CN109810894B - Group cell three-dimensional structure manipulation and construction system and method based on light-induced dielectrophoresis - Google Patents
Group cell three-dimensional structure manipulation and construction system and method based on light-induced dielectrophoresis Download PDFInfo
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- CN109810894B CN109810894B CN201910155647.4A CN201910155647A CN109810894B CN 109810894 B CN109810894 B CN 109810894B CN 201910155647 A CN201910155647 A CN 201910155647A CN 109810894 B CN109810894 B CN 109810894B
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Abstract
The invention discloses a system and a method for manipulating and constructing a three-dimensional structure of group cells based on light-induced dielectrophoresis. The invention utilizes light-induced dielectrophoresis to carry out manipulation and construction of a three-dimensional structure on the group cells in the cell suspension solution, and can accurately control the shape and the size of the three-dimensional structure of the group cells in real time. The indirect cell control mode does not need to directly contact the cells, avoids the pollution to the cells, can supplement and replace the solution in which the cells are positioned at any time, has little influence on the cells, and is favorable for obtaining the cells close to natural growth.
Description
Technical Field
The invention relates to the field of cell manipulation, in particular to the field of manipulation and construction of three-dimensional structures of group cells, and discloses a system and a method for manipulating and constructing the three-dimensional structures of the group cells based on photoinduction dielectrophoresis.
Background
The three-dimensional construction of the cells has great significance for researching the growth and differentiation of the cells and the interaction between the cells, has application in the fields of tumor biology, stem cell culture and the like, and is one of important technologies for researching the physiological activities of the cells.
Most cells in vivo are in a three-dimensional structure, and in vitro three-dimensional culture is more favorable for maintaining and playing cell functions than two-dimensional culture. Conventional technical means include cell scaffolds, porous materials, hydrogels, and the like. The requirement of the cell scaffold on scaffold materials is high; the porous material can only be manufactured into simple shapes, and has low connectivity and long time consumption; hydrogels are costly and may trigger an inflammatory response. At present, a three-dimensional construction mode with low cost, easy operation and small influence on cells is needed to provide help for further research on physiological activities of the cells.
Disclosure of Invention
The invention solves the problems: the defects of the prior art are overcome, the system and the method for manipulating and constructing the three-dimensional structure of the group cells based on the light-induced dielectrophoresis are provided, and the non-contact light-induced dielectrophoresis is used as an operation method, so that the cells are effectively prevented from being polluted. The manipulation precision can reach the micron level, and the method is favorable for accurately constructing a three-dimensional structure. And a special chip and a mechanical structure which are matched for operation are designed, so that the operation difficulty is reduced, and the operation process is simplified. The method has the advantages of low cost, simple operation, little influence on cells and capability of accurately controlling and constructing the three-dimensional structure of the group cells in real time.
The technical scheme of the invention is as follows: in order to achieve the above object, the present invention provides a system for manipulating and constructing three-dimensional structures of group cells based on light-induced dielectrophoresis, comprising: the device comprises a first computer (10), a projector (11), two positive lenses (12), a reflector (13), a three-dimensional displacement platform (14), a chip (15), an air pump (16), a signal generator (17), a microscope (18), a CCD (19) and a second computer (110); the upper part of the chip (15) is ITO glass (22) with a step structure, the lower part of the chip is flat ITO glass (24) with a step frame, and the upper surface of the chip is plated with a hydrogenated amorphous silicon layer (23); a light pattern (27) designed by a first computer (10) is projected by a projector (11), and finally converged and projected to a hydrogenated amorphous silicon layer (23) fixed at the lower part of a chip (15) of a three-dimensional displacement platform (14) through two positive lenses (12) and a reflector (13), an electric signal output by a signal generator (17) is loaded to ITO glass (22) with a step structure and flat ITO glass (24) with a step frame, the cell movement condition is observed in real time through a microscope (18), a CCD (19) and a second computer (110), the three-dimensional structure of the grouped cells can be conveniently and efficiently obtained, and an air pump (16) is used for supplementing solution to ensure the normal growth of the cells and replacing different solutions when observing the physiological activities of the cells.
The chip (15) is placed horizontally or vertically to ensure that light is projected to the hydrogenated amorphous silicon layer (23).
The upper part of the chip (15) is provided with ITO glass (22) with a step structure, the chip is a square with the side length of 30-50 mm, the whole chip is of an inverted step structure, an ITO film layer covers the lower surface, the thickness of the first step with the thinnest edge is 1-2 mm, and the steps are sequentially increased to 5-8 in the thickness of 30-50 microns; the lower part is a flat ITO glass (24) with a step frame, the ITO film layer only covers the flat glass, the ITO film layer is plated with a hydrogenated amorphous silicon layer (23) which is a square with the side length of 30-50 mm, the size of the square is the same as that of the ITO glass (22) with the step structure, the thickness of the square is 1-2 mm, the height of the step frame corresponds to the ITO glass (22) with the step structure, the thickness of the frame is 1-2 mm, and the first step with the lowest edge is 20-50 microns higher than the upper surface of the flat glass; the upper part and the lower part of the chip (15) form a closed inner cavity, and the height of the inner cavity is not more than 0.5 mm; the sheet resistance of the ITO film layer is 10 omega/sq, the light transmittance is larger than 84 percent, the thickness is 80-120 nanometers, and the thickness of the hydrogenated amorphous silicon layer (23) is 300-500 nanometers.
The horizontal section shape and size of the three-dimensional structure of the group cells are controlled by the light pattern (27), and the height of the three-dimensional structure is controlled by the stepped structure of the ITO glass (22) with the stepped structure; a layer of hydrogenated amorphous silicon is plated on an ITO film layer of the ITO glass (22) with the step structure, and the hydrogenated amorphous silicon is irradiated by another light pattern to realize the respective control of the X, Y, Z axial directions of the group cells, so that the three-dimensional structure of the group cells is obtained.
The displacement precision of three axes of the three-dimensional displacement platform (14) is 0.1-1 micron, the strokes of an X axis and a Y axis are 3-5 cm, and the stroke of a Z axis is 1-3 cm.
The invention relates to a method for manipulating and constructing a three-dimensional structure of a group cell based on light-induced dielectrophoresis, which comprises the following steps:
a. the chip (15) is fixed on a three-dimensional displacement platform (14), a cell solution is led in from an inlet (21) by an air pump (16) until the inner cavity of the chip (15) is filled, the voltage of the upper layer ITO and the lower layer ITO of the chip (15) loaded by a signal generator (17) is 3-8V, and the frequency is 1-20 kilohertz;
b. observing the group cells in real time through a microscope (18), a CCD (19) and a second computer (110), and recording the state of the group cells;
c. turning on the projector (11) and the signal generator (17) for 30-60 seconds, turning off the projector (11) for 30-60 seconds, turning off the signal generator (17) for 100-150 seconds, turning on the signal generator (17) until the cells are independent of each other, and observing the physiological activities of a large number of single cells;
d. the projector (11) projects a light pattern (27) designed by the first computer (10) to the hydrogenated amorphous silicon layer (23) through the convergence of the positive lens (12) and the deflection of the reflecting mirror (13);
e. the population of cells is manipulated by an electric field induced by the light pattern (27) to be grouped together into a three-dimensional structure, and the change of the three-dimensional structure of the population of cells is controlled by finely adjusting the shape and size of the pattern output by the first computer (10) according to the phenomenon observed by the second computer (110).
Furthermore, the two wires are respectively adhered to the ITO film layers of the upper part and the lower part of the chip by conductive silver adhesive.
Further, the electric signal output by the signal generator (17) is a sine wave.
Compared with the prior art, the invention has the advantages that:
(1) the light-induced dielectrophoresis used by the invention irradiates a photoconductive material with light to cause the conductivity of the photoconductive material to change, thereby forming a virtual electrode to change the distribution of an electric field and leading cells in a solution to be polarized to generate directional motion, and the method is an indirect control mode, does not need to directly contact the cells and avoids polluting the cells; and because the illumination is weaker, the intensity of the applied electric signal is lower, the influence on the cells is extremely small, and the light and the electricity can be turned off after the three-dimensional structure of the group cells is stable, so that the external influence is eliminated, the physiological activity of the cells can be observed more favorably, and the cells which are closer to the natural growth can be obtained.
(2) The upper part of the chip designed by the invention is transparent glass, the forming process of the three-dimensional structure of the cells is accurately observed in real time through a microscope (18), a CCD (19) and a second computer (110), and the three-dimensional structure formed by the group cells is finely adjusted through observing and adjusting light, so that the three-dimensional structure which is more in line with the expectation is obtained. The shape and size of the light determines the shape and size of the cross-section of the three-dimensional structure. Because the upper part of the chip is of a stepped structure and the lower part of the chip is of a flat plate structure, the heights of different positions in the chip are different, the height of the three-dimensional structure is determined by the position of the moving light, and the precision can reach micrometer.
(3) The cells are suspended in the solution, the air pump (16) is used for supplementing the solution to ensure the normal growth of the cells, the solution in which the cells are positioned can be replaced at any time, and each cell is fully contacted with the solution, so that the influence of the solution on the cells can be determined.
Drawings
FIG. 1 is a schematic diagram of the system of the present invention;
wherein 10 is a first computer, 11 is a projector, 12 is a positive lens, 13 is a reflector, 14 is a three-dimensional displacement platform, 15 is a chip, 16 is an air pump, 17 is a signal generator, 18 is a microscope, 19 is a CCD, and 110 is a second computer.
FIG. 2 is a schematic diagram of a chip structure according to the present invention;
wherein 21 is an inlet, 22 is ITO glass with a step structure, 23 is a hydrogenated amorphous silicon layer, 24 is flat ITO glass with a step frame, 25 is a wire interface, 26 is an outlet, and 27 is a light pattern.
FIG. 3 is a schematic view of a pooled cell;
FIG. 4 is a schematic diagram of the three-dimensional structure of a cell;
wherein 41 is a cell.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
A light-induced dielectrophoresis system shown in figure 1 is set up, in the system, a projector 11 is horizontally arranged and is positioned on the same horizontal line with the centers of a positive lens 12 and a reflector 13, the reflector 13 forms an angle of 45 degrees with the horizontal plane, a chip 15 is horizontally fixed on a three-dimensional displacement platform 14, the displacement precision of three axes of the three-dimensional displacement platform 14 is 1 micron, the travel of an X axis and a Y axis is 5 cm, the travel of a Z axis is 3 cm, the projector 11 projects a light pattern 27 designed by a first computer 10, the light pattern is projected to a hydrogenated amorphous silicon layer 23 through the convergence of the positive lens 12 and the deflection of the reflector 13, and the Z axis of the three-dimensional displacement platform 14 is adjusted to enable the hydrogenated amorphous silicon layer 23 to be positioned at the position with the maximum optical density. The outlet 26 is connected with the air pump 16, two wires led out from the wire interface 25 are respectively connected with the ITO film layer and the signal generator 17 by conductive silver adhesive, the microscope 18 and the CCD19 are vertically arranged right above the chip 15, the focus of the microscope 18 is in the solution in the inner cavity of the chip 15, and the microscope is connected with the second computer 110 to observe the cell movement condition in real time.
The structure of the chip 15 is shown in fig. 2, the upper part of the chip 15 is the ITO glass 22 with a step structure, the specification is a square with the side length of 50 mm, the whole chip is an inverted step structure, the ITO film layer covers the lower surface, the thickness of the first step with the thinnest edge is 2 mm, the thickness of the steps is sequentially increased by 50 microns, the total number is 5 steps, the flat ITO glass 24 with a step frame at the lower part is a square with the side length of 50 mm, the thickness is 2 mm, the periphery is provided with a raised frame corresponding to the step structure at the upper part, the height of the step frame is corresponding to the ITO glass 22 with the step structure at the upper part, the thickness of the frame is 1 mm, the first step with the lowest edge is 20 microns higher than the flat glass, the ITO film layer only covers the flat glass, and the hydrogenated amorphous silicon layer 23 is plated on the ITO film layer. The sheet resistance of the ITO film layer is 10 omega/sq, the light transmittance is larger than 84%, the thickness is 120 nanometers, and the thickness of the hydrogenated amorphous silicon layer 23 is 500 nanometers.
In the embodiment of the invention, yeast cells in deionized water are taken as an operation target, a cell solution is prepared, the solution containing the cells is introduced from an inlet 21 under the action of an air pump 16 and fills the whole cavity, and the output frequency and the voltage of an electric signal are adjusted, wherein the electric signal output by a signal generator 17 in the embodiment of the invention is a sinusoidal signal with the voltage of 5V and the frequency of 3.5 KHz. The projector 11 and the signal generator 17 are opened for 60 seconds, the group cells are gathered together, the projector 11 is closed, under the condition that the signal generator 17 loads an electric signal for 30 seconds, the movement speed of the cells is extremely low due to the constraint of an electric field, the electric signal is disconnected for 120 seconds, the movement speed of the cells is high, the cells are distributed more densely closer to the central cells, the electric signal is connected, the movement of the cells is accelerated and tend to be uniformly distributed, the cells are not in contact with each other and are independent of each other, and the physiological activities of a large number of single cells are observed simultaneously.
If the cell concentration is low, the light converging cells as shown in fig. 3 can be used, and a light pattern is first designed on the first computer 10, a square frame with a 45 mm length and a 5 mm width is formed on the bar, and the length of the frame is gradually reduced by 5 mm, and finally the cells are concentrated in the manipulated area. The section shape and the size of the three-dimensional structure are determined by the light pattern 27, the height of the three-dimensional structure is determined by the different positions of the light pattern 27 projected on the ITO glass 22 with the step structure, the illumination position is adjusted by the three-dimensional displacement platform 14, the cells are gradually gathered together to form the three-dimensional structure after waiting for 30 seconds, and as shown in FIG. 4, the cells 41 are gathered together to form a cylinder.
When the group cells are gathered together to form the three-dimensional structure for 5 minutes, the projector 11 is closed to eliminate the influence of light on the cells, the signal generator 17 is closed to eliminate the influence of the alternating current electric field on the cells, the group cells forming the three-dimensional structure are suspended in the solution, no external stimulation is generated, the three-dimensional structure is closer to the natural environment, the variables in the experiment are reduced, and the observation of the physiological activities of the group cells in the solution is facilitated.
Long-term observation is required to ensure that the nutrient content of the solution is sufficient, and the solution can be replenished by the air pump 16. If the three-dimensional structure needs to be put into a specific container for culturing, after the cells form the three-dimensional structure for 5 minutes, the cells are close to the outlet by moving the displacement platform, and then the cells are led out along with the solution by the air pump 16 connected with the outlet 26 and transferred into other containers. If it is desired to change the solution of the cells, it is necessary to change the solution at the inlet 21 and then slowly suck the original solution out through the air pump 16 at the outlet 26.
The method has the advantages of low cost, simple operation, small influence on cells, real-time observation and accurate control of the cells to construct the three-dimensional structure, and convenience in changing solution and leading out the three-dimensional structure.
The above examples are provided only for the purpose of describing the present invention, and are not intended to limit the scope of the present invention. The scope of the invention is defined by the appended claims. Various equivalent substitutions and modifications can be made without departing from the spirit and principles of the invention, and are intended to be included within the scope of the invention.
Claims (3)
1. A three-dimensional structure manipulation and construction system of group cells based on light-induced dielectrophoresis is characterized in that: the device comprises a first computer (10), a projector (11), two positive lenses (12), a reflector (13), a three-dimensional displacement platform (14), a chip (15), an air pump (16), a signal generator (17), a microscope (18), a CCD (19) and a second computer (110); the upper part of the chip (15) is ITO glass (22) with a step structure, the lower part of the chip is flat ITO glass (24) with a step frame, and the upper surface of the chip is plated with a hydrogenated amorphous silicon layer (23); the three-dimensional structure of the group of cells controls the horizontal section shape and the size of the three-dimensional structure by a light pattern (27); a light pattern (27) designed by a first computer (10) is projected by a projector (11) and finally converged and projected to a hydrogenated amorphous silicon layer (23) horizontally placed and fixed at the lower part of a chip (15) of a three-dimensional displacement platform (14) through two positive lenses (12) and a reflector (13), an electric signal output by a signal generator (17) is loaded to ITO glass (22) with a step structure and flat ITO glass (24) with a step frame, the cell movement condition is observed in real time through a microscope (18), a CCD (19) and a second computer (110), the three-dimensional structure of a group cell can be conveniently and efficiently obtained, and an air pump (16) is used for supplementing solution to ensure the normal growth of the cell and replacing different solutions when observing the physiological activities of the cell; the displacement precision of three axes of the three-dimensional displacement platform (14) is 0.1-1 μm, the strokes of an X axis and a Y axis are 3-5cm, and the stroke of a Z axis is 1-3 cm; the chip (15) further comprises an inlet (21) and an outlet (26), and the outlet (26) is connected with the air pump (16).
2. The light-induced dielectrophoresis-based three-dimensional structure manipulation and construction system of a group of cells according to claim 1, wherein: the upper part of the chip (15) is provided with ITO glass (22) with a step structure, the chip is a square with the side length of 30-50 mm, the whole chip is of an inverted step structure, an ITO film layer covers the lower surface, the thickness of the first step with the thinnest edge is 1-2 mm, and the steps are sequentially increased to 5-8 in the thickness of 30-50 microns; the lower part is a flat ITO glass (24) with a step frame, the ITO film layer only covers the flat glass, the ITO film layer is plated with a hydrogenated amorphous silicon layer (23) which is a square with the side length of 30-50 mm, the size of the square is the same as that of the ITO glass (22) with the step structure, the thickness of the square is 1-2 mm, the height of the step frame corresponds to that of the ITO glass (22) with the step structure, the thickness of the frame is 1-2 mm, and the first step with the lowest edge is 20-50 microns higher than the upper surface of the flat glass; the upper part and the lower part of the chip (15) form a closed inner cavity, and the height of the inner cavity is not more than 0.5 mm; the sheet resistance of the ITO film layer is 10 omega/sq, the light transmittance is larger than 84 percent, the thickness is 80-120 nanometers, and the thickness of the hydrogenated amorphous silicon layer (23) is 300-500 nanometers.
3. A method for manipulating and constructing three-dimensional structures of group cells based on light-induced dielectrophoresis, wherein the system of claim 1 or 2 is used, and the steps are as follows:
a. The chip (15) is fixed on a three-dimensional displacement platform (14), a cell solution is introduced from an inlet (21) by using an air pump (16) until the inner cavity of the chip (15) is filled, the voltage of the signal generator (17) loaded on the upper layer ITO and the lower layer ITO of the chip (15) is 3-8V, and the frequency is 1-20 KHz;
b. observing the group cells in real time through a microscope (18), a CCD (19) and a second computer (110), and recording the state of the group cells;
c. turning on the projector (11) and the signal generator (17) for 30-60 seconds, turning off the projector (11) for 30-60 seconds, turning off the signal generator (17) for 100-150 seconds, turning on the signal generator (17) until the cells are independent of each other, and observing the physiological activities of a large number of single cells;
d. the projector (11) projects a light pattern (27) designed by the first computer (10) to the hydrogenated amorphous silicon layer (23) through the convergence of the positive lens (12) and the deflection of the reflecting mirror (13);
e. the group cells are controlled by the non-uniform electric field induced by the light pattern (27) to be gathered together to form a three-dimensional structure, and the change of the three-dimensional structure of the group cells is controlled by finely adjusting the shape and the size of the pattern output by the first computer (10) or adjusting the three-dimensional displacement platform (14) according to the phenomenon observed by the second computer (110).
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