CN109810894B - Group cell three-dimensional structure manipulation and construction system and method based on light-induced dielectrophoresis - Google Patents

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

A system and method for manipulating and constructing three-dimensional structure of population cells based on light-induced dielectrophoresis

technical field

The invention relates to the field of cell manipulation, in particular to the field of three-dimensional structure manipulation and construction of cluster cells, and relates to a system and method for manipulation and construction of cluster cells three-dimensional structure based on light-induced dielectrophoresis.

Background technique

The three-dimensional construction of cells is of great significance for the study of cell growth, differentiation and cell-to-cell interactions.

Most cells in the body are in a three-dimensional structure, and three-dimensional culture in vitro is more conducive to maintaining and exerting cell functions than two-dimensional culture. Traditional technical means include cell scaffolds, porous materials, hydrogels, etc. Cell scaffolds have high requirements on scaffold materials; porous materials can only be fabricated in simple shapes, and have low connectivity and time-consuming; hydrogels are expensive and may trigger inflammatory responses. Currently, there is a need for a low-cost, easy-to-operate, three-dimensional construction method that has less impact on cells, so as to help further research on cell physiological activities.

SUMMARY OF THE INVENTION

The technology of the present invention solves the problem: overcomes the deficiencies of the prior art, provides a system and method for manipulating and constructing a three-dimensional structure of a group of cells based on light-induced dielectrophoresis, and uses non-contact light-induced dielectrophoresis as an operation method to effectively avoid contaminating cells. The manipulation precision can reach the micrometer level, which is conducive to the precise construction of three-dimensional structures. The special chip and mechanical structure for matching operation are designed, which reduces the difficulty of operation and simplifies the operation process. The method is low in cost, simple in operation, minimal impact on cells, and can precisely manipulate and construct the three-dimensional structure of group cells in real time.

Technical solution of the present invention: In order to achieve the above purpose, a light-induced dielectrophoresis-based three-dimensional structure manipulation and construction system for group cells of the present invention includes: a first computer (10), a projector (11), and two positive lenses (12), a mirror (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 an ITO glass (22) with a stepped structure, and the lower part is a flat ITO glass (24) with a stepped frame and the upper surface is plated with a hydrogenated amorphous silicon layer (23); The instrument (11) projects a light pattern (27) designed by the first computer (10), passes through two positive lenses (12) and a reflector (13), and finally collects and projects to the chip fixed on the three-dimensional displacement platform (14). (15) the hydrogenated amorphous silicon layer (23) at the lower part loads the electrical signal output by the signal generator (17) to the ITO glass (22) with a stepped structure and the flat ITO glass (24) with a stepped frame, The movement of cells is observed in real time through a microscope (18), a CCD (19), and a second computer (110), so that the three-dimensional structure of the cells can be obtained easily and efficiently. The air pump (16) is used to supplement the solution for observing the physiological activities of cells to ensure the normal growth of cells. and changing to different solutions.

The chip (15) is placed horizontally or vertically to ensure that the light is projected onto the hydrogenated amorphous silicon layer (23).

The upper part of the chip (15) is an ITO glass (22) with a stepped structure, which is a square with a side length of 30-50 mm, and is an inverted stepped structure as a whole. The thickness of each step is 1-2 mm, and the steps are gradually increased to 5-8 with a thickness of 30-50 microns; the lower part is a flat ITO glass with a stepped frame (24), the ITO film layer only covers the flat glass, and the ITO film layer A hydrogenated amorphous silicon layer (23) is plated on it, which is a square with a side length of 30-50 mm, the size is the same as the ITO glass (22) with a stepped structure, and the thickness is 1-2 mm, and the height of the stepped frame corresponds to the belt ITO glass (22) with a stepped 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 and lower parts of the chip (15) form a closed inner cavity , the height of the inner cavity does not exceed 0.5 mm; the square resistance of the ITO film layer is 10Ω/sq, the light transmittance is greater than 84%, the thickness is 80-120 nanometers, and the thickness of the hydrogenated amorphous silicon layer (23) is 300-500 nanometers.

The three-dimensional structure of the group cells is controlled by the light pattern (27) to control the horizontal section shape and size of the three-dimensional structure, and the height of the three-dimensional structure is controlled by the stepped structure of the ITO glass (22) with the stepped structure; The ITO film layer of (22) is coated with a layer of hydrogenated amorphous silicon, and is irradiated with another light pattern to realize the control of the X, Y, and Z axis directions of the group cells respectively, and obtain the three-dimensional structure of the group cells.

The displacement accuracy of the three axes of the three-dimensional displacement platform (14) is 0.1-1 micron, the travel of the X-axis and the Y-axis is 3-5 cm, and the travel of the Z-axis is 1-3 cm.

A method for manipulating and constructing a three-dimensional structure of a group of cells based on light-induced dielectrophoresis of the present invention, the steps are as follows:

a. The chip (15) is fixed on the three-dimensional displacement platform (14), and the air pump (16) is used to introduce the cell solution from the inlet (21) until the inner cavity of the chip (15) is filled, and the signal generator (17) is loaded into the chip (15). ) The voltage of the upper and lower layers of ITO is 3-8 volts, and the frequency is 1-20 kHz;

b. Observe the group cells in real time through a microscope (18), a CCD (19) and a second computer (110), and record the state of the group cells;

c. Turn on the projector (11) and the signal generator (17) for 30-60 seconds, turn off the projector (11) for 30-60 seconds, turn off the signal generator (17) for 100-150 seconds, and turn on the signal generator (17) , until the cells are independent of each other, and observe the physiological activities of a large number of single cells;

d, the projector (11) projects the 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 reflector (13);

e. The group of cells is manipulated by the electric field induced by the light pattern (27), and aggregates together to form a three-dimensional structure. According to the phenomenon observed by the second computer (110), by fine-tuning the shape and size of the pattern output by the first computer (10), Controlling changes in the three-dimensional structure of population cells.

Further, the two wires are respectively bonded to the ITO film layers of the upper and lower parts of the chip using conductive silver glue.

Further, the electrical signal output by the signal generator (17) is a sine wave.

Compared with the prior art, the present invention has the following advantages:

(1) The light-induced dielectrophoresis used in the present invention utilizes light to irradiate the photoconductive material to cause its electrical conductivity to change, thereby forming a virtual electrode to change the electric field distribution, so that the cells in the solution are polarized to generate directional motion, which is a kind of The indirect control method does not require direct contact with the cells to avoid contamination of the cells; and due to the weak light, the intensity of the applied electrical signal is low, and the impact on the cells is minimal, and the light and electricity can be turned off after the three-dimensional structure of the group of cells is stabilized. Eliminating external influences is more conducive to observing the physiological activities of cells and obtaining cells that are closer to natural growth.

(2) The upper part of the chip designed by the present invention is made of transparent glass, and the formation process of the three-dimensional structure of the cells is accurately observed in real time through the microscope (18), the CCD (19) and the second computer (110), and the light is adjusted by observing and adjusting the light to adjust the cell composition. The three-dimensional structure is fine-tuned to obtain a three-dimensional structure that is more in line with expectations. The shape and size of the light determines the shape and size of the facets of the three-dimensional structure. Since the upper part of the chip is a stepped structure and the lower part is a flat structure, the heights of different positions inside the chip are different. The height of the three-dimensional structure is determined by moving the position of the light, and the accuracy can reach microns.

(3) The cells are suspended in the solution, and the air pump (16) is used to supplement the solution to ensure the normal growth of the cells, and the solution in which the cells are located can be changed at any time, and each cell is in full contact with the solution, which is more conducive to determining the effect of the solution on the cells.

Description of drawings

1 is a schematic diagram of the system structure of the present invention;

10 is the first computer, 11 is the projector, 12 is the positive lens, 13 is the mirror, 14 is the three-dimensional displacement platform, 15 is the chip, 16 is the air pump, 17 is the signal generator, 18 is the microscope, 19 is the CCD, 110 is the second computer.

2 is a schematic diagram of a chip structure in the present invention;

21 is the inlet, 22 is the ITO glass with a stepped structure, 23 is the hydrogenated amorphous silicon layer, 24 is the flat ITO glass with a stepped frame, 25 is the wire interface, 26 is the outlet, and 27 is the light pattern.

Fig. 3 is a schematic diagram of convergent cells;

Figure 4 is a schematic diagram of the three-dimensional structure of cells;

41 of them are cells.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings.

A photoinduced dielectrophoresis system as shown in FIG. 1 is built. In the system of the present invention, the projector 11 is placed horizontally and is on the same horizontal line as the center of the positive lens 12 and the reflecting mirror 13, and the reflecting mirror 13 is 45 degrees from the horizontal plane. The chip 15 is horizontally fixed on the three-dimensional displacement platform 14 , the displacement accuracy of the three-axis displacement platform 14 is 1 micrometer, the travel of the X-axis and the Y-axis is 5 cm, the travel of the Z-axis is 3 cm, and the projector 11 projects the first computer 10 . The designed light pattern 27 is projected onto the hydrogenated amorphous silicon layer 23 through the convergence of the positive lens 12 and the deflection of the mirror 13, and the Z axis of the three-dimensional displacement platform 14 is adjusted so that the hydrogenated amorphous silicon layer 23 is located at the maximum optical density. The outlet 26 is connected to the air pump 16, the two wires drawn from the wire interface 25 are respectively connected with the ITO film layer and the signal generator 17 using conductive silver glue, the microscope 18 and the CCD 19 are vertically placed directly above the chip 15, and the focus of the microscope 18 is on the chip 15. In the lumen solution of , the second computer 110 is connected to observe the cell movement in real time.

The structure of the chip 15 is shown in Figure 2. The upper part of the chip 15 is an ITO glass 22 with a stepped structure. The specification is a square with a side length of 50 mm. The whole is an inverted stepped structure. The thickness of the first step is 2 mm, and the thickness of the steps is gradually increased by 50 microns, with a total of 5 steps. The lower flat ITO glass 24 with a step frame is a square with a side length of 50 mm and a thickness of 2 mm. There is a raised frame corresponding to the upper step structure. The height of the step frame corresponds to the ITO glass 22 with the upper step structure. 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 is only Covering the flat glass, a hydrogenated amorphous silicon layer 23 is plated on the ITO film layer. The square resistance of the ITO film layer is 10Ω/sq, the light transmittance is greater than 84%, the thickness is 120 nanometers, and the thickness of the hydrogenated amorphous silicon layer 23 is 500 nanometers.

The embodiment of the present invention takes yeast cells in deionized water as the operating target, and configures a cell solution. The solution containing cells is introduced through the inlet 21 under the action of the air pump 16 and fills the entire cavity to adjust the output frequency and voltage of the electrical signal. In the embodiment of the invention, the electrical signal output by the signal generator 17 is a sinusoidal signal with a voltage of 5V and a frequency of 3.5KHz. Turn on the projector 11 and the signal generator 17 for 60 seconds, the group of cells gather together, turn off the projector 11, and when the signal generator 17 is loaded with an electrical signal for 30 seconds, due to the restraint of the electric field, the movement of the cells is extremely slow and disconnected When the electrical signal is 120 seconds, the speed of cell movement becomes faster, and the closer the cell is to the center, the denser the distribution of cells. When connected to the electrical signal, the movement of cells is accelerated and tends to be evenly distributed, and the cells are not in contact with each other. It is used to observe the physiological activities of a large number of single cells at the same time. .

If the cell concentration is low, the light as shown in Figure 3 can be used to focus the cells. First, a light pattern is designed on the first computer 10, a square frame with a length of 45 mm and a light bar width of 5 mm, and the length of the frame is gradually reduced 5 mm, eventually focusing the cells in the manipulated area. The light pattern 27 determines the sectional shape and size of the three-dimensional structure. The light pattern 27 is projected to different positions of the ITO glass 22 with a stepped structure to determine the height of the three-dimensional structure. The illumination position is adjusted by the three-dimensional displacement platform 14. After waiting for 30 seconds, the cells will Gradually clustered together to form a three-dimensional structure, as shown in Figure 4, cells 41 clustered together to form a cylinder.

When the group of cells gather together to form a three-dimensional structure for 5 minutes, turn off the projector 11 to eliminate the influence of light on the cells, and turn off the signal generator 17 to eliminate the influence of the AC electric field on the cells. The group of cells forming the three-dimensional structure is suspended in the solution, without the outside world The stimulation is closer to the natural environment, and the variables in the experiment are reduced, which is more conducive to observing the physiological activities of the population of cells in solution.

For long-term observation, it is necessary to ensure that the nutrients in the solution are sufficient, and the air pump 16 can be used to supplement the solution. If the three-dimensional structure needs to be cultured in a specific container, when the cells form a three-dimensional structure for 5 minutes, the cells are moved close to the outlet by moving the displacement platform, and then the cells are exported with the solution through the air pump 16 connected to the outlet 26 and transferred to other containers. If you want to change the solution in which the cells are located, you need to replace the solution at the inlet 21 first, and then slowly suck out the original solution through the air pump 16 at the outlet 26 .

Using light-induced dielectrophoresis and a designed chip to manipulate and construct a three-dimensional structure of a group of cells, the method is low-cost, simple to operate, and has little impact on cells.

The above embodiments are provided for the purpose of describing the present invention only, 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 replacements and modifications made without departing from the spirit and principle of the present invention should be included within the scope of the present 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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