CN109810894A - A system and method for manipulating and constructing three-dimensional structure of population cells based on light-induced dielectrophoresis - Google Patents
A system and method for manipulating and constructing three-dimensional structure of population cells based on light-induced dielectrophoresis Download PDFInfo
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- CN109810894A CN109810894A CN201910155647.4A CN201910155647A CN109810894A CN 109810894 A CN109810894 A CN 109810894A CN 201910155647 A CN201910155647 A CN 201910155647A CN 109810894 A CN109810894 A CN 109810894A
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- 238000004720 dielectrophoresis Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 11
- 239000011521 glass Substances 0.000 claims description 27
- 229910021417 amorphous silicon Inorganic materials 0.000 claims description 19
- 238000006073 displacement reaction Methods 0.000 claims description 18
- 238000010276 construction Methods 0.000 claims description 7
- 230000001766 physiological effect Effects 0.000 claims description 7
- 239000005357 flat glass Substances 0.000 claims description 6
- 230000005684 electric field Effects 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 230000033001 locomotion Effects 0.000 claims description 2
- 239000013589 supplement Substances 0.000 claims 1
- 238000011109 contamination Methods 0.000 abstract description 2
- 239000006285 cell suspension Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 60
- 230000009087 cell motility Effects 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 210000004292 cytoskeleton Anatomy 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
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- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000002711 centrocyte Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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Abstract
本发明公开了一种基于光诱导介电泳的群细胞三维结构操纵与构建系统及方法，利用光诱导介电泳对群细胞进行三维结构操纵与构建，并设计了相应的芯片。本发明利用光诱导介电泳在细胞悬浮溶液中对群细胞进行三维结构的操纵与构建，能够实时精确控制群细胞三维结构的形状和尺寸。这种间接操控细胞的方式不需要直接接触细胞，避免了对细胞的污染，可以随时补充和更换细胞所处溶液，对细胞的影响极小，有利于获得接近于自然生长的细胞。
The invention discloses a system and method for manipulating and constructing three-dimensional structure of group cells based on light-induced dielectrophoresis. The invention utilizes light-induced dielectrophoresis to manipulate and construct the three-dimensional structure of the group cells in the cell suspension solution, and can precisely control the shape and size of the three-dimensional structure of the group cells in real time. This indirect way of manipulating cells does not require direct contact with cells, which avoids contamination of cells. The solution in which cells are located can be replenished and replaced at any time, with minimal impact on cells, which is conducive to obtaining cells that are close to natural growth.
Description
Technical field
The present invention relates to cells to manipulate field, the in particular to manipulation of group's cell three-dimensional structure and building field, is a kind of base
The manipulation of group's cell three-dimensional structure and building system and method in light-induction dielectrophoresis.
Background technique
The three-dimensional building of cell has huge meaning for the growth of research cell, differentiation and cell-cell interaction,
There is application in the fields such as oncobiology and stem cell culture, are one of the important technologies for studying cellular physiological events.
Internal most cells are in three-dimensional structure, and external dimensional culture is more advantageous to holding and performance than two dimension culture
Cell function.Traditional technological means includes cytoskeleton, porous material, hydrogel etc..Cytoskeleton wants timbering material
Ask higher；The shape that porous material can only be simple to manufacture, and connectivity is low, takes a long time；At high cost, the Ke Nengyin of hydrogel
Send out inflammatory reaction.Needing one kind at present, cost is relatively low, easy to operate, three-dimensional building mode lesser to impact cell, to thin
The further research of born of the same parents' physiological activity provides help.
Summary of the invention
The technology of the present invention solves the problems, such as: it is thin to overcome the deficiencies of the prior art and provide a kind of group based on light-induction dielectrophoresis
The manipulation of born of the same parents' three-dimensional structure and building system and method, use non-contacting light-induction dielectrophoresis as operating method, effectively avoid
Contamination of cells.Manipulation precision can achieve micron order, be conducive to accurately construct three-dimensional structure.Devise the special core of matching operation
Piece and mechanical structure, reduce operation difficulty, simplify operating process.This method is low in cost, easy to operate, to impact cell
It is minimum, it can real-time accurate manipulation and building group's cell three-dimensional structure.
The technology of the present invention solution: to achieve the above object, a kind of group based on light-induction dielectrophoresis of the invention is thin
The manipulation of born of the same parents' three-dimensional structure and building system, comprising: the first computer (10), projector (11), two positive lens (12), reflecting mirrors
(13), three-D displacement platform (14), chip (15), air pump (16), signal generator (17), microscope (18), CCD (19) and
Two computers (110)；The top of the chip (15) is the ito glass (22) with hierarchic structure, and lower part is with step edge
The plate ito glass (24) of frame and upper surface is coated with hydrogenated amorphous silicon layer (23)；First computer is projected by projector (11)
(10) light pattern (27) designed finally collects to project to and be fixed on by two positive lens (12) and a reflecting mirror (13)
The hydrogenated amorphous silicon layer (23) of chip (15) lower part of three-D displacement platform (14), the electric signal that signal generator (17) are exported
It is loaded into the ito glass (22) with hierarchic structure and the plate ito glass (24) with ladder frame, passes through microscope
(18), CCD (19), second computer (110) observe cell movement situation in real time, can be convenient and are efficiently obtained group cell three-dimensional
Structure, air pump (16) guarantee cell normal growth and replacement different solutions for make-up solution when observing cellular physiological events.
The chip (15) is horizontal positioned or places vertically, guarantees that light projects to hydrogenated amorphous silicon layer (23).
Chip (15) top is the ito glass (22) with hierarchic structure, is the pros that side length is 30-50 millimeters
Shape, generally inverted hierarchic structure, ito film layer cover lower surface, and first most thin step thicknesses of edge are 1-2 millimeters,
Step is incremented by successively to 5-8 with 30-50 microns of thickness；Lower part is the plate ito glass (24) with ladder frame, ITO
Film layer only covers plate glass, and hydrogenated amorphous silicon layer (23) are coated on ito film layer, is the square that side length is 30-50 millimeters, greatly
Small identical as with ito glass (22) of hierarchic structure, with a thickness of 1-2 millimeters, the height of ladder frame is corresponding to have hierarchic structure
Ito glass (22), frame with a thickness of 1-2 millimeters, first minimum step of edge is 20-50 higher than plate glass upper surface micro-
Rice；Two parts form a closed inner cavity to chip (15) up and down, and the height of inner cavity is no more than 0.5 millimeter；Ito film layer sheet resistance be
10 Ω/sq, light transmittance are greater than 84%, and with a thickness of 80-120 nanometers, hydrogenated amorphous silicon layer (23) is with a thickness of 300-500 nanometers.
Group's cell three-dimensional structure passes through band by the horizontal section shape and size of light pattern (27) control three-dimensional structure
There is the height of the hierarchic structure control three-dimensional structure of the ito glass (22) of hierarchic structure；In the ito glass for having hierarchic structure
(22) one layer of amorphous silicon hydride is plated on ito film layer, is irradiated with another light pattern, realizes point in group's cell X, Y, Z axis direction
It does not control, obtains group's cell three-dimensional structure.
The displacement accuracy of (14) three axis of three-D displacement platform is 0.1-1 microns, and X-axis and Y-axis stroke are 3-5 lis
Rice, Z axis stroke are 1-3 centimetres.
A kind of manipulation of group's cell three-dimensional structure and construction method based on light-induction dielectrophoresis of the invention, steps are as follows:
A, chip (15) is fixed on three-D displacement platform (14), leads cell solution by entrance (21) using air pump (16)
Enter, until being full of chip (15) inner cavity, signal generator (17) is loaded into the voltage of chip (15) upper layer and lower layer ITO and lies prostrate for 3-8,
Frequency is 1-20 kHz；
B, group's cell is observed in real time by microscope (18), CCD (19) and second computer (110), collection of record
Cell state；
C, projector (11) and signal generator (17) 30-60 seconds are opened, close projector (11) 30-60 seconds, close letter
Numbers generator (17) 100-150 seconds, opening signal generator (17) observed a large amount of individual cells until cell is mutually indepedent
Physiological activity；
D, the light pattern (27) that projector (11) designs the first computer (10) passes through the convergence of positive lens (12) and anti-
The deviation for penetrating mirror (13) projects to hydrogenated amorphous silicon layer (23)；
E, group's cell is flocked together by the electric field manipulation induced by light pattern (27) and forms three-dimensional structure, according to the
The phenomenon that two computers (110) are observed, by the pattern form and size of fine tuning the first computer (10) output, control group is thin
The variation of born of the same parents' three-dimensional structure.
Further, two conducting wires use the two-part ito film layer of conductive silver glue adhesion chip or more respectively.
Further, the electric signal of signal generator (17) output is sine wave.
The present invention has advantages in that compared with prior art
(1) light-induction dielectrophoresis that the present invention uses is that photoconductive material is mapped to using illumination to cause its conductivity to occur
Variation makes solution inner cell that polarization occur to generate directed movement, is an inter-species so that forming dummy electrodes changes field distribution
Control mode is connect, direct exposing cell is not needed, avoids pollution cell；And since illumination is weaker, applied electric signal it is strong
Spend lower, the influence to cell is minimum, and light and electricity can be closed after group's cell three-dimensional stable structure, eliminates extraneous shadow
It rings, is more advantageous to the physiological activity of observation cell, obtain closer to the cell grown naturally.
(2) the chip top that the present invention designs is transparent glass, passes through microscope (18), CCD (19) and second computer
(110) forming process for accurately observing cell three-dimensional structure in real time, the three-dimensional knot for passing through observation adjustment light to form to group's cell
Structure is finely adjusted, and acquisition more meets expected three-dimensional structure.The shape and size of light determine three-dimensional structure cross-sectional shapes and
Size.Due to chip top is hierarchic structure and lower part is slab construction, cause the height of chip interior different location different, lead to
It crosses and moves the position of light and determine that the height of three-dimensional structure, precision can achieve micron.
(3) cell is suspended in solution, guarantees cell normal growth with air pump (16) make-up solution, can also at any time more
Solution locating for cell is changed, each cell comes into full contact with solution, is more advantageous to influence of the determining solution to cell.
Detailed description of the invention
Fig. 1 is present system structural schematic diagram；
Wherein 10 be the first computer, and 11 be projector, and 12 be positive lens, and 13 be reflecting mirror, and 14 be three-D displacement platform,
15 be chip, and 16 be air pump, and 17 be signal generator, and 18 be microscope, and 19 be CCD, and 110 be second computer.
Fig. 2 is chip structure schematic diagram in the present invention；
Wherein 21 be entrance, and 22 be the ito glass with hierarchic structure, and 23 be hydrogenated amorphous silicon layer, and 24 is with ladders
The plate ito glass of frame, 25 be wire interface, and 26 be outlet, and 27 be light pattern.
Fig. 3 is convergence cell schematic diagram；
Fig. 4 is cell three-dimensional structural schematic diagram；
Wherein 41 be cell.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawing.
Build light-induction dielectrophoresis system as shown in Figure 1, in system of the invention, projector 11 it is horizontal positioned and with just
Lens 12, reflecting mirror 13 center on same horizontal line, at 45 degree, 15 level of chip is fixed on for reflecting mirror 13 and horizontal plane
Three-D displacement platform 14, the displacement accuracy of 14 3 axis of three-D displacement platform are 1 micron, and X-axis and Y-axis stroke are 5 centimetres, Z
Axis stroke is 3 centimetres, and projector 11 projects the light pattern 27 of the first computer 10 design, passes through the convergence and reflection of positive lens 12
The deviation of mirror 13 projects to hydrogenated amorphous silicon layer 23, and the Z axis for adjusting three-D displacement platform 14 makes hydrogenated amorphous silicon layer 23 be located at light
Density maximum.Outlet 26 is connected with air pump 16, and two conducting wires that wire interface 25 is drawn use conductive silver glue to connect ITO respectively
Film layer and signal generator 17, microscope 18 and CCD19 are vertically placed on right above chip 15, and the focus of microscope 18 is in chip
In 15 inner cavity solution, connection second computer 110 observes cell movement situation in real time.
As shown in Fig. 2, 15 top of chip is the ito glass 22 with hierarchic structure, specification is 15 structure of chip for side length
50 millimeters of square, generally inverted hierarchic structure, ito film layer cover lower surface, first most thin step thickness of edge
Degree is 2 millimeters, and the thickness of step is incremented by successively with 50 microns, totally 5 rank, the plate ito glass 24 with ladder frame of lower part
It is 50 millimeters of square for side length, thickness is 2 millimeters, and periphery has the raised frame of corresponding top hierarchic structure, ladder frame
Height correspond to top have hierarchic structure ito glass 22, the thickness of frame is 1 millimeter, first minimum step ratio of edge
Plate glass is 20 microns high, and ito film layer only covers plate glass, and hydrogenated amorphous silicon layer 23 is coated on ito film layer.Ito film layer side
Resistance is 10 Ω/sq, and light transmittance is greater than 84%, and with a thickness of 120 nanometers, hydrogenated amorphous silicon layer 23 is with a thickness of 500 nanometers.
The embodiment of the present invention is operation target with the yeast cell in deionized water, configures cell solution, contains cell
Solution imported under the action of air pump 16 by entrance 21 and full of entire cavity, adjust the output frequency and voltage of electric signal,
The electric signal that signal generator 17 exports in the embodiment of the present invention is that voltage is 5V, and frequency is the sinusoidal signal of 3.5KHz.It opens
Projector 11 and signal generator 17 continue 60 seconds, and group's cell is brought together, and close projector 11, add in signal generator 17
In the case where electrical signals 30 seconds, since the constraint cell movement speed of electric field is extremely slow, disconnect electric signal 120 seconds, cell movement
Speed becomes faster, more intensive closer to centrocyte distribution, connects electric signal, and cell movement is accelerated and is intended to be uniformly distributed, carefully
Intercellular does not contact, independently of each other, for observing the physiological activity of a large amount of individual cells simultaneously.
If cell concentration is lower, cell can be converged with light as shown in Figure 3, design light on the first computer 10 first
Pattern, a length are the frame for the square that 45 millimeters of striation width are 5 millimeters, and the length of frame gradually reduces 5 millimeters, finally will
Cell concentrates on being manipulated region.Light pattern 27 determines that the cross-sectional shapes and size of three-dimensional structure, light pattern 27 project to band
There is the different location of the ito glass 22 of hierarchic structure to determine the height of three-dimensional structure, light position passes through three-D displacement platform 14
It adjusts, waits 30 seconds, cell can gradually be brought together composition three-dimensional structure, the composition as shown in figure 4, cell 41 flocks together
Cylindrical body.
When group's cell aggregation is to three-dimensional structure is formed after five minutes together, closing projector 11 eliminates influence of the light to cell,
Shutdown signal generator 17 eliminates influence of the AC field to cell, and the group's cell for forming three-dimensional structure suspends in the solution, does not have
There is environmental stimuli closer to natural environment, and reduce the variable in experiment, is more advantageous to observation group cell in the solution
Physiological activity.
Observation for a long time needs to guarantee that the nutriment in solution is sufficient, can use 16 make-up solution of air pump.If desired will
Three-dimensional structure is put into special container and cultivates, when cell composition three-dimensional structure after five minutes, lean on cell by moving displacement platform
Then cell is exported by the air pump 16 of 26 connection of outlet with solution, is transferred in other containers by nearly outlet.If thinking, replacement is thin
Solution locating for born of the same parents needs first to replace the solution at entrance 21, and then original solution is slowly sucked out by the air pump 16 at outlet 26.
Using light-induction dielectrophoresis and the manipulation of the chip of design and building group's cell three-dimensional structure, this method is low in cost,
It is easy to operate, it is small to impact cell, it can observe in real time, accurate manipulation cell building three-dimensional structure, and be convenient for changing solution
And export three-dimensional structure.
Above embodiments are provided just for the sake of the description purpose of the present invention, and are not intended to limit the scope of the invention.This
The range of invention is defined by the following claims.It does not depart from spirit and principles of the present invention and the various equivalent replacements made and repairs
Change, should all cover within the scope of the present invention.
Claims (6)
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Cited By (1)
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| CN113654971A (en) * | 2021-07-21 | 2021-11-16 | 长春理工大学 | A light-induced electrode scanning microscope and method for measuring electrical properties of biological cells |
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| CN113654971B (en) * | 2021-07-21 | 2022-11-01 | 长春理工大学 | A light-induced electrode scanning microscope and method for measuring electrical properties of biological cells |
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