CN111808750A - Micro-fluidic chip device, in-situ detection method for living unicells and application - Google Patents
Micro-fluidic chip device, in-situ detection method for living unicells and application Download PDFInfo
- Publication number
- CN111808750A CN111808750A CN202010681881.3A CN202010681881A CN111808750A CN 111808750 A CN111808750 A CN 111808750A CN 202010681881 A CN202010681881 A CN 202010681881A CN 111808750 A CN111808750 A CN 111808750A
- Authority
- CN
- China
- Prior art keywords
- cell
- cells
- microfluidic
- chip device
- unit
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 22
- 239000000523 sample Substances 0.000 claims abstract description 50
- 230000000638 stimulation Effects 0.000 claims abstract description 31
- 238000004113 cell culture Methods 0.000 claims abstract description 22
- 230000008054 signal transmission Effects 0.000 claims abstract description 13
- 230000004936 stimulating effect Effects 0.000 claims abstract description 13
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- 230000004044 response Effects 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 138
- 238000002347 injection Methods 0.000 claims description 22
- 239000007924 injection Substances 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 18
- 238000006073 displacement reaction Methods 0.000 claims description 9
- 238000003384 imaging method Methods 0.000 claims description 8
- 238000005485 electric heating Methods 0.000 claims description 5
- 230000009977 dual effect Effects 0.000 claims description 2
- 238000005086 pumping Methods 0.000 claims description 2
- 230000005754 cellular signaling Effects 0.000 claims 1
- 238000007619 statistical method Methods 0.000 abstract description 3
- 239000012530 fluid Substances 0.000 description 10
- 210000000170 cell membrane Anatomy 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 238000004891 communication Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 description 5
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 229940041616 menthol Drugs 0.000 description 5
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- 208000032612 Glial tumor Diseases 0.000 description 3
- 206010018338 Glioma Diseases 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000004205 dimethyl polysiloxane Substances 0.000 description 2
- 238000001125 extrusion Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000004660 morphological change Effects 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 2
- -1 polydimethylsiloxane Polymers 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- HQMRIBYCTLBDAK-UHFFFAOYSA-M bis(2-methylpropyl)alumanylium;chloride Chemical compound CC(C)C[Al](Cl)CC(C)C HQMRIBYCTLBDAK-UHFFFAOYSA-M 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000023402 cell communication Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000011982 device technology Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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
-
- 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
- C12M23/10—Petri dish
-
- 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
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Sustainable Development (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Clinical Laboratory Science (AREA)
- Analytical Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Thermal Sciences (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mechanical Engineering (AREA)
- Cell Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a micro-fluidic chip device, a living single cell in-situ detection method and application. A microfluidic chip device comprises a microfluidic probe unit for stimulating target cells and a cell culture control unit for containing the target cells and recording stimulation signals. The micro-fluidic chip device is used for in-situ detection of living unicells, in-situ fixed-point stimulation can be carried out on the unicells on the level of the unicells, the transmission of signals among the unicells is concentrated on analysis in a short time, the heterogeneous response of the cells is subjected to statistical analysis, the interference of average analysis of the response result of cell groups is avoided, the detection method is milder, and the detection result is more accurate.
Description
Technical Field
The invention relates to a micro-fluidic chip device, a living single cell in-situ detection method and application.
Background
Cells are not independent in vivo as basic activity units of life, and often perform normal functions through close contact and communication with other cells to maintain normal life activities of human bodies. With the development of science and technology, scientists develop various detection means including fluorescence technology, capillary electrophoresis, chromatography and mass spectrometry, realize the detection of intercellular communication signals, provide important information for the research of disease detection and cell metabonomics, and play an important role in the research fields of drug research and development, disease prevention and occurrence mechanism.
The microfluidic chip is used as an effective platform for cell culture analysis, and a plurality of signal communication detection methods for cell co-culture are developed, such as a multilayer channel chip controlled by a surface tension valve, a cell droplet technology of different entrapment methods, and three-dimensional organ simulation introducing matrix materials such as hydrogel. Among these important device technologies, the multilayer channel chip device is cumbersome in fabrication process and the problem of bubbles in the channel is likely to cause damage to cells; the droplet technology has low entrapment rate and cannot reflect the real cell adherent state; the introduction of the three-dimensional matrix also has the problems of complicated manufacturing process, low utilization rate and the like. More importantly, the results obtained by the above detection techniques are the average level of the cell response behavior of the population after cell communication, and often neglect the exchange of molecules in single cells and the change of the level of organelles, and neglect the influence of cell heterogeneity on the research results. Therefore, in recent years researchers have developed different platforms for single cell based material, behavioral analysis studies, such as: the device comprises a microfluidic chip device with a micro-dam structure, a microfluidic chip droplet technology platform capable of capturing single cells and different in-situ single cell operation tools. However, in these methods, the microfluidic chip device with the micro-dam structure has high single-cell capture efficiency, but the platform is difficult to perform independent in-situ operation on each single cell; meanwhile, although the common droplet-encapsulated single cell technology on the microfluidic chip can enrich and analyze the substance concentration of single cells, the method also has the problem of cell suspension and does not conform to the real state of cell adherence; in addition, single cell in-situ signal stimulation usually exists in a mechanical stimulation mode such as extrusion, and the influence caused by mechanical damage of cells is neglected in cell behavior analysis.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a novel micro-fluidic chip device, which is used for in-situ detection of living single cells, can perform in-situ fixed-point stimulation on the single cells at the single cell level, concentrates on analyzing the transmission of signals among the single cells in a short time, performs statistical analysis on the heterogeneous response of the cells, avoids the interference of average analysis of the response result of cell groups, and compared with single cell detection methods such as mechanical extrusion, the detection method provided by the invention is milder and has more accurate detection result.
The invention provides a microfluidic chip device, which comprises a microfluidic probe unit for stimulating target cells and a cell culture control unit for accommodating the target cells and recording stimulation signals.
According to some embodiments of the device of the present invention, the cell culture manipulation unit comprises a culture dish for placing cells, a microscope for observing the cells in the culture dish, and a microscope imaging system for imaging.
According to some embodiments of the device of the present invention, the cell culture manipulation unit further comprises a thermostatic hotplate for controlling the temperature of the culture dish. Such as a 37 c thermostatic hot plate.
According to some embodiments of the device of the present invention, the cell culture manipulation unit further comprises a stage for carrying an electric hot plate. Such as an electrically controlled stage.
According to some embodiments of the apparatus of the present invention, the microfluidic probe unit comprises a microfluidic probe, and a first syringe pump and a second syringe pump communicating with both ends of the probe, wherein the first syringe pump is used for injecting the stimulation solution into one end of the target cell surface, and the second syringe pump is used for withdrawing the stimulation solution from the other end of the target cell surface.
According to some embodiments of the device of the present invention, the microfluidic probe comprises a base plate and a dual channel chip bonded to the base plate.
According to some embodiments of the device of the present invention, the microfluidic probe is preferably polydimethylsiloxane.
According to some embodiments of the device of the present invention, the microfluidic chip device further comprises a displacement platform unit.
According to some embodiments of the device of the present invention, the displacement platform comprises a holder for holding a microfluidic probe unit and a control platform for controlling the movement and positioning of the microfluidic probe unit.
According to some embodiments of the apparatus of the present invention, the bottom of the microfluidic probe is located above the culture dish, and the target cell is located directly below the microfluidic probe by adjusting the position of the electrically controlled stage.
The invention provides a method for in-situ detection of living single cells by adopting the microfluidic chip device, which comprises the steps of stimulating target cells planted in a cell culture control unit by adopting a microfluidic probe unit and recording stimulation signals.
According to some embodiments of the detection method of the present invention, the cells planted in the cell culture manipulation unit are planted at a density of 103-104One/ml. When the planting density is too dense, target living unicells suitable for detection are difficult to obtain. When the planting density is too thin, the corresponding effect of the signal presented by the formed information channel is not good enough. Therefore, the detection effect is better under the preferable planting density of the invention.
According to some embodiments of the detection method of the present invention, the cells are planted in a culture dish, and preferably, a connection structure facilitating signal communication between single cells can be formed. The attachment structure may be as shown in fig. 2.
According to some embodiments of the method of detecting of the present invention, the method of stimulating comprises injecting the stimulating solution from one end of the surface of the target cell and then withdrawing the stimulating solution from the other end of the surface of the target cell. Thereby forming a microfluidic stimulation region (channel) within the target cell.
According to some embodiments of the detection method of the present invention, the ratio of the draw flow rate to the injection flow rate is 10: 1-2. I.e. the ratio of the flow rate of the withdrawal channel to the flow rate of the injection channel is 10: 1-2.
According to some embodiments of the detection method of the present invention, the method of recording the stimulus signal comprises recording a response signal of the target cell and signal transmission between the target cell and other single cells.
According to some embodiments of the detection method of the present invention, the cells planted in the cell culture manipulation unit can be stained cells or non-stained cells, depending on experimental requirements. The dye used for staining cells also depends on the experimental requirements, and may be, for example, an actin dye, a wheat germ agglutinin dye, a cell membrane potential dye [ DiBAC4(3) ], or the like.
The third aspect of the present invention provides the application of the above microfluidic chip device or the above method for in situ detection of living single cells in cell signal transmission analysis.
Due to the adoption of the technical scheme, the invention has the following advantages:
1. the invention integrates single cell in-situ regional stimulation, continuous flow control of a stimulation solution (such as a drug stimulation solution) and online real-time monitoring of stimulation signal transmission, can perform in-situ fixed-point processing on adherent single cells in a culture dish, and realizes the transmission of molecular signals on the level of online monitoring of the single cells.
2. The cell culture control unit can keep the adherent state of the cells to the greatest extent and is closer to the real state of the response of the cells to signal stimulation.
3. The micro-fluidic chip device can stimulate single cells in situ at a single cell level, focus on analyzing the signal transmission among the single cells in a short time, perform statistical analysis on the heterogeneous response of the cells, and avoid the interference of average analysis of the response result of cell groups.
4. The stimulating solution of the present invention may be replaced with various kinds of signal stimulating solution, and may be determined based on the target cell.
5. The detection target cell can be replaced by the transmission analysis of the signals of organelles and other substances except for the molecular level, and the signal transmission of the subcellular level can be monitored in real time.
Drawings
Fig. 1 is a schematic structural diagram of a microfluidic chip device provided in embodiment 1 of the present invention;
FIG. 2 is a diagram showing the determination of the junction structure between single cells provided in example 2 of the present invention;
FIG. 3 is a time series image of the cell membrane potential change under the effect of 200. mu.M menthol according to example 3 of the present invention;
FIG. 4 is a time series image of the change in membrane potential of cells in a blank set;
FIG. 5 is a microscope photograph of the microfluidic probe provided in example 4 of the present invention applied to a single cell connected by a connecting structure;
FIG. 6 is a statistical chart of the cellular morphological area of different cells provided in example 4 of the present invention.
Description of the reference numerals
1-a cell culture manipulation unit; 2-microfluidic probe unit; 3-a displacement platform unit;
4-constant temperature electric heating plate; 5-culture dish; 6-cell staining solution;
7-a microscope; 8-a microscope imaging system; 9-cells;
10-a fixing frame; 11-a first syringe pump; 12-first syringe pump.
Detailed Description
In order that the present invention may be more readily understood, the following detailed description of the invention is given by way of example only, and is not intended to limit the scope of the invention.
[ example 1 ]
A microfluidic chip device, as shown in FIG. 1, comprises a microfluidic probe unit 2, a cell culture manipulation unit 1 and a displacement platform unit 3. The cell culture manipulation unit 1 comprises a 37 ℃ constant-temperature electric heating plate 4, a culture dish 5, a microscope 7, a microscope imaging system 8 and a stage. The microfluidic probe unit 2 comprises a microfluidic probe, the microfluidic probe comprises a bottom plate and a dual-channel chip bonded with the bottom plate, the microfluidic probe is made of polydimethylsiloxane, the microfluidic probe unit 2 further comprises a first injection pump 11 and a second injection pump 12, the first injection pump 11 is communicated with two ends of the probe, the first injection pump 11 is used for injecting stimulation solution into one end of the surface of a target cell, and the second injection pump 12 is used for pumping the stimulation solution away from the other end of the surface of the target cell. The displacement platform unit 3 comprises a fixed frame 10 and a control platform, wherein the fixed frame 10 is used for fixing the microfluidic probe unit 2, and the control platform is used for controlling the movement and the positioning of the microfluidic probe unit 2. The bottom end of the micro-fluid probe is positioned above the culture dish 5, and the target cell 9 is arranged under the micro-fluid probe by moving the objective table and the constant temperature electric hot plate 4 at 37 ℃.
[ example 2 ]
Human glioma cell U87 (labeled 9 in FIG. 1) at 103The single cells/ml are planted in a cell culture dish, and a PE rotary disc confocal microscope (purchased from platinum Elmer, Inc., and the model is Ultraview VOX) is adopted to determine images of the single cell-cell connection structure under the operation condition of taking bright field, 488nm and 561nm exciting light as light sources at the constant temperature of 37 ℃. The results are shown in FIG. 2. In FIG. 2, the A image is 103The image B is an actin staining image of the cells, the image C is a wheat germ agglutinin staining image, and the image D is a combined image of fluorescence images. As can be seen from FIG. 2, the cells are numbered 103When the single cells are planted at the bottom of the culture dish, a structure for cell signal communication and transmission (shown by an arrow in the figure) can be formed among the single cells, the structure can be determined by Actin (F-Actin) and Wheat Germ Agglutinin (WGA) staining, and the cell density is favorable for providing a certain operation space for the in-situ stimulation of the micro-fluid probe to the single cells.
[ example 3 ]
In-situ detection of single cells in vivo using the apparatus of example 1:
(1) human glioma cell U87 (labeled 9 in FIG. 1) at 103The cells are planted in cell culture dish at a density of one cell/ml to form a connection structure for signal communication between single cells, and the cell upper layer is covered with cell membrane potential dye [ DiBAC ] for analyzing potential change of target cells4(3)](cell staining solution, reference numeral 6 in FIG. 1);
(2) connecting the microfluidic probe with a first injection pump and a second injection pump, placing the microfluidic probe on a fixed frame, vertically approaching the bottom surface of a culture dish, moving an electric control objective table and a 37 ℃ constant-temperature electric heating plate, and adjusting target cells to be under the microfluidic probe through microscope observation, thereby confirming that the microfluidic probe is aligned with target single cells;
(3) the micro-adjustment displacement platform unit controls the distance between the micro-fluid probe and the bottom surface of the culture dish to be 50 mu M, the first injection pump starts an 'injection' mode, the second injection pump starts an 'extraction' mode, the ratio of the extraction flow rate to the injection flow rate is 5:1 (the extraction flow rate is 10 mu L/min, and the injection flow rate is 2 mu L/min), 200 mu M menthol solution is used as a signal stimulation drug solution (stimulation solution) and is injected from one end of the surface of a target cell and is extracted from the other end of the surface of the target cell through a micro-fluid probe channel, and therefore a micro-fluid stimulation area is formed in the target single-cell area;
(4) when the signal stimulation drug is injected, the microscope imaging recording system starts to record the signal change of the target single cell and simultaneously records the signal transmission process of another cell connected with the single cell, thereby realizing the signal transmission analysis among the single cells.
FIG. 3 is a sequence of time-series images of cell membrane potential changes (0min-10min) under the action of 200 μ M menthol under the condition of operation with 488nm excitation light as light source at constant temperature of 37 ℃ by using a PE rotary disk confocal microscope (available from platinum Elmer, Inc., model: Ultraview VOX), when 200 μ M menthol solution is acted on cells stained with cell membrane potential dye, the cells in this example show significant changes in cell membrane potential and can reach equilibrium within 0.5min compared with the blank group (time-series images of cell membrane potential changes from 0min to 10min without using 200 μ M menthol).
[ example 4 ]
In-situ detection of single cells in vivo using the apparatus of example 1:
(1) human glioma cell U87 (labeled 9 in FIG. 1) at 103The cells/ml are planted in a cell culture dish to form a connecting structure for signal communication among single cells;
(2) connecting the microfluidic probe with a first injection pump and a second injection pump, placing the microfluidic probe on a fixed frame, vertically approaching the bottom surface of a culture dish, moving an electric control objective table and a 37 ℃ constant-temperature electric heating plate, and adjusting target cells to be under the microfluidic probe through microscope observation, thereby confirming that the microfluidic probe is aligned with target single cells;
(3) the micro-adjustment displacement platform unit controls the distance between the micro-fluid probe and the bottom surface of the culture dish to be 50 mu m, the first injection pump is started to be in an injection mode, the second injection pump is started to be in an extraction mode, the ratio of the extraction flow rate to the injection flow rate is 5:1 (the extraction flow rate is 10 mu L/min, and the injection flow rate is 2 mu L/min), 0.25 wt% of pancreatin solution (purchased from Corning company) is used as signal stimulation drug solution (stimulation solution) to be injected from one end of the surface of a target cell and is extracted from the other end of the surface of the target cell through a micro-fluid probe channel, and therefore a micro-fluid stimulation area is formed in the target single-cell;
(4) when the signal stimulation drug is injected, the microscope imaging recording system starts to record the signal change of the target single cell and simultaneously records the signal transmission process of another cell connected with the single cell, thereby realizing the signal transmission analysis among the single cells.
The results are shown in FIGS. 5 and 6. FIG. 5 is a graph of the effect of a microfluidic probe on single cells connected by a linker structure using an inverted microscope (from Leica, Inc. model LEICADMI 4000B) at a constant temperature of 37 ℃ and under bright field operation conditions, (A) - (D) are changes in cell morphology at different times (0min, 1min, 2min, 10 min). As can be seen from fig. 5, in the test in which the microfluidic probe acted on the single cell connected with the connection structure, the substrate adhesion reduction treatment was performed on the single cell by injecting pancreatin, only the target single cell a acted on by the microfluidic probe underwent a significant morphological change, the cell area was significantly reduced, and the single cell b connected with it and the other cells c in the visual field all maintained good cell morphology.
FIG. 6 is a statistical chart of the cell morphology area of different cells by using Image J Image analysis processing software, which shows that the single cell acted by the micro-fluid probe has obvious morphological change, but has no influence on the connected cells and other cells in the visual field.
It can be seen from examples 2-4 that the microfluidic chip device of the present invention can be used to independently act on single cells having a connection structure, and to perform in situ detection of relatively mild signal stimulation and signal transmission.
What has been described above is merely a preferred example of the present invention. It should be noted that other equivalent variations and modifications can be made by those skilled in the art based on the technical teaching provided by the present invention, and the protection scope of the present invention should be considered.
Claims (10)
1. A microfluidic chip device comprises a microfluidic probe unit for stimulating target cells and a cell culture control unit for containing the target cells and recording stimulation signals.
2. The microfluidic chip device according to claim 1, wherein the cell culture manipulation unit comprises a culture dish for placing cells, a microscope for observing the cells in the culture dish, and a microscope imaging system for imaging;
preferably, the cell culture manipulation unit further comprises a thermostatic electric heating plate for controlling the temperature of the culture dish;
preferably, the cell culture manipulation unit further comprises a stage for carrying an electric hot plate.
3. The microfluidic chip device according to claim 1 or 2, wherein the microfluidic probe unit comprises a microfluidic probe, and a first syringe pump and a second syringe pump communicated with two ends of the probe, wherein the first syringe pump is used for injecting a stimulation solution into one end of the surface of the target cell, and the second syringe pump is used for pumping the stimulation solution out of the other end of the surface of the target cell;
preferably, the microfluidic probe comprises a base plate and a dual channel chip bonded to the base plate.
4. The microfluidic chip device according to any of claims 1 to 3, further comprising a displacement platform unit;
preferably, the displacement platform comprises a fixing frame and a control platform, wherein the fixing frame is used for fixing the microfluidic probe unit, and the control platform is used for controlling the movement and the positioning of the microfluidic probe unit.
5. A method for in-situ detection of living single cells by using the microfluidic chip device of any one of claims 1 to 4, comprising stimulating target cells planted in the cell culture manipulation unit by using the microfluidic probe unit and recording stimulation signals.
6. The method of claim 5, wherein the cells planted in the cell culture manipulation unit are planted at a density of 103-104One/ml.
7. The method of claim 5 or 6, wherein the stimulating comprises injecting the stimulating solution from one end of the target cell surface and then withdrawing the stimulating solution from the other end of the target cell surface.
8. The method of claim 7, wherein the ratio of the withdrawal flow rate to the injection flow rate is 10: 1-2.
9. A method according to any one of claims 5 to 8, wherein the method of recording the stimulus signal comprises recording the response signal of the target cell and the signal transmission between the target cell and other single cells.
10. Use of the microfluidic chip device according to any one of claims 1 to 4 or the method for in situ detection of living single cells according to any one of claims 5 to 9 in cell signaling analysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010681881.3A CN111808750A (en) | 2020-07-15 | 2020-07-15 | Micro-fluidic chip device, in-situ detection method for living unicells and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010681881.3A CN111808750A (en) | 2020-07-15 | 2020-07-15 | Micro-fluidic chip device, in-situ detection method for living unicells and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111808750A true CN111808750A (en) | 2020-10-23 |
Family
ID=72865186
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010681881.3A Pending CN111808750A (en) | 2020-07-15 | 2020-07-15 | Micro-fluidic chip device, in-situ detection method for living unicells and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111808750A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899157A (en) * | 2020-12-28 | 2021-06-04 | 中国科学院长春应用化学研究所 | Micro-fluidic chip light stimulation device, yeast single cell light regulation gene expression method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090093757A (en) * | 2008-02-29 | 2009-09-02 | 아주대학교산학협력단 | Cell-chip and automatic controlled system capable of detecting conditions for optimizing differentiation of stem cell using mechanical stimuls |
| CN103105353A (en) * | 2013-02-18 | 2013-05-15 | 西南大学 | Unicell detector based on nano fiber probe and its probe manufacturing method |
| CN107446820A (en) * | 2017-09-28 | 2017-12-08 | 清华大学 | Unicellular sampling and in situ detection mass spectrometer interface device based on micro-fluidic chip |
| CN109557163A (en) * | 2018-11-27 | 2019-04-02 | 清华大学 | A kind of Living single cell situ extracting and on-line mass spectroscopy detection device and application |
| CN209858475U (en) * | 2018-11-27 | 2019-12-27 | 清华大学 | In-situ extraction and online mass spectrum detection device for living unicells |
-
2020
- 2020-07-15 CN CN202010681881.3A patent/CN111808750A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090093757A (en) * | 2008-02-29 | 2009-09-02 | 아주대학교산학협력단 | Cell-chip and automatic controlled system capable of detecting conditions for optimizing differentiation of stem cell using mechanical stimuls |
| CN103105353A (en) * | 2013-02-18 | 2013-05-15 | 西南大学 | Unicell detector based on nano fiber probe and its probe manufacturing method |
| CN107446820A (en) * | 2017-09-28 | 2017-12-08 | 清华大学 | Unicellular sampling and in situ detection mass spectrometer interface device based on micro-fluidic chip |
| CN109557163A (en) * | 2018-11-27 | 2019-04-02 | 清华大学 | A kind of Living single cell situ extracting and on-line mass spectroscopy detection device and application |
| CN209858475U (en) * | 2018-11-27 | 2019-12-27 | 清华大学 | In-situ extraction and online mass spectrum detection device for living unicells |
Non-Patent Citations (3)
| Title |
|---|
| QIANG ZHANG等: "In Situ Partial Treatment of Single Cells by Laminar Flow in the "Open Space"", 《 ANAL. CHEM.》 * |
| SIFENG MAO等: "Chemical operations on a living single cell by open microfluidics for wound repair studies and organelle transport analysis", 《CHEM. SCI.》 * |
| SIFENG MAO等: "Measurement of Cell−Matrix Adhesion at Single-Cell Resolution for Revealing the Functions of Biomaterials for Adherent Cell Culture", 《ANAL. CHEM.》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899157A (en) * | 2020-12-28 | 2021-06-04 | 中国科学院长春应用化学研究所 | Micro-fluidic chip light stimulation device, yeast single cell light regulation gene expression method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DK170721B1 (en) | Cell Selection System and Method | |
| US7919319B2 (en) | Cultured cell and method and apparatus for cell culture | |
| Sivagnanam et al. | Exploring living multicellular organisms, organs, and tissues using microfluidic systems | |
| Bruckbauer et al. | Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking | |
| Kimlin et al. | 3D in vitro tissue models and their potential for drug screening | |
| Hutzler et al. | High-resolution multitransistor array recording of electrical field potentials in cultured brain slices | |
| JP5278913B2 (en) | Single cell capture microchannel device | |
| US9632076B2 (en) | Microfluidic device for generating neural cells to simulate post-stroke conditions | |
| Knöll et al. | Stripe assay to examine axonal guidance and cell migration | |
| JP2001500744A (en) | Method and apparatus for retaining cells | |
| JP2017063716A (en) | Cell analysis method, cell analysis chip, cell analysis reagent, cell analysis kit, and cell analysis apparatus | |
| CN108138109A (en) | Sampling system | |
| Vasaturo et al. | A novel chemotaxis assay in 3-D collagen gels by time-lapse microscopy | |
| Burks et al. | Laser direct‐write onto live tissues: a novel model for studying cancer cell migration | |
| Gross et al. | A closed flow chamber for long-term multichannel recording and optical monitoring | |
| CN111808750A (en) | Micro-fluidic chip device, in-situ detection method for living unicells and application | |
| JP2015536141A (en) | Ex vivo microfluidic analysis of biological samples | |
| JP7199427B2 (en) | An ex vivo model of inflamed human skin and its use for screening anti-inflammatory compounds | |
| WO2015192038A1 (en) | Microfluidic devices, systems, and methods for evaluating tissue samples | |
| US20240060964A1 (en) | Coordinately-ordered single cells with individual identities for high-throughput assay | |
| CN111033238A (en) | Quantitative liquid biopsy diagnostic system and method | |
| Ho et al. | Mini chamber system for long-term maintenance and observation of cultured cells | |
| Saksena et al. | The Power of CAD/CAM Laser Bioprinting at the Single-Cell Level: Evolution of Printing | |
| Schiffenbauer et al. | A cell chip for sequential imaging of individual non-adherent live cells reveals transients and oscillations | |
| Tan et al. | Skin‐ny deeping: Uncovering immune cell behavior and function through imaging techniques |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201023 |
|
| RJ01 | Rejection of invention patent application after publication |