CN113088452A - Aorta organoid chip, preparation method, chip system and application - Google Patents
Aorta organoid chip, preparation method, chip system and application Download PDFInfo
- Publication number
- CN113088452A CN113088452A CN202110450132.4A CN202110450132A CN113088452A CN 113088452 A CN113088452 A CN 113088452A CN 202110450132 A CN202110450132 A CN 202110450132A CN 113088452 A CN113088452 A CN 113088452A
- Authority
- CN
- China
- Prior art keywords
- chip
- organoid
- vacuum
- aorta
- aortic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
Abstract
The invention relates to an aorta organoid chip, a preparation method, a chip system and application, wherein the organoid chip comprises three layers of chip skeletons which are sequentially arranged from top to bottom, each layer of chip skeleton is provided with a channel, an elastic membrane is arranged between every two adjacent chip skeletons, the elastic membrane and each layer of channel form a cell culture cavity positioned in the middle and vacuum cavities positioned at two sides of the cell culture cavity, and the end part of each channel is provided with a pipeline interface for connecting with the outside. Compared with the prior art, the method has the advantages of accurately controlling the amplitude and frequency of cell stretching and the like.
Description
Technical Field
The invention relates to the technical field of biological medicines, relates to a micro-fluidic organ chip, and particularly relates to an aorta organoid chip, a preparation method, a chip system and application.
Background
In the prior art, the research on the occurrence and development mechanism of aortic lesions is mainly carried out by Two-Dimensional (2D) cell culture and animal aneurysm models. The gene modification model mouse has the defects of high lethality rate, low birth rate, difficult accurate aortic aneurysm formation rate, long experimental period, high cost and the like. Classical mouse models are widely used, but it is difficult to simulate real pathophysiological processes. In addition, species variability between animal models and humans is a gap that is difficult to overcome. Traditional in-vitro 2D cell culture models are widely applied to biological research, and the two-dimensional cell culture is carried out in a culture dish or a culture plate, so that micro environments such as human physiological stress are difficult to simulate, and complex physiological functions of human tissues and organs cannot be reflected.
The organ chip makes up for the defects of the traditional 2D cell culture and animal experiments, and establishes an in vitro model for more real, low-cost and efficient physiological research and drug development. Compared with a mouse model, the micro-fluidic organ chip combined human-derived cell disease model has unique advantages, can make up the gap caused by the huge species difference between a mouse and a human, overcomes the defects of the traditional 2D cell culture, provides a good research tool for disease mechanism research and drug screening, provides part of human body reaction basis of drug reaction for screening of drugs before clinical treatment, can reduce the dosage of experimental mice, is beneficial to animal welfare and promotes the rapid application and conversion of drugs. At present, lung chips, kidney chips, liver chips, intestinal chips, heart chips, blood vessel chips and the like are reported to be successfully constructed and used for constructing disease models and screening drugs, but at present, organ chips applied to aortic diseases are lacked. The aortic smooth muscle cells are the core link of aortic diseases and participate in various pathophysiological processes, and the study on the relevant pathogenesis of the aortic diseases by taking the aortic smooth muscle cells as experimental study objects is a hotspot of the current study; the aorta smooth muscle cells cultured in vitro are of great significance for studying physiological functions, drug reactions and pathophysiological changes under the action of various pathogenic factors.
Disclosure of Invention
The present invention aims at overcoming the defects of the prior art and providing an aorta organoid chip capable of accurately controlling the amplitude and frequency of cell stretching, a preparation method, a chip system and an application thereof.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect, the invention provides an aorta organoid chip, which comprises three layers of chip frameworks arranged from top to bottom in sequence, wherein each layer of chip framework is provided with a channel, an elastic membrane is arranged between every two adjacent chip frameworks, the elastic membrane and each layer of channels form a cell culture cavity positioned in the middle and vacuum cavities positioned at two sides of the cell culture cavity, and the end part of each channel is provided with a pipeline interface for connecting with the outside.
Further, the chip frame is made of Polydimethylsiloxane (PDMS) material.
Further, the elastic membrane is a PDMS membrane.
Further, the pipeline interface comprises a hose and a stainless steel needle tube sleeved in the hose.
In a second aspect, the present invention provides a method for preparing an aortic organoid chip as described above, comprising the steps of:
manufacturing a three-layer chip framework with a channel through a mold, and forming a through hole on the chip framework to serve as a channel inlet and outlet;
carrying out plasma surface cleaning treatment on the three-layer chip framework and the two layers of elastic films;
arranging and attaching the chip skeleton with the treated surface and the elastic film at intervals, and baking, heating and fixing;
and a pipeline interface is connected at each through hole.
Further, when the chip frame and the elastic film are subjected to bonding treatment, the edges of the channels are overlapped.
Further, the mold is made of a polymethyl methacrylate acrylic plate.
In a third aspect, the invention provides an aorta organoid chip system, which comprises the aorta organoid chip, an electromagnetic valve controller, a vacuum gas pump, a gas electromagnetic valve, a vacuum filter and a peristaltic pump, wherein a vacuum cavity of the aorta organoid chip is sequentially connected with the vacuum filter, the gas electromagnetic valve and the vacuum gas pump, the gas electromagnetic valve is connected with the electromagnetic valve controller, and a cell culture cavity of the aorta organoid chip is connected with the peristaltic pump;
cells are planted on an elastic membrane serving as the cell culture cavity, a peristaltic pump is used for replacing culture solution, and a solenoid valve controller is used for controlling the opening and closing of a gas solenoid valve and simulating physiological periodic tension applied to the cells.
Further, the vacuum gas pump comprises a pump body, a water-oil separator and a vacuum pressure regulating valve which are sequentially connected, and the vacuum pressure regulating valve is connected with the gas electromagnetic valve.
In a fourth aspect, the present invention provides a use of the aortic organoid chip system as described above in determining the effect of cyclic tension on aortic smooth muscle cell morphology, arrangement and cell phenotype.
Compared with the prior art, the invention has the following beneficial effects:
1. the aorta organoid chip of the invention has a three-layer structure, can accurately control the amplitude and the frequency of cell stretching, and can effectively simulate the physiological periodic tension on the cells of the aorta vessel wall in vivo.
2. The invention is made of dimethyl silicone polymer material, and has better gas permeability, optical performance and elasticity.
3. The invention is provided with a double-layer elastic membrane structure, and the size of the chip culture pool is increased, so that the culture area of the chip is obviously increased, and the defect that the cell amount of the microfluidic chip is not enough for complex biological experiments is overcome.
Drawings
FIG. 1 is a schematic diagram of an aortic organoid chip in accordance with an embodiment, wherein (1a) is a perspective view and (1b) is a cross-sectional view;
FIG. 2 is a schematic sectional view of an aortic organoid chip under vacuum negative pressure in an example;
FIG. 3 is a schematic view of a die used in the examples;
FIG. 4 is a simplified diagram of the fabrication of an aortic organoid chip according to an embodiment;
FIG. 5 is a schematic circuit diagram of an aorta organoid chip system provided in an embodiment;
FIG. 6 is a schematic diagram of a cross-sectional view of a chip and stretching amplitudes corresponding to different vacuum negative pressure values, wherein (6a) is a schematic diagram of a stretching strain under a previous experiment, and (6b) is a schematic diagram of stretching amplitudes under different vacuum negative pressure values;
FIG. 7 is a schematic representation of the effect of cyclic tonicity on HASMC morphology, alignment and cell phenotype;
FIG. 8 is a graph showing the effect of Mdivi-1 on the expression levels of HASMC mitochondrial fusion/division protein and the contractile phenotype SM22 and CNN1 proteins;
in the figure, 1, a solenoid valve controller, 2, a vacuum gas pump, 3, a gas solenoid valve, 4, a vacuum filter, 5, an organ chip, 6, a peristaltic pump, 501, an upper layer framework, 502, a middle layer framework, 503, a lower layer framework, 504, an elastic membrane, 505, an upper air channel, 506, a liquid channel, 507 and a lower air channel.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
Example 1
The embodiment provides an aorta organoid chip, including the three-layer chip skeleton that from top to bottom sets gradually, the passageway has been seted up to every layer of chip skeleton, is provided with the elastic membrane between adjacent chip skeleton, and the elastic membrane forms the cell culture chamber that is located the centre and is located the vacuum cavity of cell culture chamber both sides with each layer passageway, and each passageway tip is provided with the pipeline interface that is used for with external connection.
Referring to fig. 1, the aorta organoid chip of this embodiment includes an upper skeleton 501, a middle skeleton 502 and a lower skeleton 503, the upper skeleton 501 has an upper airway 505, the middle skeleton 502 has a liquid channel 506, the lower skeleton 503 has a lower airway 507, both sides of the liquid channel 506 are open, both the upper airway 505 and the lower airway 507 are open, two layers of elastic membranes 504 are disposed between the skeletons, one layer of elastic membrane and the upper airway 505 form an upper vacuum chamber, the other layer of elastic membrane and the lower airway 507 form a lower vacuum chamber, the two layers of elastic membranes and the liquid channel 506 form a cell culture chamber, the vacuum chamber is used for introducing vacuum negative pressure gas, the negative pressure gas in the two layers of chambers causes deformation of the elastic membranes, thereby simulating physiological periodic tension applied to smooth muscle cells of aortic blood vessel wall.
In this embodiment, the chip frame is made of an elastic PDMS material. The elastic membrane is a PDMS membrane. The pipeline interface comprises a hose and a stainless steel needle tube sleeved in the hose. In one embodiment, the aortic organoid chip is prepared as follows:
1. preparing PDMS glue: PDMS is a high molecular polymer material, and has good gas permeability, optical properties and elasticity. PDMS is a gel composed of solution A and solution B. The solution A is PDMS stock solution, and the solution B is PDMS solidification initiator. According to the experimental requirement, the A and B liquids are mixed according to a certain mass ratio, for example, the A: B is 5:1, 10:1, 15:1 and 20:1(W/W), the larger the ratio is, the larger the PDMS softness is, and the smaller the hardness is. Because a large amount of small bubbles appear in the PDMS gel in the stirring process, the prepared PDMS liquid is placed in a vacuum degassing tank for 0.5-1 h. The vacuum tank is maintained at a negative pressure of between-0.1 mpa and-0.8 mpa.
2. And (3) preparing an elastic film. The elastic PDMS membrane purchased from New Material science and technology, Inc. of Bauder Hangzhou is used in this embodiment, and has good elasticity, uniform thickness, convenient cutting, and the following parameters:
TABLE 1
3. Chip die: three outer frame structures with the size of 100mm multiplied by 40mm and three-layer micro-fluidic chip structures with channels with the size of 70mm multiplied by 6mm multiplied by 4mm are designed by CAD drawing software. Two pieces of outer frame structures with the size of 100mm multiplied by 40mm multiplied by 6mm are carved on a polymethyl methacrylate (PMMA) acrylic plate by a CNC carving machine tool, and three liquid channels and air channel parts with the size of 70mm multiplied by 6mm multiplied by 4mm are carved. The outer frame and the channel block carved in the above steps are attached to a flat PMMA plate by using a 3M strong double-sided adhesive tape dedicated to PMMA, and a chip mold is manufactured as shown in fig. 3.
4. Preparing and assembling a chip:
pouring the prepared PDMS glue on a PMMA chip die, and blowing small bubbles generated on the surface of the PDMS by using an ear washing ball to ensure that no bubbles exist in the PDMS glue as much as possible. The PDMS glue poured into the mould is flatly placed in an oven at 70 ℃ and heated for 2 h. The shaped PDMS block was removed from the mold, keeping the surface of the PDMS block clean during removal. And punching holes at corresponding positions by using a 1mm puncher so that the inlet and the outlet are communicated with the air channel and the liquid channel. Commercial 200 μm thick PDMS films were cut into 100mm by 40mm sizes. And (3) treating the 3 PDMS chip blocks and the 2 PDMS films prepared in the step for 5min by using a plasma cleaning instrument. From top to bottom, the air channel PDMS block-PDMS film-liquid channel PDMS block-PDMS film-air channel PDMS block, and the surface-treated PDMS block and PDMS film were attached together in the above order, as shown in fig. 4. Care was taken during the application that the edges of the three channels overlapped together to the extent possible. And (3) putting the adhered three-layer PDMS chip into an oven at 70 ℃ and heating for 1 h. A number of latex hoses having an inner diameter of 1mm and a length of 3cm were prepared. A stainless steel needle tube with the diameter of 1mm and the length of 1cm is inserted into one end of a prepared hose, and a luer connector with the size of 1.6mm is inserted into the other end of the hose to manufacture a pipeline connected with an air passage and a liquid passage interface of a chip. And inserting the prepared pipeline into the inlet and outlet of the chip air passage and the liquid passage so as to complete the complete aorta organoid chip (PDMS chip).
The embodiment designs the shape of liquid way and air flue, and wherein the liquid way includes the linear type main part and sets up in the portion of turning at linear type main part both ends, goes up the air flue and includes the linear type main part and sets up in the portion of turning of linear type main part one end, and the air flue includes the linear type main part equally and sets up in the portion of turning of linear type main part one end down, but goes up the portion of turning of air flue and the portion of turning of air flue is located different sides. When 3 PDMS chips and 2 PDMS film are adhered, the linear bodies are overlapped, and the main part of the stretched cell is the overlapped part of the liquid channel and the air channel. The liquid channel and the air channel adopt the design of turning shapes, and the advantages are that: 1. when observing cells, in order to avoid parts of the liquid channel inlet and outlet and the air channel outlet from blocking the observation visual field, the shape and biological changes of all cells are better observed; 2. the air channel outlet and the liquid channel inlet and outlet are provided with holes from the side edge of the PDMS plate, and in order to avoid the interference of the overlapping of the positions of the inlet and the outlet of the channels, a turning shape is arranged to avoid the interference between the openings of the channels.
In this example, the cell culture fluid channel length and width dimensions were increased and the bilayer doubled the cell culture area compared to the monolayer structure, resulting in a cell culture area of 7 × 0.6 × 2 — 9.8cm2, equivalent to about one well in a 6-well plate (10 cm)2) The cell culture area of the size increases the size of the chip culture pool, so that the culture area of the chip is obviously increased.
The aorta organoid chip consists of three PDMS blocks with channels and two PDMS films. The cells are cultured on the PDMS membrane in the middle, the channel in the middle of the chip is a liquid channel, and culture liquid is filled in the channel for the life activities of the cells. The upper channel and the lower channel of the chip intermittently pump air, and the air pressure difference between the upper channel and the lower channel of the chip causes the chip to generate stretching force similar to that generated on the wall of an aorta in vivo. The chip can accurately control the stretching amplitude and frequency of cells, so that the physiological periodic tension of smooth muscle cells on the wall of an aortic blood vessel is more met, the culture area of the chip is remarkably increased due to the increase of the double-layer PDMS membrane structure and the size of the chip culture pool, and the defect that the cell amount of the microfluidic chip is not enough for complex biological experiments is overcome.
The chip can be used as an organ chip for simulating the micro-physiological environment of the periodic mechanical tension of the aorta in vivo, and is applied to the research of the pathogenesis of aortic diseases and the screening of potential drugs.
Example 2
As shown in fig. 5, this embodiment provides an aorta organoid chip system, which includes the aorta organoid chip 5 of embodiment 1, a solenoid valve controller 1, a vacuum gas pump 2, a gas solenoid valve 3, a vacuum filter 4, and a peristaltic pump 6, wherein a vacuum chamber of the aorta organoid chip 5 is sequentially connected to the vacuum filter 4, the gas solenoid valve 3, and the vacuum gas pump 2, the gas solenoid valve 3 is connected to the solenoid valve controller 1, a cell culture chamber of the aorta organoid chip 5 is connected to the peristaltic pump 6, the vacuum gas pump includes a pump body, a water-oil separator, and a vacuum pressure regulating valve, which are sequentially connected, and the vacuum pressure regulating valve is connected to the gas solenoid valve. The cell is planted on an elastic membrane serving as a cell culture cavity, a peristaltic pump is used for replacing culture solution, a solenoid valve controller is used for controlling the opening and closing of a gas solenoid valve and simulating the mechanical deformation of cell contraction and relaxation, and a gas filter arranged at the outlet part of a gas source is used for avoiding the pollution of external vacuum negative pressure gas on the cell. The chip system can control the mechanical tension, rhythm and frequency of cells.
The power source of the whole system is a vacuum pump, the pumping hole of the pump is connected with the water-oil separator, and the gas is filtered to prevent the pump from being damaged. The water-oil separator is connected with a vacuum pressure regulating valve, and the pressure regulating valve is used for controlling the vacuum degree, namely controlling the size of vacuum negative pressure gas, so that the stretching degree of the elastic film is controlled. Then the chip is connected with the inlet of the electromagnetic valve, and the outlet of the electromagnetic valve is connected with the interface of the chip air passage. The electromagnetic valve is a valve which is controlled by voltage to be opened and closed. When the voltage is more than 24V, the inlet and the outlet are communicated, a low-pressure environment is caused in the air passage, and the membrane deforms; when the pressure is less than 24V, the outlet is communicated with the atmosphere, so that the pressure of the air passage is recovered to the atmospheric pressure, and the membrane is recovered to be deformed. The on-off frequency of the electromagnetic valve is controlled by a single chip microcomputer, and the single chip microcomputer with a program burned in advance controls the voltage of an output port of the single chip microcomputer according to the specified frequency. The high voltage output by the port is 5V, the low voltage is 0V, the high voltage is modulated to 24V through the modulation of the electromagnetic relay and then is connected with the electromagnetic valve, and therefore the electromagnetic valve is switched on and off according to expected frequency. The stretching amplitude can be adjusted by adjusting the opening of the pressure regulating valve, and the stretching frequency can be adjusted by changing the program of the single chip microcomputer. The culture medium is replaced or the drug is treated by a peristaltic pump.
The chip system can perform large-flux experiments, has strong repeatability, obviously improves the experimental efficiency and can reduce the experimental cost.
Example 3
In this embodiment, the aorta organoid chip system is used to perform experiments on the stretching amplitudes corresponding to different vacuum negative pressure values. Injecting 80 μ g/mL rat tail collagen (Sigma) into the prepared PDMS chip liquid channel, standing at room temperature for 0.5-1h, and extracting collagen from the liquid channel. The PDMS chip with the collagen spread thereon is placed in an oven at 60 ℃ until the collagen is dried. Placing the PDMS chip treated by the collagen in an ultraviolet disinfection cabinet, and sterilizing for 1-2 h. Placing sterilized chip into a clean bench, and digesting with 0.25% pancreatin in an incubator at 37 deg.C for 3min when the smooth muscle cells grow to above 80%. After digestion was complete, 3ml of DMEM/F12+ 10% Fetal Bovine Serum (FBS) medium was added to the dish to neutralize pancreatic enzymes, cells were repeatedly blown with a 1ml tip, and the cell suspension was collected in a 15ml centrifuge tube. Centrifuging at 1200rpm for 5min, and discarding the supernatant. 1ml of DMEM/F12+ 10% Fetal Bovine Serum (FBS) culture medium was added, and the cells were blown up and mixed well for cell counting. Cells were diluted to 2 × 105 concentration according to the number of cell counts. PBS was slowly poured into the liquid channel of the PDMS chip which had been well spread with collagen and sterilized, and the PBS was removed by suction. Diluting to 2X 105Smooth muscle cell suspension at a concentration of one/ml was slowly poured into the liquid channel. And then closing the luer joint at the inlet and the outlet of the PDMS chip. The chip was placed at 37 ℃ in 5% CO2Culturing in an incubator for 24 h. And after the cells adhere to the wall, connecting an air passage outlet of the PDMS chip to an air exhaust hole of a vacuum pump. And opening a switch of the electromagnetic valve controller. And opening the vacuum air pressure valve and adjusting the air pressure. The stretching width corresponding to the pressure measured from the cross-sectional view of the chip is shown in FIG. 6, i.e., the stretching width corresponding to 10kPa is 7.18. + -. 0.44%, and the stretching width corresponding to 15kPa is 17.28. + -. 0.91%. Control the chipSetting the stretching frequency of the system to be 1Hz and the stretching amplitude parameter to be 10kPa or 15kPa, clicking a start button, putting the chip at 37 ℃ and 5% CO2And continuously culturing in the incubator, closing the control system after stretching treatment is carried out for 24 hours, and further carrying out subsequent biological experiments.
Example 4
This example utilizes the aortic organoid chip system to study the effect of cyclic tension on morphology, arrangement and cell phenotype of primary Human Aortic Smooth Muscle Cells (HASMCs). In a static state, the cells grow without specific directionality; after the stretching treatment, the angle between the cell alignment direction and the stretching direction is close to 90 degrees, and the larger the stretching amplitude is, the closer the angle is to 90 degrees, as shown in fig. 7. Compared with the static state, the cell length is longer, the cell width is narrower, the cell length/width ratio value is obviously increased, and the larger the stretching amplitude is, the higher the cell length/width ratio value is. HASMC exhibit two distinct cellular states, contractile and secretory, under different environmental stimuli. The effect of different stretching amplitudes on the transformation of the HASMC phenotype was examined at a fixed contraction frequency (physiological heart rate) of 1 Hz. Immunofluorescent staining of the HASMC contractile phenotype markers SM22 and CNN1 revealed a significant increase in the number of SM22 positive cells and CNN1 positive cells in the stretch treated group compared to the static (0%) treated group, as shown in fig. 7. The results show that the aorta organoid chip can reproduce the real microenvironment of HASMC in human body, and HASMC shows biological characteristics similar to those in human body.
Example 5
Mitochondrial dysfunction is closely associated with the development of aortic aneurysms. In addition, the inventor found that aortic tissues of patients with bicuspid aortic disease (BAV-TAA) in which NOTCH1 is low in expression have mitochondrial dysfunction, which is associated with decreased mitochondrial fusion, as a result of previous studies on the team.
This example uses the aortic organoid chip to examine whether mitochondrial fission inhibitor (Mdivi-1) can restore the HSAMC mitochondrial fusion/fission balance and increase the contractile phenotype in patients with congenital bilobal aortic valve malformation thoracic aortic aneurysm (BAV-TAA). The primary HASMC extracted from aorta organoid chip and aorta tissue of three different BAV-TAA patients is used to screen the related drug of mitochondria. As a result, the medicine increases the expression of the mitochondrial fusion protein to different degrees, reduces the expression of the mitochondrial fission protein, and further leads the mitochondrial fusion-fission to approach the equilibrium state. At the same time, the drug increased protein expression levels of the HASMC contractile phenotype proteins SM22 and CNNN1 to varying degrees, as shown in FIG. 8.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. The utility model provides an aorta organoid chip which characterized in that, is including the three-layer chip skeleton that from top to bottom sets gradually, every layer the passageway has been seted up to the chip skeleton, is provided with the elasticity membrane between adjacent chip skeleton, the elasticity membrane forms the cell culture chamber that is located the centre with each layer passageway and is located the vacuum cavity of cell culture chamber both sides, each the passageway tip is provided with and is used for the pipeline interface with external connection.
2. The aortic organoid chip of claim 1, wherein the chip backbone is made of PDMS material.
3. The aortic organoid chip of claim 1, wherein the elastic membrane is a PDMS membrane.
4. The aortic organoid chip of claim 1 wherein the conduit interface comprises a hose and a stainless steel needle cannula disposed within the hose.
5. A method of preparing the aortic organoid chip of claim 1, comprising the steps of:
manufacturing a three-layer chip framework with a channel through a mold, and forming a through hole on the chip framework to serve as a channel inlet and outlet;
carrying out plasma surface cleaning treatment on the three-layer chip framework and the two layers of elastic films;
arranging and attaching the chip skeleton with the treated surface and the elastic film at intervals, and baking, heating and fixing;
and a pipeline interface is connected at each through hole.
6. The method of claim 5, wherein the edges of the channels overlap when the skeleton and the elastic membrane are bonded.
7. The method of claim 5, wherein said mold is made of polymethylmethacrylate acrylic plate.
8. An aorta organoid chip system, comprising the aorta organoid chip of claim 1, and a solenoid valve controller, a vacuum gas pump, a gas solenoid valve, a vacuum filter, and a peristaltic pump, wherein a vacuum chamber of the aorta organoid chip is sequentially connected to the vacuum filter, the gas solenoid valve, and the vacuum gas pump, the gas solenoid valve is connected to the solenoid valve controller, and a cell culture chamber of the aorta organoid chip is connected to the peristaltic pump;
cells are planted on an elastic membrane serving as the cell culture cavity, a peristaltic pump is used for replacing culture solution, and a solenoid valve controller is used for controlling the opening and closing of a gas solenoid valve and simulating physiological periodic tension applied to the cells.
9. The aortic organoid chip system of claim 8, wherein the vacuum gas pump comprises a pump body, a water-oil separator and a vacuum pressure regulating valve connected in sequence, and the vacuum pressure regulating valve is connected to a gas solenoid valve.
10. Use of the aortic organoid chip system of claim 8 for determining the effect of cyclic tone on aortic smooth muscle cell morphology, arrangement and cell phenotype.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110450132.4A CN113088452A (en) | 2021-04-25 | 2021-04-25 | Aorta organoid chip, preparation method, chip system and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110450132.4A CN113088452A (en) | 2021-04-25 | 2021-04-25 | Aorta organoid chip, preparation method, chip system and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113088452A true CN113088452A (en) | 2021-07-09 |
Family
ID=76679824
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110450132.4A Pending CN113088452A (en) | 2021-04-25 | 2021-04-25 | Aorta organoid chip, preparation method, chip system and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113088452A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013086502A1 (en) * | 2011-12-09 | 2013-06-13 | President And Fellows Of Harvard College | Organ chips and uses thereof |
| CN103215185A (en) * | 2013-03-29 | 2013-07-24 | 国家纳米科学中心 | Micro-fluid control device capable of simultaneously exerting mechanical stimulation and chemical stimulation |
| CN103502426A (en) * | 2011-02-28 | 2014-01-08 | 哈佛大学校长及研究员协会 | Cell culture system |
| CN107312713A (en) * | 2017-07-28 | 2017-11-03 | 中科芯瑞(苏州)生物科技有限公司 | A kind of micro-fluidic chip and its application |
| WO2019222870A1 (en) * | 2018-05-21 | 2019-11-28 | 深圳华大生命科学研究院 | Bionic intestinal organ-on-a-chip, and preparation method therefor and application thereof |
| CN110692599A (en) * | 2019-11-21 | 2020-01-17 | 复旦大学附属中山医院 | Preparation method of thoraco-abdominal aortic aneurysm mouse model |
-
2021
- 2021-04-25 CN CN202110450132.4A patent/CN113088452A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103502426A (en) * | 2011-02-28 | 2014-01-08 | 哈佛大学校长及研究员协会 | Cell culture system |
| WO2013086502A1 (en) * | 2011-12-09 | 2013-06-13 | President And Fellows Of Harvard College | Organ chips and uses thereof |
| CN103215185A (en) * | 2013-03-29 | 2013-07-24 | 国家纳米科学中心 | Micro-fluid control device capable of simultaneously exerting mechanical stimulation and chemical stimulation |
| CN107312713A (en) * | 2017-07-28 | 2017-11-03 | 中科芯瑞(苏州)生物科技有限公司 | A kind of micro-fluidic chip and its application |
| WO2019222870A1 (en) * | 2018-05-21 | 2019-11-28 | 深圳华大生命科学研究院 | Bionic intestinal organ-on-a-chip, and preparation method therefor and application thereof |
| CN110692599A (en) * | 2019-11-21 | 2020-01-17 | 复旦大学附属中山医院 | Preparation method of thoraco-abdominal aortic aneurysm mouse model |
Non-Patent Citations (3)
| Title |
|---|
| MIERADILIJIANG ABUDUPATAER ET AL.: "Aorta smooth muscle-on-a-chip reveals impaired mitochondrial dynamics as a therapeutic target for aortic aneurysm in bicuspid aortic valve disease", 《ELIFE》 * |
| NAN CHEN ET AL.: "Engineering a Human Pluripotent Stem Cell-Based in vitro Microphysiological System for Studying the Metformin Response in Aortic Smooth Muscle Cells", 《FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY》 * |
| 张良 等: "基因芯片技术筛选主动脉瘤与正常主动脉差异表达基因", 《基础医学与临床》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7200328B2 (en) | Organ-mimetic device with microchannels and methods of use and manufacture thereof | |
| AU2020202721B9 (en) | Open-top microfluidic device with structural anchors | |
| Ostrovidov et al. | Membrane-based PDMS microbioreactor for perfused 3D primary rat hepatocyte cultures | |
| Jiang et al. | Microfluidic-based biomimetic models for life science research | |
| Sun et al. | Tailoring biomaterials for biomimetic organs-on-chips | |
| CN107629997A (en) | External foam wanshing model and its application in atherosclerosis study | |
| CN113088452A (en) | Aorta organoid chip, preparation method, chip system and application | |
| CN115353951A (en) | Total aorta organoid chip model constructed based on induced pluripotent stem cells and application thereof | |
| Dermenoudis et al. | Bioreactors in tissue engineering | |
| EP3138904A1 (en) | Device and method for tissue culture comprising a hermetically sealed blood circulation | |
| WO2022104626A1 (en) | Micro-fluidic technology-based multifunctional organ chip, preparation method therefor and use thereof | |
| CN107988147A (en) | Directed differentiation based on organ chip and induced multi-potent stem cell is used for the method that 3D intends epidermis structure | |
| CN116445282B (en) | Microfluidic system and application thereof in constructing bionic organ microenvironment | |
| CN109699180A (en) | Cell co-cultures chip and preparation method thereof | |
| CN117106585A (en) | Micro-fluidic chip containing cells and preparation method and application thereof | |
| CN116731857A (en) | Organ chip for air pressure stimulation movement and cell stretching method | |
| CN114149923A (en) | Construction method of single-channel microchip model for endothelial cell-smooth muscle cell co-culture | |
| CN117701380A (en) | Microfluidic device for constructing in-vitro biological model and application thereof | |
| CN117899953A (en) | Intracranial aneurysm microfluidic vascular chip, system, preparation method and application | |
| CN111545258A (en) | Micro-fluidic chip capable of providing compression deformation and preparation method and application thereof | |
| CN108949524A (en) | The basket structure micropore culture dish and preparation method thereof formed for cell in vitro three-dimensional micro-assembly robot | |
| Hecker | Development of Functional Bioengineered Muscle Models and a Novel Micro-Perfusion System. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |