CN116179351A - Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method - Google Patents

Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method Download PDF

Info

Publication number
CN116179351A
CN116179351A CN202310293352.XA CN202310293352A CN116179351A CN 116179351 A CN116179351 A CN 116179351A CN 202310293352 A CN202310293352 A CN 202310293352A CN 116179351 A CN116179351 A CN 116179351A
Authority
CN
China
Prior art keywords
cell
microfluidic
depth
channel
length
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
Application number
CN202310293352.XA
Other languages
Chinese (zh)
Inventor
刘佛傍
杨舒淇
杨旭
何炽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xian Jiaotong University
Original Assignee
Xian Jiaotong University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Xian Jiaotong University filed Critical Xian Jiaotong University
Priority to CN202310293352.XA priority Critical patent/CN116179351A/en
Publication of CN116179351A publication Critical patent/CN116179351A/en
Pending legal-status Critical Current

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
    • 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
    • C12M23/44—Multiple separable units; Modules
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
  • Biomedical Technology (AREA)
  • Clinical Laboratory Science (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dispersion Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The invention relates to the technical field of single-cell analysis, in particular to a microfluidic single-cell chip and a gas-liquid exposure particle toxicity analysis method. The method adopts the parallel trap-chamber unit to improve the trap-chamber unit which can be accommodated in unit area, and ensures that the chip has high enough cell trapping capacity; and ensure that cells are still immersed in the culture solution in the gas-liquid exposure process, thereby meeting the application of toxicity analysis of the particles.

Description

Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method
Technical Field
The invention relates to the technical field of single-cell analysis, in particular to a microfluidic single-cell chip and a gas-liquid exposure particle toxicity analysis method.
Background
In vitro cell experiments are an important means of studying atmospheric particulate toxicity. In the traditional in vitro cell experiment means, the average response value of cell population induced by using porous plate cell culture to detect the atmospheric particulates is used for evaluating the toxicity of the atmospheric particulates, and single cell response value cannot be analyzed. However, cellular heterogeneity is a ubiquitous biological phenomenon, and single or several cells that vary most significantly often reveal important biological effects. Thus, studying the heterogeneity of atmospheric particulate-induced single cells at multiple levels, such as genotype and phenotype, is critical to understanding the toxicity and biological effects of atmospheric particulates.
Flow cytometry can analyze single cell signals but cannot provide a dynamic response for a particular single cell. The development of microfluidic technology provides a good technical means for capturing single cells and tracking the time dynamic response of single cells. Microfluidic technology is a technology for manipulating and controlling microfluidics in microscale channels, and has the advantages of microscale, high efficiency, high throughput, and automation. At the same time, micromachining techniques provide a method of fabricating microstructures similar to single cell sizes, providing the potential for accurate capture and identification of single cells.
Patent CN105441307a describes a single cell capture chip comprising a fluidic layer, an elastic membrane layer and a driving structure, which by presetting cell cavities, achieves a high efficiency of single cell capture and the captured cells are not easily detached from the capture sites. However, the chip needs to be provided with an elastic film and a driving structure, and the micromachining process is complex and has high cost. Patent CN110004043B describes a single-cell capturing microfluidic chip, which has a single layer, simple processing technology, and can realize uniform sample injection of cell carrier fluid by arranging reasonable structures such as a dispersion column, a buffer column, a capturing trap and the like. The cells can be firmly in the trap under the condition of fluid shearing force, and are not easy to flow out. But the device has a limited capture unit per unit area of chip that can accommodate, resulting in limited cell capture throughput. The relevant literature describes a single cell capture array chip device (Environmental Science & Technology,2020,54,13121-13130) having a plurality of cells containing capture wells and chambers, each cell consisting of a front capture well and a rear chamber. The chip can capture cells at the position of the capture trap by utilizing the hydrodynamics principle, and can fix the cells in the rear chamber by applying a certain pressure at the inlet, so that the cells are not lost when the cells are stimulated by using the particle solution later. But during the pushing of the cells from the trap into the chamber, the cells tend to escape from the flow conduit behind the chamber, resulting in a lower efficiency of single cell capture by the chip device.
In addition, in the prior art, no toxicity analysis method of gas-liquid exposure particles is involved, the single-cell capture microstructure design of the method cannot ensure that cells are still immersed in a cell culture solution in the gas-liquid exposure process, and the technical limitation easily causes larger deviation of toxicity analysis results.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a microfluidic single-cell chip and a gas-liquid exposure particle toxicity analysis method.
The invention is realized by the following technical scheme:
the microfluidic single-cell chip comprises a liquid inlet, a gas inlet and a plurality of single-cell capturing arrays which are arranged in parallel, wherein the single-cell capturing arrays share an outlet, each single-cell capturing array comprises a main channel which is arranged in a snake shape, a plurality of capturing trap-chamber units are arranged between two adjacent rows of sections, each capturing trap-chamber unit comprises a cell capturing trap, a front limiting channel and a cell culture chamber which are communicated, the inlet of each cell capturing trap is communicated with the section of the previous row, the middle part of each cell culture chamber is communicated with a rear limiting channel, and each rear limiting channel is communicated with the section of the next row.
Preferably, the rear limiting channel is provided with two.
Preferably, the two rear limiting channels are symmetrically arranged.
Preferably, the inlets of the rear restricted passage are located at the same level.
Preferably, let the transverse length of the cell capture trap be H1, the longitudinal length be H2, the depth be H3, the dimensions are as follows: the depth H3 of the cell capture well is greater than the transverse length H1 of the cell capture well, and both the transverse length H1 and the depth H3 are greater than the diameter of a single cell, and the longitudinal length H2 is greater than or equal to the transverse length H1.
Preferably, the lateral length H4 of the front confinement channel is less than the lateral length H1 of the cell capture well, the longitudinal length H5 of the front confinement channel is less than the longitudinal length H2 of the cell capture well, the depth H6 of the front confinement channel is less than the depth H3 of the cell capture well, and the depth H6 of the front confinement channel is less than the diameter of a single cell.
Preferably, the lateral length H7 of the cell culture chamber is greater than the lateral length H1 of the cell capture trap, the longitudinal length H8 of the cell culture chamber is such that H8 is greater than the longitudinal length H2 of the cell capture trap, and the depth H9 of the cell culture chamber is such that H9 is greater than the diameter of a single cell.
Preferably, the lateral length H10 of the confinement channel is less than the lateral length H1 of the cell capture well, and the longitudinal length H11 of the post-confinement channel satisfies H11 less than the longitudinal length H2 of the cell capture well; the depth H13 of the rear confinement channel satisfies that H13 is less than H3 of the rear confinement channel and less than the diameter of a single cell.
An application of a microfluidic single-cell chip in gas-liquid exposure particle toxicity analysis.
Compared with the prior art, the invention has the following beneficial effects:
according to the microfluidic single-cell chip, the method of adopting the parallel trap-chamber units is adopted, and the trap-chamber units which can be accommodated in unit area are improved, so that the chip is ensured to have high enough cell trapping capacity.
The microfluidic single-cell chip is provided with two flow path inlets for gas and liquid, so that unnecessary errors such as inlet rupture caused in the flow path switching process are avoided. Meanwhile, the chip captures single cells by utilizing shearing force according to the fluid dynamics principle, and has the advantages of simple operation, low cost and better function realization.
According to the microfluidic single-cell chip, the culture solution of the cell culture chamber flows out from two sides in the middle of the chamber, so that cells can be immersed in the culture solution in the gas-liquid exposure process.
According to the microfluidic single-cell chip, the culture solution in the cell chamber flows out from two sides in the middle of the chamber, so that the cells can be prevented from escaping from the chamber in the process of pushing the cells into the chamber from the capture well, and the capture efficiency of the cells is improved. Meanwhile, the arrangement can also realize that in the process of introducing the particles through gas-liquid exposure, the contact between cells and the particles can be ensured, the cells can be immersed in the cell culture solution in the process, and the deviation of toxic response results caused by the sub-health state of the cells due to no culture solution can be avoided.
Drawings
FIG. 1 is a schematic diagram of a microfluidic single-cell chip according to the present invention;
fig. 2 is a top view of a trap-chamber unit in a microfluidic single-cell chip according to the present invention.
Fig. 3 is a three-dimensional structure diagram of a trap-chamber unit in a microfluidic single-cell chip provided by the invention.
In the figure, 11, liquid inlet; 12. a gas inlet; 13. a single cell capture array; 14. an outlet; 111. a trap; 112: front limiting channel, 113: cell culture chamber, 114: the sides limit the channels.
Detailed Description
The invention will now be described in further detail with reference to specific examples, which are intended to illustrate, but not to limit, the invention.
The invention discloses a microfluidic single-cell chip, referring to fig. 1, comprising a liquid inlet, a gas inlet and a plurality of parallel single-cell capturing arrays, wherein the single-cell capturing arrays share an outlet, each single-cell capturing array comprises a main channel which is arranged in a snake shape with a plurality of rows of sections, a plurality of capturing trap-chamber units are arranged between two adjacent rows of sections, each capturing trap-chamber unit comprises a cell capturing trap, a front limiting channel and a cell culture chamber which are communicated, the inlet of the cell capturing trap is communicated with the section of the upper row, the middle part of the cell culture chamber is communicated with a rear limiting channel, and the rear limiting channel is communicated with the section of the lower row. The two rear limiting channels are symmetrically arranged, and inlets of the rear limiting channels are located at the same horizontal height.
Assuming that the segments are provided with n rows, the inlet of the liquid is provided with a head of the segment in the flow direction of the liquid, the outlet of the liquid is provided with a tail of the segment, the tail of the segment of row 1 is communicated with the head of the segment of row 2, the tail of the segment of row 2 is communicated with the head of the segment of row 3, and so on.
Referring to fig. 2 and 3, let the transverse length of the cell capture trap be H1, the longitudinal length be H2, the depth be H3, and the dimensions satisfy: the depth H3 of the cell capture well is greater than the transverse length H1 of the cell capture well, and both the transverse length H1 and the depth H3 are greater than the diameter of a single cell, and the longitudinal length H2 is greater than or equal to the transverse length H1.
The lateral length H4 of the front confinement channel is less than the lateral length H1 of the cell capture trap, the longitudinal length H5 of the front confinement channel is less than the longitudinal length H2 of the cell capture trap, the depth H6 of the front confinement channel is less than the depth H3 of the cell capture trap, and the depth H6 of the front confinement channel is less than the diameter of a single cell.
The transverse length H7 of the cell culture chamber is greater than the transverse length H1 of the cell capture well, the longitudinal length H8 of the cell culture chamber satisfies H8 greater than the longitudinal length H2 of the cell capture well, and the depth H9 of the cell culture chamber satisfies H9 greater than the diameter of a single cell.
The transverse length H10 of the limiting channel is smaller than the transverse length H1 of the cell capture well, and the longitudinal length H11 of the rear limiting channel is smaller than the longitudinal length H2 of the cell capture well; the depth H13 of the rear confinement channel satisfies that H13 is less than H3 of the rear confinement channel and less than the diameter of a single cell.
An application of a microfluidic single-cell chip in gas-liquid exposure particle toxicity analysis is characterized by comprising the following steps:
s1, injecting cell suspension liquid into a microfluidic chip from a liquid inlet; the height of the liquid inlet of the microfluidic chip is higher than that of the outlet, and the flow rate of the cell suspension is 5-10 mu l/min.
S2, a cell carrier fluid enters the single-cell capturing array from a liquid inlet, a cell capturing trap intercepts target cells, and a waste liquid is discharged from the microfluidic chip from an outlet, and the specific operation is as follows: a200. Mu.l tip was inserted at the liquid inlet and a polyethylene tube was attached at the outlet. The cell suspension was loaded from the tip to the microfluidic chip by placing the polyethylene tube end at a lower position to create a height difference between the inlet and the outlet of the polyethylene tube, thereby creating a gravity driven flow of liquid. The cell carrier fluid enters the single cell capturing array through the cell suspension inlet, so that cells flow along the main channel, the cells can be trapped by the capturing trap under the action of shearing force, and the residual waste liquid is discharged out of the microfluidic chip through the outflow opening.
S3, after the cell capture trap captures cells, removing the cell suspension at the liquid inlet, and adding the cell culture solution; and then connecting a first injector at the liquid inlet, pushing the first injector, observing whether cells in the microfluidic chip enter the cell culture chamber from the cell capture trap through the pre-limiting channel under a microscope, disconnecting the first injector after the cells enter the cell culture chamber, and culturing the chip to obtain the culture chip.
During culturing, the chip is placed in saturated humidity at 37deg.C and 5% CO 2 Culturing for 24h in an incubator, and placing the waste liquid outlet and the chip at the same height.
S4, performing gas-liquid exposure particle toxicity evaluation test by using the culture chip, connecting a second injector at the gas inlet, and introducing gas carrying particles to be tested into the culture chip. The second syringe is placed in the syringe pump to ensure that the syringe has been filled with the gas loaded with the particulate matter to be tested. The cell culture chamber designed by the invention can still ensure that cells are immersed in the cell culture liquid within the gas injection speed range of 20-40 mu l/min and the gas-liquid exposure experiment lasts for 4 hours.
In the gas-liquid exposure test, after 1-4 hours of clean air is introduced, the survival rate of cells is not significantly different from that of a negative control group without air, which indicates that the chip is completely suitable for the gas-liquid exposure particle toxicity evaluation test. Fluorescent indicators for assessing particulate toxicity may be injected into the chip to stain cells before or after the gas-liquid exposure test, as desired by the experiment.
The preparation process of the microfluidic single-cell chip comprises the following steps:
(1) Pretreatment of a silicon wafer: cleaning the surface of the silicon wafer by degreasing, polishing, washing with isopropanol and washing with water, placing the silicon wafer on an electric heating plate at 150 ℃ for 20min, and drying;
(2) Gluing: and a layer of uniform photoresist is coated on the surface of the treated silicon wafer, and the spin coating speed can be set according to the depth of the required pattern. In the embodiment of the invention, the spin coating speed is set to 3000rpm, and the spin coating is operated for 30 seconds.
(3) Pre-baking: at a certain temperature, the solvent of the photoresist is volatilized. The pre-bake temperature and time are set by the properties of the photoresist used. In the embodiment of the invention, a silicon wafer is placed on an electric heating plate at 80 ℃ for min.
(4) Exposure: the photoresist is irradiated by ultraviolet rays through the photoetching mask. The exposure time is set according to the power of the exposure machine, the thickness of the adhesive film, the thickness of the photoetching film and the distance between the light source and the silicon wafer. In the embodiment of the invention, the exposure time is 60s.
It should be noted that: the mould is manufactured by adopting a three-step photoetching process, the first two steps are used for manufacturing a cell culture chamber and two limiting channels on two sides of the middle of the chamber, and the third step is used for manufacturing a cell capture trap and a main channel.
(5) Post-baking: and taking out the template after exposure to carry out post-baking treatment for a short time. In the embodiment of the invention, the temperature during post-baking is set to be 100 ℃ for 2min.
(6) Developing: and (3) putting the silicon wafer into propylene glycol monomethyl ether acetate developer to remove unexposed photoresist, wherein the development time is determined according to the type of the developer and the development temperature, and developing for 2-3 times in the process. In the embodiment of the invention, the developing temperature is set to 25 ℃ and the developing time is set to 60 seconds.
(7) Hardening: and cleaning the developed substrate, and baking at 100 ℃ to thoroughly remove the solvent or water remained in the adhesive film after development, so that the adhesive film and the substrate are tightly adhered to prevent the adhesive layer from falling off. The temperature of the hardening process is set according to the photoresist thickness. The temperature at which the film was hardened in the examples of the present invention was 100 ℃.
(8) And (3) developing and detecting: the developed photoresist pattern was checked for differences from the standard pattern. So far, the die processing is completed.
(9) Surface coupling: evaporating the coupling medicine to the surface of the silicon wafer in a closed vacuum space to form a coupling layer, wherein the coupling time is at least 60min. The coupling medicine can be tridecafluoro-1, 2-tetrahydrooctyl-1-trichlorosilane or (3-acryloxypropyl) -trichlorosilane.
(10) And (3) preparing PDMS adhesive: the PDMS matrix and the curing agent are stirred uniformly in proportion, and then degassing treatment is carried out in a closed vacuum space. The proportion of the matrix and the curing agent is configured according to the required hardness of the PDMS material. In the embodiment of the invention, the ratio of the matrix to the curing agent is 15:1.
(11) Pouring glue: and fixing the silicon chip in a culture dish, and uniformly pouring the prepared PDMS glue into the dish to ensure that no bubbles exist in the glue.
(12) And (3) heat treatment: and (5) placing the PDMS glue into a constant temperature drying oven at 75 ℃ for heat treatment for at least 2 hours.
(13) Stripping: after cooling, the cured PDMS was peeled from the wafer using a knife.
(14) Cutting: cutting along the outer frame of the chip by using a scalpel, wiping the part with the pattern of the chip with the adhesive tape Gao Yinxing, and then continuing to adhere the invisible adhesive tape.
(15) Punching: and punching holes at the inlet and outlet of the chip by using a puncher. The diameter of the hole puncher is selected according to the diameter of a pipeline connecting the inlet and the outlet of the chip. The diameter of the puncher in the embodiment of the invention is 1.5mm.
(16) Bonding: the bonding surface of the PDMS substrate and the glass slide are modified by using a plasma machine or a corona discharge instrument, and the treated two surfaces are bonded and compacted.
(17) Baking: the chip was baked on a 110℃hotplate for 10min.
The preparation method of the cell suspension in the invention comprises the following steps:
(1) Cell culture: alveolar macrophages are grown in cell culture medium and placed under saturated humidity at 37 ℃ and 5%CO 2 In an incubator. The cell culture medium consists of RPMI-1640 medium, 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics.
(2) Cell extraction: when the cells grow to 80% -90% fusion, taking out the cells from a carbon dioxide incubator, adding trypsin to digest the cells, separating the adherent cells, using 3ml of trypsin for a single time, centrifuging the separated mixed solution for 10min at 1000r/min, sucking the supernatant, adding 5ml of culture medium, and fully and uniformly mixing to obtain the cell suspension.
Example 1
Microfluidic single cell chips and configured cell suspensions were prepared using the above described manner. The size parameters of the microfluidic single-cell chip are as follows: h1 =15 μm, h2=12 μm, h3=20 μm, h4=10 μm, h5=10 μm, h6=10 μm, h7=20 μm, h8=36 μm, h9=20 μm, h10=8 μm, h11=8 μm, h12=6 μm. Deionized water and phosphate buffer solution are sequentially introduced into the washing machine by a syringe for washing. And (3) introducing the cell suspension into the chip from the liquid inlet, regulating the height difference between the liquid inlet and the waste liquid outlet pipeline, controlling the inflow speed of the cell suspension to be 10 mu l/min, and observing the capturing condition of single cells in the capturing trap under a microscope. After the cells are captured in the cell capture trap in front of the respective trap-chamber unit, the cell suspension at the liquid inlet is removed and the cell culture liquid is added. A syringe is connected to the liquid inlet to push the cells into the cell culture chamber. After the cells were cultured in a carbon dioxide incubator for 24 hours, a gas-liquid exposure experiment was performed. Air is introduced into the gas inlet of the chip for 1h, and the gas injection speed is controlled to be 40 mu l/min.
In example 1, the trapping efficiency of single cells in the trapping trap reached 82%, and the survival rate of cells after 1h of gas-liquid exposure experiment was 96%, which was not significantly different from that of the negative control group without air.
Example 2
Microfluidic single cell chips and configured cell suspensions were prepared using the above described manner. The size parameters of the microfluidic single-cell chip are as follows: h1 =15 μm, h2=12 μm, h3=20 μm, h4=10 μm, h5=10 μm, h6=10 μm, h7=20 μm, h8=36 μm, h9=20 μm, h10=8 μm, h11=8 μm, h12=6 μm. Deionized water and phosphate buffer solution are sequentially introduced into the washing machine by a syringe for washing. And (3) introducing the cell suspension into the chip from the liquid inlet, regulating the height difference between the liquid inlet and the waste liquid outlet pipeline, controlling the inflow speed of the cell suspension to be 5 mu l/min, and observing the capturing condition of single cells in the capturing trap under a microscope. After the cells are captured in the cell capture trap in front of the respective trap-chamber unit, the cell suspension at the liquid inlet is removed and the cell culture liquid is added. A syringe is connected to the liquid inlet to push the cells into the cell culture chamber. After the cells were cultured in a carbon dioxide incubator for 24 hours, a gas-liquid exposure experiment was performed. Air is introduced into the gas inlet of the chip for 2 hours, and the gas injection speed is controlled to be 30 mu l/min.
In example 2, the single cell has a trapping efficiency of 88% in the trap, and the survival rate of the cell after 2h of gas-liquid exposure experiment is 93% without significant difference from the negative control group without air.
Example 3
Microfluidic single cell chips and configured cell suspensions were prepared using the above described manner. The size parameters of the microfluidic single-cell chip are as follows: h1 =15 μm, h2=12 μm, h3=20 μm, h4=10 μm, h5=10 μm, h6=10 μm, h7=20 μm, h8=36 μm, h9=20 μm, h10=8 μm, h11=8 μm, h12=6 μm. Deionized water and phosphate buffer solution are sequentially introduced into the washing machine by a syringe for washing. And (3) introducing the cell suspension into the chip from the liquid inlet, regulating the height difference between the liquid inlet and the waste liquid outlet pipeline, controlling the inflow speed of the cell suspension to be 8 mu l/min, and observing the capturing condition of single cells in the capturing trap under a microscope. After the cells are captured in the cell capture trap in front of the respective trap-chamber unit, the cell suspension at the liquid inlet is removed and the cell culture liquid is added. A syringe is connected to the liquid inlet to push the cells into the cell culture chamber. After the cells were cultured in a carbon dioxide incubator for 24 hours, a gas-liquid exposure experiment was performed. Air is introduced into the gas inlet of the chip for 4 hours, and the gas injection speed is controlled to be 20 mu l/min.
In example 3, the single cell has a trapping efficiency of 85% in the trap, and the survival rate of the cell after 4h of the gas-liquid exposure experiment is 92% without significant difference from the negative control group without air.
The foregoing description of the preferred embodiment of the present invention is not intended to limit the technical solution of the present invention in any way, and it should be understood that the technical solution can be modified and replaced in several ways without departing from the spirit and principle of the present invention, and these modifications and substitutions are also included in the protection scope of the claims.

Claims (9)

1. The microfluidic single-cell chip is characterized by comprising a liquid inlet, a gas inlet and a plurality of single-cell capturing arrays which are arranged in parallel, wherein the single-cell capturing arrays share an outlet, each single-cell capturing array comprises a main channel which is arranged in a snake shape with a plurality of sections, a plurality of capturing trap-cavity units are arranged between two adjacent rows of sections, each capturing trap-cavity unit comprises a cell capturing trap, a front limiting channel and a cell culture cavity which are communicated, the inlet of each cell capturing trap is communicated with the section of the previous row, the middle part of each cell culture cavity is communicated with a rear limiting channel, and each rear limiting channel is communicated with the section of the next row.
2. The microfluidic single cell chip according to claim 1, wherein two post-restriction channels are provided.
3. The microfluidic single cell chip according to claim 2, wherein the two post-restriction channels are symmetrically arranged.
4. The microfluidic single cell chip according to claim 2, wherein the inlets of the rear restriction channels are located at the same level.
5. The microfluidic single cell chip according to claim 1, wherein the cell capture well has a lateral length H1, a longitudinal length H2, a depth H3, and a size: the depth H3 of the cell capture well is greater than the transverse length H1 of the cell capture well, and both the transverse length H1 and the depth H3 are greater than the diameter of a single cell, and the longitudinal length H2 is greater than or equal to the transverse length H1.
6. The microfluidic single-cell chip according to claim 5, wherein the lateral length H4 of the front confinement channel is smaller than the lateral length H1 of the cell capture well, the longitudinal length H5 of the front confinement channel is smaller than the longitudinal length H2 of the cell capture well, the depth H6 of the front confinement channel is smaller than the depth H3 of the cell capture well, and the depth H6 of the front confinement channel is smaller than the diameter of a single cell.
7. The microfluidic single-cell chip according to claim 5, wherein the lateral length H7 of the cell culture chamber is greater than the lateral length H1 of the cell capture well, the longitudinal length H8 of the cell culture chamber satisfies H8 greater than the longitudinal length H2 of the cell capture well, and the depth H9 of the cell culture chamber satisfies H9 greater than the diameter of the single cell.
8. The microfluidic single-cell chip and gas-liquid exposed particle toxicity analysis method according to claim 5, wherein the lateral length H10 of the limiting channel is smaller than the lateral length H1 of the cell capture well, and the longitudinal length H11 of the rear limiting channel satisfies that H11 is smaller than the longitudinal length H2 of the cell capture well; the depth H13 of the rear confinement channel satisfies that H13 is less than H3 of the rear confinement channel and less than the diameter of a single cell.
9. Use of the microfluidic single cell chip according to any one of claims 1 to 8 in gas-liquid exposure particle toxicity analysis.
CN202310293352.XA 2023-03-23 2023-03-23 Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method Pending CN116179351A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310293352.XA CN116179351A (en) 2023-03-23 2023-03-23 Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310293352.XA CN116179351A (en) 2023-03-23 2023-03-23 Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method

Publications (1)

Publication Number Publication Date
CN116179351A true CN116179351A (en) 2023-05-30

Family

ID=86438550

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310293352.XA Pending CN116179351A (en) 2023-03-23 2023-03-23 Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method

Country Status (1)

Country Link
CN (1) CN116179351A (en)

Similar Documents

Publication Publication Date Title
Lin et al. A microfluidic dual-well device for high-throughput single-cell capture and culture
CN110004043B (en) Single cell capture micro-fluidic chip
Ziolkowska et al. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage
Bruzewicz et al. Fabrication of a modular tissue construct in a microfluidic chip
US20150204763A1 (en) System for analyzing biological sample material
US9885059B2 (en) Ultrahigh throughput microinjection device
CN105713834B (en) A kind of micro-fluidic chip and its preparation method and application
CN110437992B (en) Large-scale and rapid digital liquid-phase sample decomposition chip and use method thereof
JP2008054521A (en) Cell-culturing device and cell-culturing method
KR101691049B1 (en) Microfluidic perfusion cell culture apparatus, method for manufacturing the same and method of cell culture
CN105420103A (en) Simple micro-fluidic chip and cell analysis method
CN103087915A (en) Bubble eliminating device for high-throughput microfluidics cell chip and operation method thereof
CN112920951B (en) Cell screening chip and manufacturing and cell screening and collecting method thereof
JP2018525003A (en) Hydrodynamic shuttling chip instrument and method for capturing isolated single cells
US20230053160A1 (en) Selective and High-Resolution Printing of Single Cells
KR101118087B1 (en) Lab-on-a-chip and Manfacturing Method and Cell Cultivation Method for Cell Cultivation and Observation Using Transparency Macromolecule Material
CN116179351A (en) Microfluidic single-cell chip and gas-liquid exposure particle toxicity analysis method
CN112063510A (en) Structure of high-flux cell culture chip and manufacturing and using method thereof
CN116121031B (en) Multistage microfluidic chip for single cell screening and preparation method thereof
Ko et al. Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel
CN115466680A (en) Micro-fluidic chip for monitoring immune killing of organoid in real time and method thereof
CN113862151A (en) Microfluidic chip device for cell co-culture and cell co-culture method
CN111019827A (en) Hydrogel oocyte in-vitro three-dimensional culture micro-fluidic chip based on different cross-linking degrees and application thereof
US20210260576A1 (en) Single Cell Isolation and Processing System With Reversible Well Shape
CN220246114U (en) Concentration gradient micro-fluidic chip for drug screening

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