CN114836314A - High-throughput microfluidic system for researching mechanical and biochemical signal induced single cell kinetic response and using method thereof - Google Patents
High-throughput microfluidic system for researching mechanical and biochemical signal induced single cell kinetic response and using method thereof Download PDFInfo
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Abstract
A high-flux microfluidic system for researching mechanical and biochemical signal induced single cell kinetic response and a using method thereof belong to the field of cell biology experimental devices. The system comprises a micro-fluidic chip and a peripheral loading, detecting and controlling device. The loading device is combined with the control device, can capture or control a large number of single cells, and accurately loads different dynamic mechanical and biochemical stimulation signals to the cells. The control device receives dynamic image information such as single cell deformation, motion tracks, biochemical signal space-time distribution and the like observed in real time from the detection device and pressure/flow values in the loading device, so that the loading device is fed back and controlled, the single cell deformation process is controlled, the microchannel flow field and biochemical signal transmission are accurately controlled, and the in-vivo cell dynamic mechanics and biochemical microenvironment are accurately simulated. The invention can realize the high-efficiency capture and control of a large number of single cells and is used for analyzing the dynamic response of the single cells under the stimulation of different dynamic mechanical and biochemical signals, the mechanism thereof and other cell biological researches.
Description
Technical Field
The invention belongs to the field of cell biology experimental devices, and relates to a high-throughput microfluidic system which is designed based on hydrodynamics and microfluidic chip technology, consists of a microfluidic chip and a peripheral loading, detecting and controlling device, and is used for quantitatively researching dynamic mechanics and biochemical signal induced single cell kinetic response.
Background
In a complex dynamic flow microenvironment formed by peripheral histiocytes, intercellular substance and body fluid, cells are stimulated not only by mechanical signals such as fluid shear force, positive pressure and tension force in the microenvironment, but also by the concentration signals of biochemical substances such as biochemical factors, hormones and neurotransmitters in the microenvironment. The cell can recognize mechanical and biochemical signals from an extracellular microenvironment, transduces the signals through various signal pathways and transmits the signals to the interior of the cell, so as to cause a series of cell events such as change of intracellular second messenger concentration and transcription level, gene expression, protein synthesis and the like, and present multi-scale time and space dynamic responses, namely the dynamic responses of the cell, wherein the dynamic responses are closely related to functions and behaviors such as cell division, differentiation, proliferation, apoptosis and the like.
Unlike classical single cell biology which focuses on studying the molecular omics and the signs, the morphology, the subcellular structure and the functions of organelles of cells, the cytodynamics study regards mechanical signals and biochemical signals in an extracellular microenvironment as input, cell stress deformation, intracellular biochemical signal dynamics response and the like as output, and a cellular mechanical and biochemical signal system as a power system, develops the study from the viewpoint of system dynamics, and emphasizes the dynamic changes of cellular events and the influence of internal and external environmental factors on the cellular structure, the functions and the overall behaviors. Particularly, single cell dynamics taking single cells as a research object emphasizes the difference between single cell mechanical properties and single cell signal dynamics characteristics among individuals, is expected to be developed into important new content and new means of single cell analysis, and provides more comprehensive dynamic quantitative information for the aspects of mechanism exploration, disease diagnosis, drug evaluation and the like of occurrence and development of human serious diseases.
Due to extremely complex in-vivo microenvironment, a plurality of interference factors which are difficult to control exist in-vivo research of cell dynamics, and in-vitro model research can exclude the interference factors to a great extent so as to realize parameter controllable observation, so that the in-vitro model research is a currently accepted feasible research means in the field. Under the common efforts of many researchers, a plurality of in vitro experiment technology platforms such as a parallel plate flow cavity, an atomic force microscope, optical tweezers, micro tube sucking and the like are proposed. The cell dynamics research based on the parallel flat plate flow cavity system mostly takes a cell colony as a research object, input mechanical signals (mostly approximate to the shear stress of the wall surface at the bottom of the flow cavity) are not accurate stress of cells, output signals are average values of a group of cell responses, and the individual differences of mechanical properties and kinetic signal characteristic parameters of heterogeneous or homogeneous cells cannot be accurately analyzed; while the atomic force microscope, the optical tweezers, the micro-tube sucking and the like can realize the high-precision measurement of the mechanical property of the single cell, the mechanical and biochemical stimulation environment close to the human body is not easy to provide, and the defects of complex operation, low detection flux and the like exist at the same time, so that the high-flux single cell kinetic experiment measurement cannot be carried out. In recent years, the micro-fluidic chip technology provides a physiological environment more close to the human body by using a specific fluid dynamic control system, and can load mechanical and biochemical signals simultaneously and accurately control the movement of single cells with high flux, so that the micro-fluidic chip technology becomes one of the mainstream platforms for accurately controlling single cells and the microenvironment thereof at present, and is suitable for single cell mechanical and mechanical biological analysis and single cell signal dynamics research. The existing high-throughput microfluidic chips for single cell kinetic analysis are mainly divided into two types: one is a flow cytometry class, which is based on the flow cytometry principle to extrude a single cell through a narrow single channel along with fluid, and realizes the high-flux control of the single cell by increasing the fluid speed, thereby being very suitable for the high-flux detection of the stress deformation of the single cell, but mainly used for the mechanical signal loading of the single cell and the research under the cooperative stimulation of biochemical factors lacking; the other type is microarray type, which is based on the microstructure array to carry out high-throughput capture on single cells, and realizes single cell sucking or biochemical signal transmission to realize dynamic biochemical signal loading by controlling single cell external flow field and pressure distribution. Therefore, a high-throughput microfluidic platform capable of accurately and quantitatively researching the single-cell kinetic response under the synergistic stimulation of different dynamic mechanical and biochemical signals is urgently needed.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip which can accurately load a large number of single cells and is stimulated by dynamic mechanical and biochemical signals, and a micro-fluidic system which can be used for realizing high-throughput and quantitative single cell dynamics research is constructed by integrating peripheral loading, detection and control devices. The invention combines the fluid mechanics principle and the micro-fluidic technology, takes a space-time concentration gradient generator formed by combining a Christmas tree and a Y-shaped channel and a single-cell control micro-fluidic chip designed based on the standing-point flow principle and the channel boundary effect as main bodies, realizes the stable capture or movement control of a large number of single cells by improving the structural design and the geometric dimension of different micro-channels and regulating and controlling inlet solution and flow thereof by utilizing a loading device, and quantitatively loads the stimulation of mechanical and biochemical signals with different space-time distributions. The control device receives dynamic image information such as single cell deformation, motion tracks, biochemical signal space-time distribution and the like measured by the detection device and pressure/flow values of all injection pumps in the loading device, so that the loading device is fed back and controlled, the single cell deformation process is controlled, a micro-channel flow field and biological signal transmission are accurately controlled, dynamic mechanics and biochemical microenvironment of cells in a body are accurately simulated, and multi-form combined stimulation of mechanical and biochemical signals is accurately loaded on the captured single cells.
The technical scheme of the invention is as follows:
a high-throughput microfluidic system for researching mechanical and biochemical signal induced single cell kinetic response is a microfluidic system which is combined with a microfluidic chip and a peripheral loading, detecting and controlling device and is used for high-throughput single cell kinetic research, and is shown in figure 1. The micro-fluidic chip comprises a space-time concentration gradient generator formed by combining a Christmas tree and a Y-shaped channel, a cell suspension inlet, a single cell control unit and a chip outlet, as shown in figure 2. The outlet of the space-time concentration gradient generator and the inlet of the cell suspension liquid, which are formed by combining the Christmas tree and the Y-shaped channels, are communicated with the inlet of the single cell control unit, and the outlet of the chip is connected with the output channel of the single cell control unit.
The space-time concentration gradient generator (simply referred to as the space-time concentration gradient generator) formed by the combination of the Christmas tree and the Y-shaped channel comprises a biochemical stimulation solution inlet 1, a biochemical stimulation solution inlet 2, a Christmas tree-shaped micro-channel and a dynamic buffer solution inlet; the biochemical stimulation solution inlet 1 and the biochemical stimulation solution inlet 2 are both communicated with the inlet end of the Christmas tree type microchannel, and the outlet end of the Christmas tree type microchannel is converged and then converged with the dynamic buffer solution inlet and then connected with the inlet of the single cell control unit;
the single cell control unit comprises a cell flow cavity, a cell deformation micro-channel array, a resistance channel 1, a resistance channel 2 and an output channel; cell flow chamber is by curve boundary 1, curve boundary 2, the nearly triangle-shaped chamber that straight line boundary back end and straight line boundary anterior segment enclose in proper order, and curve boundary 1 and straight line boundary anterior segment intersection are equipped with the export that accesss to resistance passageway 1, and curve boundary 2 and straight line boundary back end intersection are equipped with the export that accesss to resistance passageway 2, and resistance passageway 1 and resistance passageway 2 collect output channel to the centre, and cell deformation microchannel array is in between output channel and the cell flow chamber, promptly: the cell flow cavity comprises an inlet, an upper outlet, an array outlet and a lower outlet, the inlet is communicated with the outlet of the space-time concentration gradient generator and the cell suspension inlet, the upper outlet and the lower outlet are respectively communicated with the resistance channel 1 and the resistance channel 2, and the array outlet is communicated with the cell deformation micro-channel array; the cell deformation micro-channel array is composed of a plurality of cell deformation micro-channels, and is merged with the resistance channel 1 and the resistance channel 2 to an output channel communicated with an outlet, as shown in fig. 3.
The cell injection and dynamic mechanical and biochemical signal loading device comprises 4 groups of programmable injection pumps and injectors, wherein the 4 injectors are respectively communicated with a biochemical stimulation solution inlet 1, a biochemical stimulation solution inlet 2, a dynamic buffer solution inlet and a cell suspension liquid inlet and are used for injecting cells into the chip and injecting a biochemical stimulation solution and a cell culture medium;
the single cell dynamics experiment detection device comprises an inverted fluorescence microscope, a pressure sensor and a flow sensor, and is used for monitoring dynamic image information of single cell deformation, a motion track, biochemical signal space-time distribution and pressure/flow values of each injection pump in the loading device in real time;
the control device is a computer, is respectively connected with the loading device and the experiment detection device, and is used for receiving cell images, fluorescence signals and sensor data and driving a programmable injection pump in the loading device to accurately control the injection flow.
The micro-fluidic system is mainly used for realizing the following functions, namely, the efficient capture and control of a large number of single cells, and the accurate application of dynamic mechanical and biochemical signal stimulation to each captured single cell so as to explore and analyze the dynamic response of the single cell. In order to realize the functions, the specific design principle is as follows:
efficient capture and manipulation of single cells
The single cell capture and control are mainly realized by a single cell control unit. Because the height of the cell flow cavity is far smaller than the width and the length of the cell flow cavity, and the size is in the micrometer level, according to the fluid mechanics principle, the liquid flow in the cell flow cavity is mainly influenced by the pressure gradient and the friction force of the upper and lower parallel flat plates, the influence of the friction force of the side boundary is negligible, and the average flow speed after averaging along the height direction of the cell flow cavity
Can be found by a method similar to that for processing the planar potential flow. The thin lines can be determined according to the shapes of streamline and equipotential lines determined by known complex potentialThe cell flow cavity is formed by the boundary of the cell flow cavity and has a fluid stagnation point.
Taking the coordinate system as shown in FIG. 4, complex potential is introduced on the Z-x + iy plane
In the formula
And phi (x, y) are the average flow velocities, respectively
The potential function and the flow function of (c),
selecting the more common flow complex potential W (Z) AZ n (where A is a real number, n > 1), and Z-x + iy-re iθ R (cos θ + isin θ). Wherein r is the modulus of Z and θ is the argument of Z. When n is 2, the potential function
The sum flow function phi (x, y) will satisfy
And
φ(x,y)＝Ar 2 sin(2θ) (3)
thus, the equipotential and streamline are respectively
r 2 cos(2θ)＝const (4)
And
r 2 sin(2θ)＝const (5)
FIG. 4 shows the complex potential W (Z) -AZ 2 Determining the distribution condition of the planar potential flow field, wherein the solid line is a streamline, the dotted line is an equipotential line, and the flow velocity at the coordinate origin is zeroI.e., the fluid stagnation point, to thereby construct the cell flow lumen shown in the figure. The two curved boundaries 1 and 2 of the flow chamber coincide with the streamlines that are symmetrical along the x-axis, satisfying the equation
And
in the formula
Wherein the corresponding coordinate system is based on complex potential W (Z) ═ AZ 2 ＝A(x+iy) 2 ＝A(re iθ ) 2 Determined coordinate system, L c Length of cell flow lumen, W c For the cell flow lumen entrance width, θ and r are the polar angle and radius of the point in the polar coordinate system, r 0 Is the polar diameter, θ, of the right end of the curve boundary 1 0 Is the polar angle, θ, of the end point to the right of the curve boundary 1 1 Is the polar angle of the left end point of the curved boundary 1.
The front section and the rear section of the linear boundary coincide with two streamline passing through the original point to satisfy the equation
Further, the linear boundary of the cell flow chamber is communicated with a plurality of cell deformation micro-channels arranged in parallel (as shown in fig. 3), and the curved boundary 1 and the curved boundary 2 symmetrically extend along the central axis of the cell flow chamber and are respectively communicated with the resistance channel 1 and the resistance channel 2, so that the whole cell flow chamber is formedThe somatic single-cell manipulation unit can be equivalent to the circuit diagram shown in fig. 5. Assuming that the total flow rate into the cell flow chamber is Q, the flow rates through the resistance channel 1 and the resistance channel 2 are Q, respectively 1 And Q 2 The total flow through each cell deformation microchannel is Q m Thus there are
Q＝Q 1 +Q m +Q 2 (11)
In the formula (I), the compound is shown in the specification,
n is the total number of cell deformation micro-channels, Q mi The flow through the ith cell was modified by the flow through the microchannel.
Analogizing to the circuit theory, in the flow field, the pressure difference Δ P between each fluid stagnation point (i.e. each cell deformation micro-channel inlet position) and the resistance channel outlet and the flow rate of each channel satisfy the following relation
Q 1 R 1 ＝Q m1 R m1 ＝…＝Q mi R mi ＝…＝Q mN R mN ＝Q 2 R 2 ＝ΔP (12)
Wherein R is 1 And R 2 Flow resistances, R, of the resistance channel 1 and the resistance channel 2, respectively mi The flow resistance of the microchannel is deformed for the ith cell. When the aspect ratio of the rectangular microchannel is large (i.e. W > H or W < H), the flow resistance of the microchannel is
Therefore, the geometric dimensions (i.e., length L, width W, and height H) of the channel-tunable microchannel adjust the amount of flow resistance of the microchannel.
When single cell capture or control is carried out, the biochemical stimulation solution inlet 1, the biochemical stimulation solution inlet 2 and the dynamic buffer solution inlet are closed, and the cell suspension inlet is opened. According to the relationship between flow conservation and flow resistance, the specific capture mechanism is as follows: by adjusting the geometric dimensions of the resistance channel and the cell deformation micro-channel, the initial cell deformation micro-channel is enabledFlow resistance R mi Less than resistance channel flow resistance (R) 1 Or R 2 ) After the cell suspension flows into the cell flow cavity, the cell will flow to a certain cell deformation micro-channel i along the streamline direction of the flow field in the flow cavity, and because the size of the entrance of the cell deformation micro-channel is smaller than that of the cell, under the condition of proper entrance flow or pressure, the single cell will be captured at the entrance of the deformation micro-channel i, so that the cell deformation micro-channel is closed, and the flow resistance R is low mi Increase, resulting in a flow rate Q mi Decrease, flow rate Q mk (k ≠ i) increases, and since R mk Is also less than resistance channel flow resistance (R) 1 Or R 2 ) Therefore, the rest cells flow to the deformed micro-channel of other cells to be captured, thereby realizing the capture of a large number of single cells; when all the cell deformation micro-channels are closed, the rest of the cells will flow out of the two resistance channels. When the flow field in the cell flow cavity is changed by external disturbance, the captured cells deviate from the original capture points or pass through the cell deformation micro-channel due to deformation, the cell deformation micro-channel is opened at the moment, the flow is increased, the deviated cells are pulled back to the set cell capture points again or other cells continue to flow to the micro-channel to be captured, and therefore the efficient capture of the single cells can be realized. When each cell deformation micro-channel is blocked by cells, a fluid stagnation point is formed at the inlet position of each cell deformation micro-channel, and then the distribution conditions of the flow field around the cells are basically consistent.
Because the cell can be deformed, the inlet flow or pressure is regulated by the control device, the cell can be captured at the inlet position of the cell deformation micro-channel and can also pass through the micro-channel, and therefore, the micro-fluidic system can realize the single cell control.
(II) quantitative loading and control of dynamic mechanical and biochemical signals
After the single cell is captured, closing the cell suspension inlet, opening the biochemical stimulation solution inlet 1, the biochemical stimulation solution inlet 2 and the dynamic buffer solution inlet, and quantitatively loading mechanical and biochemical signals to the captured cell for stimulation.
According to the relation between the flow and the flow resistance, because the proportional relation between the flow resistance and the flow resistance is known after the geometric dimension of the micro-channel is fixed, the mechanical signal received by the captured cell is mainly determined by the total flow of the inlet, so the force acting on the cell can be quantized; the flow value of each injection pump in the loading device is controlled by the control device to change in any form along with time, so that mechanical signals with different waveforms are applied to the captured cells; when the cell deformation micro-channels are completely the same, the mechanical signals received by each cell are also the same. In addition, as described above, by adjusting the inlet flow rate or pressure, the cell can be passed through the cell deformation micro-channel, so as to study the single cell deformation and dynamic response of the cell in the process of passing through the micro-channel, and in this case, by changing the geometrical shape of the cell deformation micro-channel, different mechanical stimulation modes can be applied to the single cell in the process of passing through. As shown in FIG. 6, when the cell deformation micro-channel is a straight channel, the mechanical stimulation mode of the cell in the process of passing through is constant; when the cell deformation micro-channel is a variable cross-section channel, the mechanical stimulation is gradually changed, or gradually increased, or gradually decreased; and a periodically-changed mechanical stimulation mode can be realized, and mechanical stimulation in a form of existence, absence, presence and absence is realized.
Likewise, the loading means in combination with the control means may load the captured cells with different spatiotemporal distributions of biochemical signal stimuli. In order to enhance the mixing effect of biochemical stimulators in the Christmas tree type micro-channel, the Christmas tree type micro-channel is designed into a structure which is similar to a structure that wide channels and narrow channels are in cross circulation in a shape of a Chinese chikungunya; in order to ensure the linear effect of the concentration gradient formed at the outlet of the Christmas tree type microchannel in the transverse direction, the outlet width of the last stage branch channel of the Christmas tree type microchannel is designed according to a certain proportion according to the quantitative relation between the expected concentration and the space distance, as shown in the detail enlargement of figure 2. The method for loading the capture cells with dynamic biochemical signals is as follows: biochemical stimulation solution with constant and equal flow and cell culture medium without biochemical factors are respectively introduced into a biochemical stimulation solution inlet 1 and a biochemical stimulation solution inlet 2 of the space-time concentration gradient generator, and biochemical stimulation concentration gradients which are linearly and spatially distributed along the transverse direction can be generated at a Christmas tree type micro-channel outlet (communicated with a dynamic buffer solution inlet) according to the fluid mechanics and material transmission principle; and the cell culture medium with the flow changing along with the time is introduced into the dynamic buffer solution inlet, and dynamic biochemical signals with linear space distribution can be generated at the outlet of the space-time concentration gradient generator (the inlet of the cell flow cavity), so that biochemical signal stimulation with different space-time distribution can be accurately applied to each captured cell. In addition, the biochemical stimulation solution with constant and equal flow can be introduced into the biochemical stimulation solution inlet 1 and the biochemical stimulation solution inlet 2, the cell culture medium with the flow changing along with time is introduced into the dynamic buffer solution inlet, and at the moment, a dynamic biochemical signal without space gradient can be generated at the outlet of the space-time concentration gradient generator, so that the same dynamic biochemical signal stimulation is applied to the captured cells. Further, the flow value of the corresponding injection pump in the loading device is controlled by the control device to change in a desired function form along with time, so that different biochemical signals are applied to the captured cells.
In addition, in order to prevent the impurities such as external micro foreign matters, cell debris or PDMS debris from blocking the microchannel or adhering to the side wall of the microchannel to affect the internal flow environment, the four inlets of the microfluidic chip, i.e., the biochemical stimulation solution inlet 1, the biochemical stimulation solution inlet 2, the dynamic buffer solution inlet and the cell suspension inlet, are all connected to an impurity filter, and the filter structure is shown in fig. 7.
The invention has the advantages that the invention can efficiently capture and control a large number of single cells, apply different dynamic mechanical and biochemical signal stimulation to the captured cells, can be used for researching the dynamic response of the single cells under the stimulation of different dynamic mechanical and biochemical signals, and can also be widely used for researching the cell biology research experiment of the dynamic cell environment regulation and control of the biological behavior and mechanism of the isolated cells.
Drawings
FIG. 1 is a schematic diagram of a high throughput microfluidic system for studying the mechanical and biochemical signal-induced single cell kinetic responses.
Fig. 2 is a top view of the structure of the microfluidic chip.
FIG. 3 is a schematic diagram of a single cell manipulation unit.
FIG. 4 is a graph based on the complex potential W (Z) AZ 2 Determined planar potential flow field distribution structureSchematic diagram of the cell flow chamber.
FIG. 5 is an equivalent schematic diagram of a single cell trapping mechanism.
FIG. 6 is an example of three cell deformation microchannels; wherein, (1) cell deformation microchannel example 1: straight channel, (2) cell deformation microchannel example 2: variable cross-section channel, (3) cell deformation microchannel example 3: the channels are periodically changed.
FIG. 7 is a top view of a filter construction; the white areas are obstacles.
In the figure: (A) the device comprises a micro-fluidic chip, (B) a cell injection and dynamic mechanical and biochemical signal loading device, (C) a single cell dynamics experiment detection device and (D) a control device; a space-time concentration gradient generator formed by combining an I 'Christmas tree' and a Y-shaped channel, and a II single cell control unit; an S1 biochemical stimulation solution inlet 1, an S2 biochemical stimulation solution inlet 2, a B1 dynamic buffer solution inlet, a C1 cell suspension inlet and an O1 chip outlet; a cell flow cavity, a cell deformation micro-channel array, a resistance channel 1, a resistance channel 2 and an output channel, wherein the 1-1 curve boundary 1, the 1-2 curve boundary 2, the 1-3a straight line boundary front section and the 1-3b straight line boundary rear section are arranged in the output channel; w c Cell flow lumen inlet width, L c Cell flow lumen length; q 1 Flow through resistance channel 1, Q 2 Flow through the resistance channel 2, Q mi Flow through the ith cell deformation microchannel, R c Flow resistance of cell flow lumen, R 1 Resistance channel 1 flow resistance, R 2 Resistance channel 2 flow resistance, R mi Flow resistance, R, of the ith cell-deforming Microchannel out The output channel is flow resistant.
Detailed Description
As shown in figure 1, the invention is a microfluidic system for high-throughput single cell dynamics research, which comprises a microfluidic chip A, a cell injection and dynamic mechanics and biochemical signal loading device B, a single cell dynamics experiment detection device C and a control device D.
The microfluidic chip A is composed of a space-time concentration gradient generator I formed by combining a Christmas tree + Y-shaped channels, a cell suspension inlet C1, a single cell control unit II and a chip outlet O1, and is shown in figure 2. Wherein, the space-time concentration gradient generator I formed by the combination of the Christmas tree and the Y-shaped channel comprises a biochemical stimulation solution inlet 1S1, a biochemical stimulation solution inlet 2S2, a Christmas tree-shaped micro-channel and a dynamic buffer solution inlet B1; the single cell control unit II consists of a cell flow cavity I, a cell deformation micro-channel array II, a resistance channel 1, a resistance channel 2 and an output channel V; the cell flow cavity (I) is surrounded by a curve boundary (11-1), a curve boundary (21-2), a straight boundary front section (1-3a) and a straight boundary rear section (1-3b) and comprises two inlets, an upper outlet, an array outlet and a lower outlet, the inlets are communicated with an outlet of a space-time concentration gradient generator and an inlet of cell suspension, the upper outlet and the lower outlet are respectively communicated with a resistance channel (1) and a resistance channel (2), and the array outlet is communicated with a cell deformation micro-channel array (II); the cell deformation micro-channel array is composed of 70 straight channel cell deformation micro-channels, and is converged to an output channel (fifthly) communicated with an outlet O1 together with a resistance channel 1 and a resistance channel 2, as shown in figure 3. The four inlets of the microfluidic chip, namely the biochemical stimulation solution inlet 1S1, the biochemical stimulation solution inlet 2S2, the dynamic buffer solution inlet B1 and the cell suspension inlet C1, are all connected to an impurity filter, and the structure of the filter is shown in fig. 7.
All the microchannels of the microfluidic chip A are manufactured by adopting a standardized micromachining method and using PDMS (polydimethylsiloxane), and finally, the microchannels are permanently bonded with a clean cover glass to form a sealed and transparent glass-PDMS type chip with good biocompatibility. The structural parameters of the microchannel are as follows: the width of narrow channel and wide channel of "Christmas tree" type microchannel is 40 micrometers and 100 micrometers respectively, and the length L of cell flow cavity c And an inlet width W c 5 mm and 500 micron, the length and width of the straight channel in the cell deformation micro-channel array are 50 micron and 10 micron, the length of the resistance channel 1 and the length of the resistance channel 2 are 3.4 cm and the width are 150 micron, the length and width of the output channel are 7.5 mm and 2 mm, the side length of the square barrier in the filter is 10 micron, 45 micron and 120 micron, and the height of all micro-channels is 30 micron.
The cell injection and dynamic mechanical and biochemical signal loading device B is composed of 4 groups of programmable injection pumps and injectors, wherein the 4 injectors are respectively communicated with a biochemical stimulation solution inlet 1S1, a biochemical stimulation solution inlet 2S2, a dynamic buffer solution inlet B1 and a cell suspension liquid inlet C1, and are used for injecting cells into the chip and injecting biochemical stimulation solution and cell culture medium.
The single cell dynamics experiment detection device C comprises an inverted fluorescence microscope, a pressure sensor and a flow sensor, and is used for monitoring dynamic image information such as single cell deformation, motion tracks, biochemical signal space-time distribution and the like in real time and pressure/flow values of all injection pumps in the loading device.
The control device D is a computer, is respectively connected with the loading device B and the experiment detection device C, and is used for receiving cell images, fluorescence signals and sensor data and driving a programmable injection pump in the loading device B to accurately control the injection flow.
To study the kinetic response of single cells under stimulation by mechanical and biochemical signals, the experimental procedure was as follows:
step one, efficient capture and control of a large number of single cells
After all devices in the microfluidic system are communicated, a cell suspension inlet C1 is opened, a biochemical stimulation solution inlet 1S1, a biochemical stimulation solution inlet 2S2 and a dynamic buffer solution inlet B1 are closed, cell suspension is injected into a cell flow cavity I, an injection pump connected with a cell suspension injector in a loading device B is controlled by a control device D, and the flow is regulated to realize that a large number of single cells are efficiently captured in a cell deformation microchannel array II or controllably pass through the cell deformation microchannel.
Step two, quantitative loading and control of different dynamic mechanical and biochemical signals, and dynamic response to real-time measurement and monitoring of single cells
After the cells are captured, cell suspension inlet C1 is closed, and biochemical stimulation solution inlet 1S1, biochemical stimulation solution inlet 2S2, and dynamic buffer inlet B1 are opened. The biochemical stimulation solution with the same constant and equal flow rate is introduced into the biochemical stimulation solution inlet 1S1 and the biochemical stimulation solution inlet 2S2, and the cell culture medium with the flow rate dynamically changing along with time is introduced into the dynamic buffer solution inlet B1, so that the same dynamic mechanical and biochemical signal stimulation can be applied to each captured cell; or the biochemical stimulation solution and the cell culture medium with constant and equal flow rates are respectively introduced into the biochemical stimulation solution inlet 1S1 and the biochemical stimulation solution inlet 2S2, and the cell culture medium with the flow rate dynamically changing along with time is introduced into the dynamic buffer solution inlet B1, so that the same dynamic mechanical signal stimulation and the dynamic biochemical signal with linear spatial distribution can be applied to each captured cell. The single cell dynamics response process under the stimulation of different dynamic mechanics and biochemical signal signals is monitored and recorded in real time through a fluorescence microscope, and cell images, fluorescence signals and the like are fed back to the control device D.
In conclusion, the invention can efficiently capture and control a large number of single cells, load different dynamic mechanical and biochemical signal stimuli to the single cells, and can be used for analyzing the biological behavior of the micro-flow environment and quantitatively regulating and controlling the isolated cells and the cell biology research of the mechanism of the isolated cells under the single cell level.
Claims (5)
1. A high-throughput microfluidic system for researching mechanical and biochemical signal induced single cell kinetic response is characterized by comprising a microfluidic chip (A), a cell injection and dynamic mechanical and biochemical signal loading device (B), a single cell kinetic experiment detection device (C) and a control device (D);
the micro-fluidic chip (A) comprises a space-time concentration gradient generator (I) formed by combining a Christmas tree + Y-shaped channel, a cell suspension inlet (C1), a single cell control unit (II) and a chip outlet (O1), wherein the space-time concentration gradient generator (I) outlet and the cell suspension inlet (C1) formed by combining the Christmas tree + Y-shaped channel are communicated with the inlet of the single cell control unit (II), and the chip outlet (O1) is connected with an output channel (fifth) of the single cell control unit (II);
the space-time concentration gradient generator (I) formed by combining the Christmas tree and the Y-shaped channel comprises a biochemical stimulation solution inlet 1(S1), a biochemical stimulation solution inlet 2(S2), a Christmas tree-shaped microchannel and a dynamic buffer solution inlet (B1), wherein the biochemical stimulation solution inlet 1(S1) and the biochemical stimulation solution inlet 2(S2) are communicated with the inlet end of the Christmas tree-shaped microchannel, the outlet ends of the Christmas tree-shaped microchannel are converged and then converged with the dynamic buffer solution inlet (B1), and then the outlet ends of the Christmas tree-shaped microchannel are connected with the inlet of the single-cell control unit (II);
the single cell control unit (II) comprises a cell flow cavity (I), a cell deformation micro-channel array (II), a resistance channel 1 (III), a resistance channel 2 (II) and an output channel (fifth), wherein the cell flow cavity (I) is a nearly triangular cavity which is sequentially enclosed by a curve boundary 1(1-1), a curve boundary 2(1-2), a straight line boundary rear section (1-3b) and a straight line boundary front section (1-3a), an outlet leading to the resistance channel 1 (III) is arranged at the intersection of the curve boundary 1(1-1) and the straight line boundary front section (1-3a), an outlet leading to the resistance channel 2 (IV) is arranged at the intersection of the curve boundary 2(1-2) and the straight line boundary rear section (1-3b), the resistance channel 1 (III) and the resistance channel 2 (IV) are converged to the output channel (fifth) towards the middle, the cell deformation micro-channel array (II) is positioned between the output channel (fifth) and the cell flow cavity (first); the cell deformation micro-channel array (II) is composed of a plurality of cell deformation micro-channels;
the cell injection and dynamic mechanical and biochemical signal loading device (B) comprises 4 groups of programmable injection pumps and injectors, wherein the 4 injectors are respectively communicated with a biochemical stimulation solution inlet 1(S1), a biochemical stimulation solution inlet 2(S2), a dynamic buffer solution inlet (B1) and a cell suspension inlet (C1) and are used for injecting cells into the chip and injecting biochemical stimulation solution and cell culture medium;
the single cell dynamics experiment detection device (C) comprises an inverted fluorescence microscope, a pressure sensor and a flow sensor, and is used for monitoring dynamic image information of single cell deformation, a motion track, biochemical signal space-time distribution and pressure/flow values of each injection pump in the loading device in real time;
and the control device (D) is a computer, is respectively connected with the loading device (B) and the experiment detection device (C), and is used for receiving cell images, fluorescence signals and sensor data and driving a programmable injection pump in the loading device (B) to accurately control the injection flow.
2. High throughput according to claim 1The microfluidic system is characterized in that the boundary of the cell flow cavity (r) is based on the complex potential W (Z) -AZ 2 And (3) constructing the determined planar potential flow field distribution, wherein the curve boundary 1(1-1) and the curve boundary 2(1-2) satisfy the formula:
and
in the formula
Wherein the corresponding coordinate system is based on complex potential W (Z) ═ AZ 2 ＝A(x+iy) 2 ＝A(re iθ ) 2 Determined coordinate system, L c The length of the cell flow lumen (r), W c Is the inlet width of the cell flow lumen (r), theta and r are the polar angle and the polar diameter of a point in a polar coordinate system, r 0 Is the polar diameter, θ, of the end point on the right side of the curve boundary 1(1-1) 0 Is the polar angle, θ, of the end point on the right side of the curve boundary 1(1-1) 1 Is the polar angle of the left end point of the curve boundary 1 (1-1);
the front section (1-3a) of the straight line boundary, the rear section (1-3b) of the straight line boundary and two streamline passing through the origin are coincided to satisfy the formula
3. The high-throughput microfluidic system according to claim 1 or 2, wherein the cell deformation microchannel array (ii) is located on the straight line boundary of the cell flow cavity (i), and when the cell is captured, a fluid stagnation point is formed at the entrance of the cell deformation microchannel; the structure of the cell deformation micro-channel can be adjusted according to research needs, and the cell deformation micro-channel can comprise a straight channel, a variable cross-section channel and a width periodic variation channel.
4. The high-throughput microfluidic system according to claim 1 or 2, wherein the biochemical stimulation solution inlet 1(S1), the biochemical stimulation solution inlet 2(S2), the dynamic buffer solution inlet (B1) and the cell suspension inlet (C1) of the microfluidic chip (a) are connected to an impurity filter.
5. The method of using the high throughput microfluidic system of any one of claims 1-4, wherein the steps are as follows:
step one, efficient capture and control of a large number of single cells
After all devices in the high-flux microfluidic system are communicated, opening a cell suspension inlet (C1), closing a biochemical stimulation solution inlet (S1), a biochemical stimulation solution inlet (S2) and a dynamic buffer solution inlet (B1), injecting the cell suspension into a cell flow cavity (I), controlling an injection pump connected with a cell suspension injector in a loading device (B) through a control device (D), and adjusting the flow rate to realize the efficient capture of a large number of single cells in a cell deformation microchannel array (II) or the controlled passing of the single cells through the cell deformation microchannel;
step two, quantitative loading and control of different dynamic mechanical and biochemical signals, and dynamic response to real-time measurement and monitoring of single cells
After the cells are captured, closing the cell suspension inlet (C1), and opening the biochemical stimulation solution inlet (S1), the biochemical stimulation solution inlet (S2), and the dynamic buffer solution inlet (B1); introducing the same constant and equal biochemical stimulation solution into the biochemical stimulation solution inlet (S1) and the biochemical stimulation solution inlet (S2), and introducing a cell culture medium with the flow dynamically changing along with time into the dynamic buffer solution inlet (B1), so that the same dynamic mechanical and biochemical signal stimulation can be applied to each captured cell; or respectively introducing a biochemical stimulation solution and a cell culture medium with constant and equal flow rates into the biochemical stimulation solution inlet (S1) and the biochemical stimulation solution inlet (S2), and introducing a cell culture medium with a flow rate dynamically changing along with time into the dynamic buffer solution inlet (B1), so that the same dynamic mechanical signal stimulation and dynamic biochemical signals with linear spatial distribution can be applied to each captured cell; the single cell dynamics response process under the stimulation of different dynamic mechanics and biochemical signal signals is monitored and recorded in real time through a fluorescence microscope, and cell images, fluorescence signals and the like are fed back to a control device (D).
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