500 mu m, is used for supporting the hydrogel 'nest' layer and separating the hydrogel 'nest' layer from the perfusion layer, and allows nutrient substances and other small molecular substances in the perfusion process to pass through the porous membrane to reach the middle-layer chamber; the second porous membrane has a pore size of 10-50 μm and serves to separate the stem cell culture layer from the hydrogel "nest" layer and to form microchannels that allow stem cell migration.

And the upper-layer cavity and the middle-layer cavity are respectively connected with a gold/platinum electrode, the upper-layer cavity is connected with negative electricity, the middle-layer cavity is connected with positive electricity, and the voltage is 0-2.0 v, so that the stem cell homing is promoted by simulating electrical stimulation. The middle layer cavity is embedded with a 3D nest structure formed by mixing bioactive hydrogel and acellular matrixes of mammal organs corresponding to target organs to form biological, physical and chemical factors for creating a microenvironment for inducing stem cells to differentiate, so that the behaviors of differentiation, proliferation, migration and the like of the stem cells and the phenotype of the stem cells in the upper layer cavity are determined, and the middle layer cavity is embedded with a composite hydrogel three-dimensional structure with bioactivity and loose pores to create a specific microenvironment to create a stem cell 'nest'.

The upper cavity is used for culturing pluripotent stem cells, embryonic stem cells, mesenchymal stem cells or other stem cells with differentiation potential, which can be induced to differentiate into a target organ. Can directionally and directionally migrate to a target area through a porous membrane with certain pore size, and can be planted and survive in a hydrogel 'nest' of a middle-layer cavity after a directional induced differentiation process.

Example 4

The embodiment provides a blood brain barrier and brain organ homing bionic microfluidic chip.

The preparation of the barrier chip comprises a four-layer structure, namely a substrate layer, a clamping groove layer and a porous membrane from bottom to top in sequence. The interface layer substrate layer is made of glass, and a chemical etching method is used for etching the channel and the cavity; the clamping groove layer is made of PDMS, the middle circular clamping groove is formed by reverse molding of a 3D printing mold. The porous membrane is made of PET material and has a pore diameter of 3 μm. The interface layer is made of PC material through injection molding, and the interface is designed into a spiral interface which can be inserted into a luer external-rotation female connector. And a gold electrode bar is embedded into the central top end of the interface layer cavity, and the electrode interface is fixed and sealed by hot melt adhesive.

Preparation of stem cell homing chip (operating between dust-free): the stem cell homing unit structures sequentially comprise a lower perfusion layer, a first porous membrane, a middle hydrogel 'nest' layer, a second porous membrane, an upper stem cell culture layer and an interface layer from bottom to top. The perfusion layer at the lower layer is a glass channel, and the channel is etched by using a chemical etching method. The first porous membrane is made of PDMS (polydimethylsiloxane), the aperture is 100 mu m, the hydrogel nest layer of the middle layer is PDMS, and the cavity is formed by reverse molding through a 3D printing mold. The second porous film is made of PDMS and has a pore size of 20 μm. And the stem cell culture layer cavity and the observation window on the upper layer are formed by reverse molding through a 3D printing mold. The interface layer is made of PC material. And pressing the electrode wires on the stem cell culture layer and the hydrogel nest layer on the surface before the PDMS is not completely molded, and then continuously curing and molding. According to the structural design, each layer of cavity corresponds to an inlet and an outlet, and the independent liquid feeding and taking operations are allowed.

Chip sterilization: before the chip is used, all elements are disinfected, the high-temperature resistant material is sterilized by moist heat, other elements are soaked in 75% alcohol for sterilization, or a gaseous sterilization device is used for sterilization. Chip assembly and cell culture and subsequent liquid feeding and taking experiments are all operated on a sterile operating platform.

Chip embedding and fluid path connection: the surface of an upper cavity in the stem cell homing chip is coated with 0.1mg/ml of Matrigel and 0.2mg/ml of coating solution with the type I collagen mixing ratio of 2:1, and the subsequent cell culture is carried out after the incubation for 2h in a 5% CO2 cell culture box at 37 ℃. Embedding pig brain matrix in the middle chamber, and mixing with GELMA with 200 μm aperture.

The preparation method of the pig brain matrix comprises the following steps: porcine brain tissue was cut into 5mm by 5mm sized particles and washed in DW solution containing 1% penicillin/streptomycin (P/S) for 48h to remove blood water and impurities. The tissue was washed for 24h in a solution containing 0.5% Sodium Dodecyl Sulfate (SDS) and 1% P/S, and the solution was changed every 12h to remove cell membranes and a portion of intracellular stroma. The tissue was washed for 12h in a solution containing 50U/ml DNase and 1M NaCl and the cytogenetic material was removed by enzymatic digestion and hypertonic salt action. The tissue was washed in a solution containing 0.5% Triton X-100 for 24-96h until the pig brain tissue turned off-white, clearing most of the cellular components. Washing the tissue in a solution containing 0.1% peracetic acid and 4% ethanol for 2h, and sterilizing the tissue; the tissue was washed with 1X PBS solution for 1h to remove residual decellularized reagent from the tissue. Freezing the treated pig brain tissue at-80 deg.C for overnight, and lyophilizing for over 72h to obtain pig brain acellular matrix.

The first inlet of the barrier chip is connected with the first multifunctional liquid feeding unit, the second inlet of the barrier chip is connected with the second multifunctional liquid feeding unit, the first outlet of the barrier chip is connected with the multifunctional liquid collecting unit, the second outlet of the barrier chip is connected with the two-way valve, one way of the two separated ways is connected with the multifunctional liquid collecting unit, and the other way of the two separated ways is connected with the third inlet of the stem cell chip.

The third inlet of the stem cell homing chip (in one unit as an example) is connected with the second outlet of the barrier chip. The fourth inlet and the fifth inlet are respectively connected with a third multifunctional liquid feeding unit. All outlets are connected to a multi-functional liquid collection unit. And check valves are arranged on all the outlets of the barrier-stem cell homing bionic microfluidic chip and the passages connected with the multifunctional liquid collecting unit to prevent the backflow of the fluid. All multi-functional liquid collection units are provided with knobs, and liquid can be taken out in time and used for connecting a downstream analysis system or carrying out further experiments or preservation on cells.

Chip cell culture: mixing human brain microvascular endothelial cells hBMEC and human brain perivascular cells HBVP in a ratio of 1:1, and inoculating at a concentration of 1 × 10 ⁶ cells/mL, human astrocytes, plated at a concentration of 1X 10 ⁶ Respectively mixing pig brain matrix with 5mg/mL Matrigel hydrogel, injecting into lower and upper cavities of barrier chip and middle cavity of stem cell chip, placing in 37 deg.C 5% CO ₂ The incubator lasts 30 min. Stem cells are inoculated in an upper-layer cavity of the stem cell homing chip, cultured by using an mTeSR Plus Kit culture Kit, kept stand overnight for culture, and then perfused.

The perfusion rate of the lower layer of the barrier chip was 10. mu.l/min, and EBM-2 medium supplemented with Single-spots (hydrocortisone, ascorbic acid and heparin), antibiotic antifungal solution and 20% fetal bovine serum was used as the culture medium. The barrier chip upper perfusion rate was 0.15 μ L/min, the culture was made using mTeSR Plus Kit medium and consisting of RPMI 1640, supplemented with 5% human serum, 1% glutamine and 1% penicillin/streptomycin medium 10: 1 mixed perfusion culture.

On-line monitoring and multifunctional liquid feeding operation of cells on a chip: the growth state of the cells was observed every 12 h. The barrier chip can be observed in real time under an optical microscope. The stem cell homing chip respectively establishes observation windows in the upper chamber and the middle chamber to allow real-time observation under an optical microscope.

The barrier function of the barrier chip was determined every 12h by measuring the transmembrane resistance value and Fluorescein Isothiocyanate (FITC) -dextran, 70kDa, 20kDa and 4kDa, 0.5 mg/ml. The stem cell homing chip intermittently applies 0-2 v of alternating current to different units every day, the induced differentiation condition of stem cells cultured for 3, 7, 12 and 18 days is respectively detected in the different units, and the induced differentiation condition of the stem cells is measured by an immunostaining method.

In addition, the bionic micro-fluidic chip can be used for carrying out a drug test for promoting stem cell induced differentiation. According to two kinds of medicines with different penetration rates through the barrier, the liquid is fed through a lower channel of the barrier chip, the medicine is directly fed in the barrier, the liquid is fed through an upper channel of the stem cell homing chip, and after the administration concentration and the administration time are determined in a pre-experiment, the barrier penetration rate of the medicine, the influence of the medicine on the blood brain barrier, the pharmacokinetics of the different medicines and the like can be verified so as to carry out medicine screening.

Example 5

Similar to the preparation method and the cell culture method, the invention provides the application of the barrier-stem cell homing bionic microfluidic chip, wherein an upper layer cavity of the barrier chip is used for culturing astrocytes, a lower layer cavity of the barrier chip is used for culturing microvascular endothelial cells and pericytes, an upper layer cavity of the stem cell homing chip is used for culturing multipotent stem cells or stem cells with brain organoid differentiation potential, and a middle layer cavity forms a 'nest' environment by mammal brain acellular matrixes corresponding to target organs and is used for the blood brain barrier-brain organoid bionic microfluidic chip.

The application of the barrier-stem cell homing bionic microfluidic chip is characterized in that an upper layer chamber of the barrier chip is used for culturing alveolar epithelial cells, a lower layer chamber of the barrier chip is used for culturing microvascular endothelial cells and pericytes, an upper layer cell of the stem cell homing chip is used for culturing multipotent stem cells and stem cells with lung organoid differentiation potential, and a middle layer chamber is used for forming a 3D 'nest' by a mammal lung acellular matrix corresponding to a target organ and is used for the blood-gas barrier-organoid bionic microfluidic chip.

The utility model provides an application of barrier-stem cell homing bionical micro-fluidic chip, barrier chip upper chamber cultivates renal capsule epithelial cells, barrier chip lower floor cavity cultivates microvascular endothelial cell, the stem cell homing chip upper chamber cultivates multipotential stem cell or has the stem cell of kidney class organ differentiation potential, middle level cavity is by the 3D "nest" of the cell matrix formation of mammal's kidney that the target organ corresponds, is used for hematuria barrier-class kidney organ bionical micro-fluidic chip.

The utility model provides an application of barrier-stem cell homing bionical micro-fluidic chip, the upper chamber of barrier chip cultivates intestinal epithelial cells, and the lower chamber of barrier chip cultivates capillary endothelial cell, and the upper chamber of stem cell homing chip cultivates multipotent stem cell or has the stem cell of intestines class organ differentiation potential, and middle level cavity forms 3D "nest" by the cell matrix is taken off from the intestines of mammal that the target organ corresponds for intestinal vascular barrier-intestines class organ bionical micro-fluidic chip.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (16)

1. A barrier-stem cell homing bionic microfluidic chip comprises a barrier chip and a stem cell homing chip, and is characterized in that the barrier chip is cultured in a layered manner from a lower layer to an upper layer according to the sequence of cell composition of a barrier structure of a target organ; the upper layer cavity of the stem cell homing chip is used for culturing stem cells, the middle layer cavity is embedded with a 3D 'nest' formed by mammal acellular matrixes corresponding to target organs, and the lower layer cavity is a perfusion cavity which is perfused by cell culture solution flowing out from the upper layer cavity of the barrier chip.

2. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, wherein the barrier chip is separated from the upper chamber and the lower chamber by a porous membrane, and a first inlet and a first outlet are provided at an interface layer of the barrier chip and are communicated with the upper chamber; and the barrier chip interface layer is provided with a second inlet and a second outlet which are communicated with the lower-layer cavity.

3. The barrier-stem cell homing bionic microfluidic chip according to claim 2, wherein the barrier chip perfuses a culture solution for culturing cells in the lower layer to the chamber in the lower layer, and perfuses a mixed culture solution for culturing cells in the upper layer to the chamber in the upper layer and culturing stem cells in the upper layer in the stem cell homing chip.

4. The barrier-stem cell homing biomimetic microfluidic chip according to claim 2, wherein the porous membrane is adapted to the size of critical functional interface cells of a target organ, and the pore size is 3-10 μm.

5. The barrier-stem cell homing biomimetic microfluidic chip according to claim 3, wherein electrodes are respectively inserted into the upper chamber and the lower chamber of the barrier chip for measuring transmembrane resistance.

6. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, wherein the stem cell homing chip is separated into a lower chamber and a middle chamber by a first porous membrane, separated into a middle chamber and an upper chamber by a second porous membrane, wherein a third inlet and a third outlet are provided on the interface layer of the stem cell homing chip and communicated with the lower chamber, a fourth inlet and a fourth outlet are provided on the interface layer of the stem cell homing chip and communicated with the middle chamber, and a fifth inlet and a fifth outlet are provided on the interface layer of the stem cell homing chip and communicated with the upper chamber.

7. The barrier-stem cell homing biomimetic microfluidic chip according to claim 6, wherein a 3D nest structure formed by mixing bioactive hydrogel and acellular matrix of a mammalian organ corresponding to a target organ is embedded in a middle layer cavity of the stem cell homing chip, and a pluripotent stem cell, an embryonic stem cell, a mesenchymal stem cell or a stem cell with differentiation potential capable of inducing differentiation into the target organ is cultured in an upper layer cavity.

8. The barrier-stem cell homing biomimetic microfluidic chip according to claim 6, wherein the pore size of the first porous membrane of the stem cell homing chip is 100-500 μm; the pore diameter of the second porous membrane is 10 to 50 μm.

9. The barrier-stem cell homing biomimetic microfluidic chip according to claim 6, wherein the stem cell homing chip is connected with gold/platinum electrodes in the upper layer chamber and the middle layer chamber respectively, the upper layer chamber is connected with negative electricity, the middle layer chamber is connected with positive electricity, and the voltage is 0-2.0 v.

10. The barrier-stem cell homing biomimetic microfluidic chip according to claim 6, wherein the stem cell homing chip is provided with an upper-layer chamber observation window and a middle-layer chamber observation window on the stem cell homing chip.

11. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, wherein a multifunctional liquid supply unit is arranged at an upstream of a liquid path of the barrier chip and the stem cell homing chip, and a fluid driving system is connected to the liquid path; and a multifunctional liquid collection unit is connected with the downstream of the liquid paths of the barrier chip and the stem cell homing chip.

12. The barrier-stem cell homing biomimetic microfluidic chip according to claim 1, wherein the stem cell homing chip is a high-throughput biomimetic microfluidic chip, and a plurality of the barrier chips are connected with the same stem cell homing chip for perfusion at the same time.

13. The use of the barrier-stem cell homing bionic microfluidic chip of claim 1, wherein 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 organoid differentiation potential, and the middle chamber forms a "nest" environment by mammalian brain acellular matrix corresponding to a target organ for the blood brain barrier-brain organoid bionic microfluidic chip.

14. The use of the barrier-stem cell homing biomimetic microfluidic chip according to claim 1, wherein 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 cultures pluripotent stem cells or stem cells with lung organoid differentiation potential, and the middle chamber forms a 3D "nest" from mammalian lung acellular matrix corresponding to a target organ for the blood-gas barrier-lung organoid biomimetic microfluidic chip.

15. The use of the barrier-stem cell homing biomimetic microfluidic chip according to claim 1, wherein the upper chamber of the barrier chip cultures the renal vesicle epithelial cells, the lower chamber of the barrier chip cultures the microvascular endothelial cells, the upper chamber of the stem cell homing chip cultures the pluripotent stem cells or the stem cells with renal organoid differentiation potential, and the middle chamber forms a 3D "nest" from the mammalian renal acellular matrix corresponding to the target organ for the hematuria barrier-renal organoid biomimetic microfluidic chip.

16. The use of the barrier-stem cell homing bionic microfluidic chip of claim 1, wherein the upper chamber of the barrier chip is used for culturing intestinal epithelial cells, the lower chamber of the barrier chip is used for culturing microvascular endothelial cells, the upper chamber of the stem cell homing chip is used for culturing pluripotent stem cells or stem cells with intestinal organoid differentiation potential, and the middle chamber is used for forming 3D 'nest' by the intestinal acellular matrix of mammals corresponding to a target organ and is used for the intestinal vascular barrier-intestinal organoid bionic microfluidic chip.

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