CN117025359A - Mechanically stretchable annular organ chip and method - Google Patents
Mechanically stretchable annular organ chip and method Download PDFInfo
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- CN117025359A CN117025359A CN202311072147.7A CN202311072147A CN117025359A CN 117025359 A CN117025359 A CN 117025359A CN 202311072147 A CN202311072147 A CN 202311072147A CN 117025359 A CN117025359 A CN 117025359A
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Classifications
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- 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
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
-
- 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
- B01L3/502707—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 characterised by the manufacture of the container or its components
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- 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
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- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/10—Perfusion
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- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/14—Means for pressure control
Abstract
The application belongs to the technical field of microfluidic chips, and particularly discloses a mechanically stretchable annular organ chip and a mechanically stretchable annular organ method, wherein the mechanically stretchable annular organ chip comprises a film layer, a flow channel layer and a bottom layer which are arranged on two sides of the film layer, a culture cavity is arranged on the flow channel layer, the culture cavity is of an end-to-end groove structure, and the culture cavity is connected with a liquid storage bin through a cell channel; the runner layer and the film layer are provided with communicated air holes, and the bottom layer is provided with an air cavity communicated with the air holes; the air cavity is of a groove structure, and the projection of the edge of the air cavity on the flow channel layer is positioned between the inner edge and the outer edge of the culture cavity; the application provides a mechanically stretchable annular organ chip and a method for forming an experiment group and a control group in the same chip, wherein in the control chip and the experiment group chip, the influence of uneven mixing of cell suspension on an experiment result is avoided; the cells can be uniformly stretched, and the growth trend of the cells can be conveniently observed.
Description
Technical Field
The application belongs to the technical field of microfluidic chips, and particularly relates to a mechanically stretchable annular organ chip and a mechanically stretchable annular organ method.
Background
The microfluidic chip uses the chip as an operation platform and life science as a main application object at present. Organ chips are one of the major applications of microfluidics, and microfluidic cell culture systems are manufactured by micromachining processes, aimed at reproducing physiological and pathological features of organs in vivo by constructing in vitro models.
Although the organ-chip is more difficult to handle at some levels than other culture methods, its advantages are significant. The 2D model cannot accurately describe and simulate abundant environment and complex processes observed in vivo, the animal model and the human body are easy to have larger deviation due to different species, so that the test medicine is invalid, and the organ chip can reach the tissue and organ function level which cannot be reached by the conventional 2D or conventional 3D culture system, so that more accurate test data can be obtained.
However, a key problem faced by current organ-chips is how to build an in vitro microenvironment close to that in vivo for cell growth. The behavior of cells in physiological states is affected by a number of physical stimulus signals, such as electrical stimulus signals, fluid shear forces, mechanical stretching, etc., which are important influencing factors for the cells to maintain physiological functions or to initiate lesions, and it is far from sufficient to simply grow cells in organ-chips. Some cells, such as heart cells, muscle cells and the like, are naturally in a moving growth environment, whether the cells depend on such an environment for growth or not, whether the proliferation growth of the cells is promoted after the movement stimulation or not, and how the effect is brought to the body of people cannot be known.
At present, the stimulation of cells in an organ chip mainly comprises gas pressure stimulation, electric stimulation and the like. Adri' anL (pez-Canosa 1 et al) combines electrospun nanofibers with electrical stimulation in a micromachining system to control the degree of anisotropy of cardiac tissue within a microdevice; annaMarsano et al designed a heart chip, built a pneumatically actuated system of uniaxial cyclic strain, and predicted signs of hypertrophic changes in cardiac phenotype by deforming PDMS by pressurizing the bottom compartment.
However, the existing chip has some disadvantages, such as the possibility of uneven mixing of the control chip and the experimental chip during the injection of the cell suspension, which leads to obvious deviation of the cell number; the mechanical stretching of the attached cells on the membrane in the straight culture chamber is uneven, and the growth trend is difficult to observe because of different stretching directions of each cell; the external injection system is difficult to fix on the air hole of the organ chip, so that the organ chip is turned over, the needle head slides out and the like.
Disclosure of Invention
The application aims to provide a mechanically stretchable annular organ chip and a mechanically stretchable annular organ method for forming an experiment group and a control group in the same chip, wherein in the control chip and the experiment group chip, the influence of uneven cell suspension mixing on an experiment result is avoided; the cells can be uniformly stretched, and the growth trend of the cells can be conveniently observed.
Based on the above purpose, the application adopts the following technical scheme:
the mechanically stretchable annular organ chip comprises a film layer, a flow channel layer and a bottom layer, wherein the flow channel layer and the bottom layer are arranged on two sides of the film layer, a culture cavity is arranged on the flow channel layer, the culture cavity is of a groove structure connected end to end, and the culture cavity is connected with a liquid storage bin through a cell channel; the runner layer and the film layer are provided with communicated air holes, and the bottom layer is provided with an air cavity communicated with the air holes; the air cavity is of a groove structure, and the projection of the edge of the air cavity on the runner layer is positioned between the inner edge and the outer edge of the culture cavity.
Further, the runner layer, the film layer and the bottom layer are relatively fixed, the runner layer and the film layer are sealed by bonding/pasting, and the film layer and the bottom layer are sealed by bonding/pasting.
Further, the air hole is communicated with the center of the air cavity.
Further, the culture cavity is of a circular groove structure, the air cavity is of a circular groove structure, and the air hole is of a circular hole structure. The diameter of the air cavity is larger than the inner diameter of the culture cavity and smaller than the outer diameter of the culture cavity.
Further, the film layer is a biocompatible transparent impermeable film.
Further, the thickness of the runner layer is 4-5 mm, the thickness of the bottom layer is 2-3 mm, and the thickness of the film layer is 100-300 mu m.
Further, the diameters of the through holes and the air holes are 1-1.5 mm, and the diameters of the air cavities are 1.5-2.5 mm.
Further, the cell channel comprises a pair of culture channels communicated with the culture cavity, and further comprises two through holes formed in the flow channel layer and communicated with the flow channel, a coiled pipe is connected between each culture channel and each through hole, and the coiled pipe is of a groove structure; one end of each through hole far away from the coiled pipe is connected with a liquid storage bin.
Further, the length of the coiled pipe is 4-5 mm, the length of the culture channel is 10-12 mm, and the width of the culture channel is 0.5mm.
A cell stretching method using the mechanically stretchable annular organ chip described above, comprising the steps of:
step 1, injecting a collagen modified cell channel from one through hole until collagen overflows from the other through hole after passing through a coiled pipe, a runner and a culture cavity, and then placing an organ chip into an incubator.
Step 2, injecting the prepared cell suspension from the through hole, then placing the organ chip into an incubator for culturing for 6-12 hours, and taking out the organ chip after the cells grow in close contact with the film layer so as to prevent the injected culture solution from flushing the cells; adding culture solution from the through hole, and placing the organ chip into an incubator.
And 3, communicating the injection pump with the air hole, starting the injection pump to perform continuous pumping, performing periodic stretching stimulation on cells on the film layer, and observing the result.
Further, in step 3, cells corresponding to the region within the air cavity and cells corresponding to the region outside the air cavity are classified into an experimental group and a control group when the result is observed.
Further, in step 2, a cell suspension is prepared using endothelial cells of human umbilical vein, the concentration of endothelial cells in the cell suspension being 0.8X10 6 ～1.5×10 6 /ml。
Further, in step 2, the volume of the culture solution added into the two liquid storage bins is 6:1.
Further, in step 3, the syringe pump is connected to the air hole through a metal tube needle.
Compared with the prior art, the application has the following beneficial effects:
1. the size of the air cavity is larger than the inner edge of the culture cavity and smaller than the outer edge of the culture cavity, namely the air cavity divides the culture cavity into an inner ring and an outer ring. Cells of the inner ring are attached to the film and are positioned above the air cavity, and the cells of the area can be stretched under the influence of the air cavity; the cells of the outer ring are attached to the membrane and bonded with the bottom layer outside the air cavity, so that the effect of mechanical stimulation on the growth of the cells can be expressed in the same chip without being influenced by the air cavity, and a control group is directly formed in the chip.
2. Some annular air cavities are provided with perfusion ports from the side ways to perfuse the annular air cavities, so that the cells above the air cavities cannot be stimulated uniformly. According to the application, the air is injected from the center of the air cavity, so that the cells in the culture cavity are mechanically stimulated, and the stimulation is more uniform.
3. The growth direction of the cells affected by the mechanical stimulation is consistent with the stretching direction, the growth direction of the cells can be affected by the stimulation, the cells in the culture cavity are mechanically stimulated from the air cavity, the growth direction of the cells can be in a certain radial direction, and the air cavity is circular, so that the subsequent observation is easier.
4. The injection pump is used for injecting and sucking gas through the metal pipe needle head, and the culture cavity is more uniform than other rectangular runner structures in force from the middle stimulation, so that uniform pressure stimulation can be realized on cells of the upper runner layer, and effective continuous movement of the cells is realized. Because the metal tube needle is positioned in the middle of the chip, and the thicker thickness of the flow channel layer on the chip can provide support, the metal tube needle is not easy to fall off from the chip.
The application provides a mechanically stretchable annular organ chip which is used for stimulating cells under the condition of not directly contacting the cells, and has the advantages of simple structure, convenient operation, easy chip manufacture and good biocompatibility.
Drawings
FIG. 1 is a schematic diagram of embodiment 1 of the present application;
FIG. 2 is a schematic view showing the internal structure of embodiment 1 of the present application;
FIG. 3 is an exploded view of example 1 of the present application;
FIG. 4 is a schematic view of the lower surface of the runner layer of embodiment 1 of the present application;
FIG. 5 is a schematic diagram of the perfusion of the culture solution according to the embodiment 2 of the present application;
FIG. 6 is a schematic view of a needle cannula according to embodiment 2 of the present application;
FIG. 7 is a schematic illustration of the connection of a syringe and a syringe pump according to embodiment 2 of the present application;
FIG. 8 is a schematic diagram of cells before pneumatic stimulation according to example 3 of the present application;
FIG. 9 is a schematic diagram of cells after pneumatic stimulation according to example 3 of the present application;
FIG. 10 is a schematic diagram of embodiment 5 of the present application;
FIG. 11 is an exploded view of example 5 of the present application;
FIG. 12 is a schematic diagram of embodiment 6 of the present application;
FIG. 13 is an exploded view of example 6 of the present application;
FIG. 14 is a schematic view of embodiment 7 of the present application;
fig. 15 is an exploded view of example 7 of the present application.
In the figure: the liquid storage device comprises a runner layer 1, a film layer 2, a bottom layer 3, a liquid storage bin 11, a culture cavity 12, a coiled pipe 13, a runner 14, an air cavity 31, a first through hole 111, a second through hole 112, an air hole 113, a third through hole 121 and a fourth through hole 122.
Detailed Description
Example 1
1-4, a mechanically stretchable annular organ chip comprises a middle film layer 2, a runner layer 1 and a bottom layer 3 which are arranged on the upper side and the lower side of the film layer 2, wherein the runner layer 1, the film layer 2 and the bottom layer 3 are horizontally arranged and relatively fixed, and the runner layer 1 and the film layer 2 and the bottom layer 3 are connected through bonding or a PDMS glue method; the material of the film layer 2 is PDMS film.
The lower surface of the runner layer 1 is provided with a culture cavity 12, the culture cavity 12 is of a circular groove structure, and the culture cavity 12 is connected with a liquid storage bin 11 through a cell channel; the flow channel layer 1 and the film layer 2 are provided with communicated air holes 113, the air holes 113 are of a circular hole-shaped structure, the upper surface of the bottom layer 3 is provided with an air cavity 31 communicated with the air holes 113, and the air cavity 31 is of a circular groove structure; culture chamber 12, air hole 113 and air chamber 31 are coaxially arranged; the diameter of the air chamber 31 is larger than the inner diameter of the culture chamber 12 and smaller than the outer diameter of the culture chamber 12.
As shown in fig. 4, the cell channel comprises a pair of culture channels communicated with the culture cavity 12, and further comprises two through holes communicated with the flow channel 14, which are formed on the flow channel layer 1, wherein the through holes are of a stepped hole structure with the lower diameter larger than the upper diameter, the upper parts of the two through holes are respectively provided with a first through hole 111 and a second through hole 112, and the lower parts of the two through holes are respectively provided with a third through hole 121 and a fourth through hole 122; the through holes and the air holes 113 are vertically arranged; a coiled pipe 13 is connected between each culture channel and each through hole, the coiled pipe 13 is of a coiled groove structure, and the coiled pipe 13 and the culture channels are all arranged on the lower surface of the runner layer 1; one end of each through hole far away from the coiled pipe 13 is connected with a liquid storage bin 11, the liquid storage bin 11 is arranged on the upper surface of the runner layer 1, and the liquid storage bin 11 is a glass pipe coaxial with the through hole. The depth of the culture channel was 100. Mu.m, the depth of the air cavity 31 was 100. Mu.m, the diameter of the glass tube was 8mm, and the height was 15mm.
Example 2
A cell stretching method using the mechanically stretchable annular organ chip of example 1, comprising the steps of:
step 1, cells (human umbilical vein endothelial cells HUVEC as the experimentFor example), the culture state should reach a confluence rate of about 70% -80%, cells are changed from an adherent state to a floating state by pancreatin, and are stopped by a solution containing 20% of high sugar, centrifuged, and the supernatant is extracted and added into a culture solution to prepare the cell density of 1X 10 6 Cell suspension per ml.
An organ chip not connected with a syringe pump is selected for sterilization, 10 mu l of collagen is injected into a cell channel of the sterilized organ chip from one through hole until collagen overflows from the other through hole after passing through the coil 13, the runner 14 and the culture cavity 12, and then the organ chip is placed into a cell incubator with the temperature of 37 ℃ and the carbon dioxide concentration of 5% for 10min.
And 2, injecting the prepared cell suspension into the cell channel from a through hole, wherein the cell suspension can be uniformly distributed in the upper flow channel layer 1, and no fluorescence reaction in the air cavity proves that no cell suspension leaks. Placing the organ chip into an incubator for culturing for 6 hours, observing that cells grow closely to the film layer 2, and then taking out the organ chip to prevent the cells from being washed away by the injected culture solution; injecting 1200 mu l and 200 mu l of EGM-2 culture solution into the two glass tube liquid storage bins 11 respectively, and placing the organ chip into an incubator; the flow of the culture medium through the cell channels is shown in FIG. 5.
Step 3, as shown in fig. 6-7, placing a metal tube needle into the air hole 113, placing a 2ml needle tube on the injection pump for clamping, starting the injection pump for continuous drawing and pushing, setting the flow to be 500 μl, the unidirectional feeding time to be 6s, continuously operating in a continuous mode, drawing and pushing in the operation direction, periodically drawing and stimulating cells on the film layer 2, and observing the result; during observation, the organ chip can be taken out from the incubator and photographed under a microscope.
The injection pump pumps and pushes air to the air cavity 31 through the needle tube, the needle head and the air hole 113, so that the air pressure in the air cavity 31 is changed, the film layer 2 above the air cavity 31 is driven to deform, and cells on the film layer 2 above the air cavity 31 are stretched; the air hole 113 is inflated from the center of the air cavity 31, so that the film layer 2 corresponding to the air cavity 31 is uniformly deformed, and cells are uniformly stimulated. In the observation, the cells on the thin film layer 2 corresponding to the region in the air cavity 31 were used as the experimental group, and the cells on the thin film layer 2 corresponding to the region outside the air cavity 31 outside the outer ring of the culture cavity were used as the control group.
Step 4, changing liquid in the glass tube liquid storage bin 11 in the organ chip every other day, wherein the specific operation is as follows: the original culture solution was aspirated by using a glass pipette, and then a new culture solution was injected, and the injection amounts of the culture solutions in the two glass tubes were alternately changed. If 1.2ml of culture solution is added to the left liquid storage bin 11 on the first day and 0.2ml of culture solution is added to the right liquid storage bin 11, 1.2ml of culture solution is added to the right liquid storage bin 11 on the next day and 0.2ml of culture solution is added to the left liquid storage bin 11.
Example 3
Other portions of this embodiment are the same as embodiment 2 except that: the concentration of the injected cell suspension was 1.5X10 6 And each ml. After 16 hours of cell adhesion, the syringe pump was started to perform continuous pumping, and the flow rate was set at 400. Mu.l.
As shown in FIGS. 8-9, it is evident that the number of cells increases after stretching for 9 hours, the cells aggregate and grow toward the middle ring (outline of bottom air cavity 31), and the cell growth direction on the middle ring is mostly along the diameter direction, and the cell growth direction on the outer side is irregular.
Example 4
A method for preparing a mechanically stretchable annular organ chip, comprising the steps of:
step 1, manufacturing and selecting a film layer 2; the film layer 2 is preferably a PDMS film with the thickness of 50 μm, specifically, PDMS prepolymer and curing agent are uniformly mixed in a ratio of 10:1, vacuumized and poured on a cover of a culture dish, and spin-coated and dried to obtain the PDMS film.
Step 2, manufacturing an integral structure of the organ chip; the structure of the designed runner layer 1 and the bottom layer 3 is drawn into a drawing by using AutoCAD software, the drawing is processed into a mask, a silicon wafer with a micro-channel structure (the depth of a selected culture channel is 100 mu m) is manufactured by a soft photoetching method, and then the silicon wafer is stuck in a culture dish by using double-sided adhesive to manufacture a mould. And uniformly mixing Polydimethylsiloxane (PDMS) prepolymer and a curing agent in a ratio of 10:1, pouring into a mold, and performing operations such as vacuumizing, bubble blowing, drying, stripping and the like to obtain a PDMS sheet with a micro-channel structure, thereby obtaining a runner layer 1 and a bottom layer 3. Then, punching is performed at the position of the through hole of the runner layer 1, bonding is performed with the PDMS film (or bonding is performed by using PDMS glue), after bonding (bonding), punching is performed on the air hole 113 from one side of the film, and then the bottom layer 3 is bonded with the film to obtain the chip (two through holes penetrate through the upper runner layer 1, and the air hole 113 penetrates through the upper runner layer 1 and the film layer 2). Finally, dipping a small amount of PDMS (polydimethylsiloxane) by using a glass tube, adhering the PDMS to the two through holes, putting the chip into a drying box for drying for 2 hours at 65 ℃ to obtain the organ chip with the glass tube liquid storage bin 11. The chip is sterilized at high temperature and ultraviolet rays in advance before the experiment, and is put into a clean bench for preservation.
Step 3, a mechanical stretching system connected with the injection pump system; the organ chip manufactured is placed at the bottom of a culture dish and is connected with a syringe pump system through a metal tube needle head and a latex tube. When in use, the metal tube needle head adhered to one side of the latex tube is inserted from the air hole 113 on the upper surface of the runner layer 1 to the air cavity 31, and the upper runner layer 1 is thicker, and the retaining position of the metal tube needle head is positioned at the center of the chip, so that the metal tube needle head is supported and stands in the chip, and the other side of the latex tube is connected with the 2ml needle tube.
Example 5
Other portions of this embodiment are the same as embodiment 1 except that: as shown in fig. 10-11, the runner layer 1 is connected with the bottom layer 3 without the film layer 2; the diameter of the air chamber 31 is slightly smaller than the inner diameter of the culture chamber 12.
The organ chip of the embodiment is used for observing the growth form of cells under negative pressure, when the organ chip is used, the needle head of a 2ml needle tube is connected with the air hole 113, the needle tube is used for extracting the gas in the cavity, the negative pressure is applied in the air cavity, the cells react to the micro-change of the environment, the culture cavity is stretched by the negative pressure, the deformation towards the middle is generated, and the cell form in the culture cavity is changed; the negative pressure time of each time is 6-8 hours, and the organ chip can be taken out from the incubator and photographed under a microscope during observation.
The specific operation of determining the existing negative pressure state of the cavity is rebound of the piston in the needle tube after the hand is released. Therefore, after the gas in the cavity is extracted, a buckle is needed to be arranged in the piston and the pipe wall of the needle pipe, so that the piston is prevented from rebounding.
Example 6
Other portions of this embodiment are the same as embodiment 1 except that: as shown in fig. 12 to 13, the reservoir 11 is not provided, and a through hole is used as the reservoir 11. When the chip is prepared, a 2mm puncher is used for punching the through holes, and the formed through holes serve as the own liquid storage bin 11 for supplying culture solution to cells in the flow channel 14.
Example 7
Other portions of this embodiment are the same as embodiment 1 except that: as shown in fig. 14-15, the serpentine tube is modified to other configurations of channels, such as straight channels, split channels, etc.
Example 8
Other portions of this embodiment are the same as embodiment 1 except that: the film layer 2 is directly formed by other non-porous films with biological compatibility, such as PC film, PET film and the like.
Claims (10)
1. The mechanically stretchable annular organ chip comprises a film layer, a flow channel layer and a bottom layer which are arranged on two sides of the film layer, and is characterized in that a culture cavity is arranged on the flow channel layer, the culture cavity is of a groove structure with a head connected with a tail, and the culture cavity is connected with a liquid storage bin through a cell channel; the runner layer and the film layer are provided with communicated air holes, and the bottom layer is provided with an air cavity communicated with the air holes; the air cavity is of a groove structure, and the projection of the edge of the air cavity on the runner layer is located between the inner edge and the outer edge of the culture cavity.
2. The mechanically stretchable annular organ-chip of claim 1, wherein the air hole communicates with a central location of the air cavity.
3. The mechanically stretchable annular organ chip according to claim 1, wherein the culture chamber is a circular groove structure, the air chamber is a circular groove structure, and the air holes are circular hole structures.
4. The mechanically stretchable annular organ chip according to claim 1, wherein the membrane layer is a biocompatible transparent impermeable membrane.
5. The mechanically stretchable annular organ chip according to claim 1, wherein the thickness of the flow channel layer is 4 to 5mm, the thickness of the bottom layer is 2 to 3mm, and the thickness of the thin film layer is 100 μm to 300 μm.
6. The mechanically stretchable annular organ chip according to any one of claims 1-5, wherein the cell channel comprises a pair of culture channels communicating with the culture chamber, and further comprises two through holes communicating with the flow channel formed in the flow channel layer, wherein a serpentine tube is connected between each culture channel and each through hole, and the serpentine tube has a groove structure; and one end of each through hole, which is far away from the coiled pipe, is connected with a liquid storage bin.
7. A cell stretching method using the mechanically stretchable annular organ chip of any one of claims 1-6, comprising the steps of:
step 1, injecting a collagen modified cell channel from one through hole until collagen overflows from the other through hole after passing through a coiled pipe, a runner and a culture cavity, and then placing an organ chip into an incubator;
step 2, injecting the prepared cell suspension from the through hole, then placing the organ chip into an incubator for culturing, taking out the organ chip after the cells grow closely to the film layer, adding a culture solution into a liquid storage bin, and placing the organ chip into the incubator;
and 3, communicating the injection pump with the air hole, starting the injection pump to perform continuous pumping, performing periodic stretching stimulation on cells on the film layer, and observing the result.
8. The method of stretching cells according to claim 7, wherein in step 3, cells corresponding to the region within the air cavity and cells corresponding to the region outside the air cavity are classified into an experimental group and a control group when the result is observed.
9. The cell stretching method of claim 7, wherein in step 2, the cell suspension is prepared using human umbilical vein endothelial cells.
10. The cell stretching method according to claim 7, wherein in the step 2, the culture solution is added to the two reservoirs in a volume of 6:1.
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| CN202311072147.7A CN117025359A (en) | 2023-08-24 | 2023-08-24 | Mechanically stretchable annular organ chip and method |
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