CN118146946A - Gradient light stimulation and drug concentration gradient combined regulation and control device and preparation method - Google Patents
Gradient light stimulation and drug concentration gradient combined regulation and control device and preparation method Download PDFInfo
- Publication number
- CN118146946A CN118146946A CN202410329277.2A CN202410329277A CN118146946A CN 118146946 A CN118146946 A CN 118146946A CN 202410329277 A CN202410329277 A CN 202410329277A CN 118146946 A CN118146946 A CN 118146946A
- Authority
- CN
- China
- Prior art keywords
- capturing
- culture
- gradient
- chip layer
- light intensity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003814 drug Substances 0.000 title claims abstract description 26
- 229940079593 drug Drugs 0.000 title claims abstract description 26
- 230000000638 stimulation Effects 0.000 title claims abstract description 26
- 230000033228 biological regulation Effects 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 39
- 238000012258 culturing Methods 0.000 claims abstract description 19
- 238000002347 injection Methods 0.000 claims abstract description 14
- 239000007924 injection Substances 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims description 19
- 230000008569 process Effects 0.000 claims description 17
- 230000003287 optical effect Effects 0.000 claims description 15
- 238000002174 soft lithography Methods 0.000 claims description 9
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Natural products C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 claims description 8
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 6
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 6
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 6
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 6
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 6
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000009832 plasma treatment Methods 0.000 claims description 3
- 229920000642 polymer Polymers 0.000 claims description 3
- 238000007639 printing Methods 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims 3
- 239000000243 solution Substances 0.000 abstract description 16
- 238000002835 absorbance Methods 0.000 abstract description 13
- 230000004069 differentiation Effects 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 63
- 239000002609 medium Substances 0.000 description 9
- 239000000126 substance Substances 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 241000191291 Abies alba Species 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 210000005056 cell body Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000010146 3D printing Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a gradient light stimulation and drug concentration gradient combined regulation and control device and a preparation method thereof, wherein the device comprises a light stimulation light intensity gradient chip layer and a cell capturing culture chip layer; the cell capturing culture chip layer is provided with a main sample injection section and a capturing culture channel; the capturing culture channels are provided with a plurality of groups of capturing culture cavities in parallel, and a plurality of capturing structures are arranged in the capturing culture cavities; the light stimulation light intensity gradient chip layer is provided with a main liquid inlet section and a light intensity adjusting channel; also comprises the preparation steps of S1-S4, etc. The light-stimulated light intensity gradient chip layer generates different light intensity gradients through different absorbance of solution channels with different concentrations; the cell capturing and culturing chip layer is provided with a plurality of groups of capturing and culturing cavities, and capturing structures arranged in an array are arranged in the capturing and culturing cavities and used for capturing and culturing cells; the invention can realize the differentiation process and behavior regulation of the target cells cultured on the microfluidic chip under the multi-factor conditions of the drug concentration gradient and the gradient light intensity gradient.
Description
Technical Field
The invention relates to the field of cell culture chips, in particular to a gradient light stimulation and drug concentration gradient combined regulation device and a preparation method thereof.
Background
Microfluidic chip technology originates from the micro-total analysis system proposed in Manz 1990 in its paper, with the aim of maximally transferring the functions of an analysis laboratory into a portable analysis device, even onto a centimeter-sized chip, through miniaturization and integration of chemical analysis devices. The microfluidic chip can be designed into multiple channels, liquid is split into multiple units through a network structure, the channels are not interfered with each other, and biochemical experiments of the multiple units can be performed simultaneously. The microchip structure is controllable, and control of microscopic objects (e.g., microparticles, cells) such as population cell studies can be achieved through design processing of the microstructures. The two-dimensional cell culture in the microfluidic chip has been widely applied to research on cell response and cell activity, and a cell culture system based on a microfluidic platform provides a relatively stable microenvironment for two-dimensional cell culture in a static and continuous perfusion state.
Although microfluidic chip cell culture techniques have been successfully used in drug screening and pathology research, there are some problems. In particular, in the aspect of microfluidic in-vitro culture of tumor cells, the cells need to be captured and cultured in a positioning way, and meanwhile, various physical and chemical influence factors need to be loaded, so that the growth and proliferation process of target cells is monitored, and subsequent metabolic products and genomics analysis are carried out. In the prior art, when the cells of the microfluidic chip are cultured on the in-vitro sheet, various physical and chemical influence factors cannot be loaded, so that better research on cell body differentiation and behavior response cannot be performed. Therefore, it is necessary to combine the micro-fluidic chip and the more efficient cell capturing structure to realize efficient and stable cell capturing and positioning culture, and a combined regulation and control device for loading various physical and chemical influence factors is provided.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a gradient light stimulation and drug concentration gradient combined regulation and control device and a preparation method thereof, which are used for solving the problems that in the prior art, when cells are cultured on an in-vitro sheet based on a microfluidic chip, a plurality of factors cannot be loaded and regulated, and research on cell body differentiation and behavior response cannot be realized.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
The device comprises a light-stimulated light intensity gradient chip layer and a cell capturing culture chip layer which are sequentially arranged from top to bottom; the cell capturing and culturing chip layer is provided with a main sample injection section and capturing and culturing channels in series, the main sample injection section is provided with a plurality of capturing and culturing channels in parallel, and the number of the main sample injection section is smaller than that of the capturing and culturing channels; each capturing culture channel is provided with a capturing culture section, a plurality of groups of capturing culture cavities are arranged on the capturing culture sections in parallel, each group of capturing culture cavities comprises two capturing culture cavities which are connected in parallel, and a plurality of capturing structures are arranged in each capturing culture cavity; the light stimulation light intensity gradient chip layer is provided with a main liquid inlet section and a light intensity adjusting channel in series, the main liquid inlet section is provided with a plurality of light intensity adjusting channels in parallel, and the number of the main liquid inlet section is smaller than that of the light intensity adjusting channels; and each light intensity adjusting channel is provided with a light emphasis section, and the light emphasis sections correspond to the positions of the capturing culture cavities.
Further, catch the structure and include the spill main part, be provided with two places openings on the spill main part, two places openings set up for the symmetry axis symmetry of spill main part.
Further, two sample inlets are arranged on the inlet side of the main sample inlet section in parallel, sample outlets are arranged on the outlet side of the plurality of capturing culture channels in parallel, the sample inlets are positioned at the top of the cell capturing culture chip layer, and the sample outlets are positioned at the bottom of the cell capturing culture chip layer; the inlet side of the main liquid inlet section is provided with two liquid inlets in parallel, and the outlet side of the sub liquid inlet section is provided with a liquid outlet in parallel.
Further, the capturing culture channel further comprises a sample dividing and feeding section and a sample dividing and discharging section, and the sample dividing and feeding section, the capturing culture section and the sample dividing and discharging section are connected in sequence; the light intensity adjusting channel further comprises a liquid separating and feeding section which is communicated with the light intensity adjusting section.
Further, the sample separating and feeding section and the liquid separating and feeding section are all serpentine micro-channels.
Further, the main sample injection section and the main liquid inlet section are both serpentine micro-channels.
Further, the number of main sampling sections is smaller than the number of capturing culture channels, and the number of main liquid inlet sections is smaller than the number of light intensity adjusting channels.
Further, the capturing culture cavity is a rectangular cavity, the inlet and outlet of the capturing culture cavity are arranged at diagonal positions, a plurality of rows of capturing structures are arranged along the width direction of the capturing culture cavity, a plurality of columns of capturing structures are arranged along the length direction of the capturing culture cavity, and capturing structures located in two adjacent columns are staggered.
A gradient light stimulation and drug concentration gradient combined regulation device and a preparation method thereof comprise the following steps:
S1: preparing a light-stimulated light intensity gradient chip layer:
preparing a printing light stimulus light intensity gradient chip layer by using a 3D printer;
s2: preparing a cell capturing culture chip layer;
Constructing a cell capturing and culturing chip layer by utilizing a PDMS polymer through a soft lithography process and a reverse mould process;
S3: performing ultrasonic treatment and plasma cleaning on the light-stimulated light intensity gradient chip layer and the cell capturing culture chip layer, wherein the plasma treatment time is 15-20s;
S4: bonding the light-stimulated light intensity gradient chip layer and the cell capturing culture chip layer at the bonding temperature of 100-120 ℃.
The preparation of the cell capturing culture chip layer in the step S2 specifically comprises the following steps:
the soft lithography process and the reverse process in the step S2 specifically comprise the following steps:
s21: processing a cell capturing culture chip layer mould with the structure height of 20-25 mu m by utilizing a soft lithography process;
s22: fixing a cell capturing culture chip layer mould on an acrylic mould;
s23: pouring PDMS mixed glue, standing and vacuumizing;
S24: placing the acrylic mould into an oven for heating and curing, wherein the temperature of the oven is 60-80 ℃;
S25: and (5) reversing the mould to finish the preparation of the cell capturing culture chip layer.
The beneficial effects of the invention are as follows:
According to the technical scheme, cells to be captured enter a main sample injection section through a sample injection port, a cell movement track is controlled to a capturing structure in a capturing culture cavity by utilizing fluid resistance under the control of a capturing culture channel and flow, and then pulse signals are applied to perform intermittent electrical stimulation; in this process, the cells are prevented from being washed away by the fluid due to the double slit arrangement of the capturing structure.
The uppermost layer of the invention is composed of light stimulus light intensity gradient chip layers of solutions with different concentrations based on absorbance regulation, the chip layer is prepared by a 3D printer, the upper end of the chip layer is composed of Christmas tree model snake-shaped micro-channels composed of a main liquid inlet section and a sub liquid inlet section, the solutions are passively mixed to generate solutions with four concentration gradients of 0, 1/3C and 2/3C, C, the solutions are introduced into four light emphasis sections, and the light stimulus with different light intensities can be generated in the 4 light emphasis sections based on different absorbance of the solutions with different concentrations.
The middle layer of the invention is composed of a cell capturing culture chip layer, the concentration gradients of 0, 1/3C and 2/3C, C are still generated by a Christmas tree model, the chip of the layer is provided with a 4 multiplied by 4 array chamber culture structure, and a single culture chamber is internally provided with two rows of single cell capturing structures; the intermediate layer chip can realize the in vitro culture of cells under different drug concentration gradients.
The light-stimulated light intensity gradient chip layer generates different light intensity gradients through different absorbance of solution channels with different concentrations; the cell capturing and culturing chip layer is provided with a plurality of groups of capturing and culturing cavities, and capturing structures arranged in an array are arranged in the capturing and culturing cavities and used for capturing and culturing cells.
The invention can realize the differentiation process and behavior regulation of the target cells cultured on the microfluidic chip under the multi-factor conditions of the drug concentration gradient and the gradient light intensity gradient.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a gradient optical stimulation and drug concentration gradient combined regulation device;
FIG. 2 is a physical diagram of a gradient optical stimulus and drug concentration gradient combined regulation device;
FIG. 3 is a layered diagram of a cell capture culture chip layer and a light intensity gradient chip layer;
FIG. 4 is a schematic diagram of the structure of a cell capture culture chip layer;
FIG. 5 is a schematic diagram of the structure of a light intensity gradient chip layer;
FIG. 6 is a partial enlarged view A of FIG. 4;
FIG. 7 is a schematic structural view of a capture structure;
FIG. 8 is a photograph of CMTPX dye-verified light gradient chip layer four-intensity gradient generation experiment;
FIG. 9 is a graph of experimental data generated by CMTPX dye-verified optical gradient chip layer four-intensity gradient;
FIG. 10 is a schematic diagram of a gradient light stimulus and drug concentration gradient combined control device of different height light emphasis segments.
The main component symbols in the drawings are described as follows:
1. A cell capture culture chip layer; 11. a sample inlet; 12. a main sample injection section; 13. capturing a culture channel; 131. dividing a sample introduction section; 132. separating out sample sections; 133. capturing a culture section; 134. capturing a culture cavity; 135. a capture structure; 1351. a concave body; 1352. a notch; 14. a sample outlet;
2. a light-stimulated light intensity gradient chip layer; 21. a liquid inlet; 22. a main liquid inlet section; 23. a light intensity adjusting channel; 231. a liquid inlet section is divided; 232. a light emphasizing segment; 24. and a liquid outlet.
Detailed Description
The following is a detailed description of embodiments, but embodiments of the invention are not limited thereto. The technical means used in the following embodiments are conventional means well known to those skilled in the art unless otherwise specified; the test methods used are all conventional methods; the materials, reagents, and the like used are all commercially available.
As shown in fig. 1,2 and 3, a gradient optical stimulation and drug concentration gradient combined regulation device and a preparation method thereof comprise an optical stimulation light intensity gradient chip layer 2 and a cell capturing culture chip layer 1 which are sequentially arranged from top to bottom, and fig. 2 is a layered diagram of the cell capturing culture chip layer and the optical stimulation light intensity gradient chip layer.
As shown in fig. 4, a main sample injection section 12 and a capturing culture channel 13 are serially arranged on the cell capturing culture chip layer 1, a plurality of main sample injection sections 12 are parallelly connected, and a plurality of capturing culture channels 13 are parallelly connected. The number of main injection sections 12 is smaller than the number of capture culture channels 13. In this embodiment, the main sample section 12 is preferably provided with three positions, and the capturing culture channels 13 are preferably provided with four positions, and the three main sample sections 12 and the four capturing culture channels 13 are in a christmas tree structure. Each capturing culture channel 13 is provided with a capturing culture section 133, a plurality of groups of capturing culture cavities 134 are arranged on the capturing culture section 133 in parallel, each group of capturing culture cavities 134 comprises two capturing culture cavities 134 which are connected in parallel, and a plurality of capturing structures 135 are arranged in each capturing culture cavity 134. The capturing culture channel 13 further comprises a sample separating section 131 and a sample separating section 132, and the sample separating section 131, the capturing culture section 133 and the sample separating section 132 are sequentially connected. The light intensity adjusting channel 23 further comprises a liquid-separating and feeding section 231, and the liquid-separating and feeding section 231 is communicated with the light intensity adjusting section 232. The main sample section 12, the sub-sample section 131 and the sub-sample section 132 are all serpentine micro-channels.
As shown in fig. 5, a main liquid inlet section 22 and a light intensity adjusting channel 23 are serially arranged on the light stimulation light intensity gradient chip layer 2, a plurality of light intensity adjusting channels 23 are parallelly arranged on the main liquid inlet section 22, a plurality of light intensity adjusting channels 23 are parallelly arranged on each light intensity adjusting channel 23, a light intensity emphasizing section 232 is arranged on each light intensity adjusting channel 23, the light intensity adjusting sections 232 correspond to the positions of the capturing culture cavities 134, the light intensity adjusting sections 232 are rectangular micro channels, the length of each rectangular micro channel is 12000 μm, the width of each rectangular micro channel is 1000 μm, and the depth of each rectangular micro channel is 500 μm; the solution is passively mixed to generate four concentration gradient solutions of 0, 1/3C and 2/3C, C, and the four concentration gradient solutions are introduced into the rectangular micro-channels, so that light stimuli with different light intensities are generated in the 4 rectangular micro-channels based on different absorbance of the solutions with different concentrations. The split intake section 231 and the main intake section 22 are serpentine micro-channels. Two liquid inlets 21 are arranged on the inlet side of the main liquid inlet section 22 in parallel, and a liquid outlet 24 is arranged on the outlet side of the sub liquid inlet section 231 in parallel.
In this embodiment, two sample inlets 11 are arranged in parallel on the inlet side of the main sample section 12, and sample outlets 14 are arranged in parallel on the outlet side of the plurality of capturing culture channels 13, the sample inlets 11 are positioned at the top of the cell capturing culture chip layer 1, and the sample outlets 14 are positioned at the bottom of the cell capturing culture chip layer 1. The two sample inlets 11 of the cell capturing culture chip layer 1 are connected with the main sample inlet section 12, the channel width is 100-150 mu m, the main sample inlet section 12 is a serpentine micro-channel, the main sample inlet section 12 can realize drug concentration gradients of 0, 1/3C and 2/3C, C, and the serpentine part of the multiple serpentine channels can realize better solution mixing effect. The diameters of the sample inlet 11 and the sample outlet 14 are 2-5 mm, and the sizes of the sample inlet 11 and the sample outlet 14 are designed mainly for matching with the external pressure pump pipeline required by the test. The number of the main liquid inlet sections 22 is smaller than that of the light intensity adjusting channels 23, the main liquid inlet sections 22 are preferably three, the light intensity adjusting channels 23 are preferably four, and the three main liquid inlet sections 22 and the four light intensity adjusting channels 23 form a Christmas tree structure.
In the present embodiment, the height of the cell-capturing culture chip layer 1 is 20 to 25 μm, and setting the height of the cell-capturing culture chip layer 1 to 20 to 25 μm can ensure smooth flow of cells without occurrence of a phenomenon in which a plurality of cells overlap in the longitudinal direction to affect counting.
As shown in fig. 6, the capturing culture chambers 134 are rectangular chambers, the inlet and outlet of the capturing culture chambers 134 are arranged at diagonal positions, a plurality of capturing culture chambers 134 are in a 4×4 array structure, two rows of single-cell capturing structures 135 are arranged in a single capturing culture chamber 134, each row of cell capturing structures 135 is provided with 5 columns, the capturing structures 135 are provided with 4 rows along the width direction of the capturing culture chambers 134, the capturing structures 135 are provided with 4 columns along the length direction of the capturing culture chambers 134, and the capturing structures 135 positioned in two adjacent columns are staggered, and the staggered displacement is 50-60 μm. To ensure that the flow rate of the fluid to each row of chambers is the same, the width of the capture culture channels 13 is preferably set to 100-150 μm, and cells can be uniformly dispersed to the sample inlet of each capture culture chamber 134.
As shown in FIG. 7, the cell capture culture chip layer 1 can realize in vitro cell culture under different drug concentration gradients. The capturing structure 135 comprises a concave main body 1351, wherein two openings 1352 are formed in the concave main body 1351, and the two openings 1352 are symmetrically arranged relative to the symmetry axis of the concave main body 1351. The inclination angle of the opening of the concave main body 1351 is preferably 55-65 degrees, the inclination angle of the opening has an important influence on the cell capturing efficiency, and the cell capturing efficiency is reduced due to the fact that the inclination angle is too large or too small, so that the cell quantity consistency inside each cavity is low during culture, and in addition, the cell can be prevented from escaping from a capturing structure under the action of a flow field through the adjustment of the inclination angle of the opening, so that the stable capturing of the cell is realized. The capturing structures 135 have a transverse spacing of 40-50 μm and a longitudinal spacing of 70-80 μm, and by adjusting the row-column spacing of the capturing structures 135, the capturing structures have a shunting effect in the capturing process, so that the cells can conveniently shuttle to the corresponding capturing structures according to the expected streamline. Two openings 1352 are formed in the concave main body 1351, the width of each opening 1352 is 5-10 μm, the flow resistance of fluid is matched through the openings with the double-slit width of 5-10 μm, the cells can be ensured to smoothly enter the capturing structure 135 and clamped at the positions of the openings 1352, and redundant cells flow to the capturing structure 135 which is emptied next.
In this embodiment, the capturing culture cavity 134 has a diagonal rectangular geometry, the length is set to 1000-1200 μm, the width is set to 600-800 μm, and the inlet and outlet of the capturing culture cavity 134 are designed to be diagonal in and out, so that a capturing load with spatial distribution is provided, the spatial distribution can be more uniformly captured, the spatial distribution of the verified cells after capturing is more uniform, and the capturing uniformity is better.
The specific implementation principle of the gradient light stimulation and drug concentration gradient combined regulation device is as follows: the cells to be captured enter the channel through the two sample inlets 11, enter the capturing culture cavity 134 through the main sample inlet section 12 and the capturing culture channel 13, capture the cells through the fluid resistance, count after the cell suspension sample inlet process is finished, and then add culture medium into the cell capturing culture chip layer 1 through a continuous perfusion mode, wherein the flow rate is controlled at 4 mu l/min. As shown in fig. 8 and 9, experimental photographs and experimental data graphs for generating four light intensity gradients of the CMTPX dye-verified light gradient chip layer.
As shown in fig. 10, according to the light emphasis segments 232 of different heights, light stimuli of different light intensities are generated in 4 rectangular micro-channels based on the difference in absorbance of solutions of different concentrations; the Beer-Lambert law relates to the definition of the relationship between the intensity of light in an optical medium and the concentration of a substance in a medium, in which when monochromatic light passes through a homogeneous, non-luminescent solution or gas, the intensity of light transmitted through the medium will decrease as the concentration of the substance in the medium increases, and its change is proportional to the length of the medium. When the incident intensity of light is I0, the light intensity after passing through the medium is I, the concentration of the medium is C, and the length of the medium is l, there is a relationship of i=i0e (-epsilon cb) where epsilon is a proportionality constant called absorbance coefficient or molar absorbance, and b and C represent the length and the concentration of the medium, respectively. Absorbance (a) =ε×b×c, where a is absorbance, which is the logarithmic ratio of the intensity of absorbed light, and is typically measured by a spectrophotometer, ε is a constant representing the absorbance coefficient of a substance, b is the optical path length, i.e., the actual distance traveled by light in a sample, and c is the concentration of a solution, i.e., the molar concentration of a substance. The light-absorbing solution with different concentration gradients is combined with the design of channels with different height differences to prepare the light-gradient layer chip, the chip is irradiated by using a visible light source, and four light spots with different light intensity gradients are generated to irradiate the lower culture layer chip due to different absorbance of each channel after the light-gradient layer is transmitted, so that the regulation and control of the proliferation and differentiation processes of the C2C12 cells by multi-gradient light stimulation are realized. The heights of the four light emphasis sections 232 are respectively 2000-2500 μm, 1500-2000 μm, 1000-1500 μm and 500-1000 μm, and the optical lengths b of the four light emphasis sections 232 are different, so that the absorbance of different gradients can be generated.
A gradient light stimulation and drug concentration gradient combined regulation device and a preparation method thereof comprise the following steps:
s1: preparation of a light-stimulated light intensity gradient chip layer 2:
preparing a printing light stimulus light intensity gradient chip layer 2 by using a 3D printer; the printed light stimulus light intensity gradient chip layer 2 can also be prepared by soft lithography, 3D printing and other technologies;
s2: preparing a cell capturing culture chip layer 1;
constructing a cell capturing and culturing chip layer 1 by using a PDMS polymer through a soft lithography process and a reverse mould process, and specifically;
s21: processing a cell capturing culture chip layer mould with the structure height of 20-25 mu m by utilizing a soft lithography process;
s22: fixing a cell capturing culture chip layer mould on an acrylic mould;
s23: pouring PDMS mixed glue, standing and vacuumizing;
S24: placing the acrylic mould into an oven for heating and curing, wherein the temperature of the oven is 60-80 ℃;
s25: and (5) reversing the mould to finish the preparation of the cell capturing culture chip layer 1.
S3: performing ultrasonic treatment and plasma cleaning on the light-stimulated light intensity gradient chip layer 2 and the cell capturing culture chip layer 1, wherein the plasma treatment time is 15-20s;
s4: bonding the light-stimulated light intensity gradient chip layer 2 with the cell capturing culture chip layer 1 at a bonding temperature of 100-120.
Claims (9)
1. The gradient light stimulation and drug concentration gradient combined regulation and control device is characterized by comprising a light stimulation light intensity gradient chip layer (2) and a cell capturing culture chip layer (1) which are sequentially arranged from top to bottom;
The cell capturing and culturing chip layer (1) is provided with a main sample injection section (12) and capturing and culturing channels (13) in series, the main sample injection section (12) is provided with a plurality of capturing and culturing channels (13) in parallel, and the number of the main sample injection sections (12) is smaller than the number of the capturing and culturing channels (13); each capturing culture channel (13) is provided with a capturing culture section (133), a plurality of groups of capturing culture cavities (134) are arranged on the capturing culture sections (133) in parallel, each group of capturing culture cavities (134) comprises two capturing culture cavities (134) which are connected in parallel, and a plurality of capturing structures (135) are arranged in each capturing culture cavity (134);
The light stimulation light intensity gradient chip layer (2) is provided with a main liquid inlet section (22) and light intensity regulating channels (23) in series, the main liquid inlet section (22) is provided with a plurality of light intensity regulating channels (23) in parallel, and the number of the main liquid inlet sections (22) is smaller than the number of the light intensity regulating channels (23); and each light intensity adjusting channel (23) is provided with a light emphasis section (232), and the light intensity adjusting sections (232) correspond to the positions of the capturing culture cavities (134).
2. The gradient light stimulation and drug concentration gradient combined regulation and control device according to claim 1, wherein the capturing structure (135) comprises a concave main body (1351), two openings (1352) are arranged on the concave main body (1351), and the two openings (1352) are symmetrically arranged relative to the symmetry axis of the concave main body (1351).
3. The gradient optical stimulation and drug concentration gradient combined regulation and control device according to claim 1, wherein two sample inlets (11) are arranged on the inlet side of the main sample inlet section (12) in parallel, sample outlets (14) are arranged on the outlet side of the plurality of capturing culture channels (13) in parallel, the sample inlets (11) are positioned at the top of the cell capturing culture chip layer (1), and the sample outlets (14) are positioned at the bottom of the cell capturing culture chip layer (1); two liquid inlets (21) are arranged on the inlet side of the main liquid inlet section (22) in parallel, and a liquid outlet (24) is arranged on the outlet side of the sub liquid inlet section (231) in parallel.
4. The gradient optical stimulation and drug concentration gradient combined control device according to claim 1, wherein the capturing culture channel (13) further comprises a sample separating section (131) and a sample separating section (132), and the sample separating section (131), the capturing culture section (133) and the sample separating section (132) are sequentially connected; the light intensity adjusting channel (23) further comprises a liquid separating and feeding section (231), and the liquid separating and feeding section (231) is communicated with the light intensity adjusting section (232).
5. The gradient optical stimulus and drug concentration gradient combined control device according to claim 4, wherein the sample separation and feeding section (131), the sample separation and feeding section (132) and the liquid separation and feeding section (231) are all serpentine micro-channels.
6. The gradient optical stimulus and drug concentration gradient combined control device according to claim 1, wherein the main sample introduction section (12) and the main liquid inlet section (22) are both serpentine micro-channels.
7. The gradient light stimulation and drug concentration gradient combined regulation and control device according to claim 1, wherein the capturing culture cavity (134) is a rectangular cavity, an inlet and an outlet of the capturing culture cavity (134) are arranged at diagonal positions, the capturing structure (135) is provided with a plurality of rows along the width direction of the capturing culture cavity (134), the capturing structure (135) is provided with a plurality of columns along the length direction of the capturing culture cavity (134), and the capturing structures (135) positioned in two adjacent columns are staggered.
8. A method for preparing the gradient optical stimulus and drug concentration gradient combined regulation device as claimed in claims 1-7, which is characterized by comprising the following steps:
S1: preparing a light-stimulated light intensity gradient chip layer (2):
preparing a printing light stimulus light intensity gradient chip layer (2) by using a 3D printer;
S2: preparing a cell capturing culture chip layer (1);
Constructing a cell capturing and culturing chip layer (1) by utilizing a PDMS polymer through a soft lithography process and a reverse mould process;
s3: performing ultrasonic treatment and plasma cleaning on the light-stimulated light intensity gradient chip layer (2) and the cell capturing culture chip layer (1), wherein the plasma treatment time is 15-20s;
S4: bonding the light-stimulated light intensity gradient chip layer (2) with the cell capturing culture chip layer (1) at the bonding temperature of 100-120 ℃.
9. A method for preparing the gradient optical stimulation and drug concentration gradient combined regulation device as claimed in claim 8, which is characterized in that the preparation of the cell capturing culture chip layer (1) in the step S2 specifically comprises the following steps:
the soft lithography process and the reverse process in the step S2 specifically comprise the following steps:
s21: processing a cell capturing culture chip layer mould with the structure height of 20-25 mu m by utilizing a soft lithography process;
s22: fixing a cell capturing culture chip layer mould on an acrylic mould;
s23: pouring PDMS mixed glue, standing and vacuumizing;
S24: placing the acrylic mould into an oven for heating and curing, wherein the temperature of the oven is 60-80 ℃;
S25: and (5) reversing the mould to finish the preparation of the cell capturing culture chip layer (1).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410329277.2A CN118146946A (en) | 2024-03-21 | 2024-03-21 | Gradient light stimulation and drug concentration gradient combined regulation and control device and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410329277.2A CN118146946A (en) | 2024-03-21 | 2024-03-21 | Gradient light stimulation and drug concentration gradient combined regulation and control device and preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118146946A true CN118146946A (en) | 2024-06-07 |
Family
ID=91284935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410329277.2A Pending CN118146946A (en) | 2024-03-21 | 2024-03-21 | Gradient light stimulation and drug concentration gradient combined regulation and control device and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118146946A (en) |
-
2024
- 2024-03-21 CN CN202410329277.2A patent/CN118146946A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Keenan et al. | Biomolecular gradients in cell culture systems | |
| Young et al. | Fundamentals of microfluidic cell culture in controlled microenvironments | |
| CN110004043B (en) | Single cell capture micro-fluidic chip | |
| US20130068310A1 (en) | Method and Apparatus for a Microfluidic Device | |
| Gao et al. | A microfluidic localized, multiple cell culture array using vacuum actuated cell seeding: integrated anticancer drug testing | |
| CN105170208A (en) | Preparation method of microarray chip and product thereof | |
| CN110586210B (en) | Concentration gradient generation device for gel 3D cell culture, preparation and application method | |
| Somaweera et al. | On-chip gradient generation in 256 microfluidic cell cultures: simulation and experimental validation | |
| CN210765350U (en) | Array micro-control chip for single cell capture and tumor ball culture | |
| CN116590147A (en) | Device, system and method for multi-factor detection of in vitro skin tissue culture | |
| CN112430542B (en) | Integrated microfluidic three-dimensional tumor chip and bionic method | |
| CN118146946A (en) | Gradient light stimulation and drug concentration gradient combined regulation and control device and preparation method | |
| CN107236668B (en) | Microfluidic chip for breast cancer stem cell culture and drug analysis | |
| CN110747102B (en) | Single cell separation device and method based on micro-fluidic chip | |
| CN110283724B (en) | High-throughput organoid phenotype screening system and method | |
| CN116083234A (en) | Organoid array chip and drug screening method and application thereof | |
| US20210339242A1 (en) | Microfluidic device with embedded cell culture chambers for high throughput biological assays | |
| Zhu et al. | A microfluidic single-cell array for in situ laminar-flow-based comparative culturing of budding yeast cells | |
| CN214529079U (en) | Micro-fluidic bionic model | |
| WO2017091043A1 (en) | Microfluidic perfusion cell culture system | |
| CN118146947A (en) | Optical stimulation, electrical stimulation and concentration gradient combined regulation and control device and preparation method | |
| CN103599722B (en) | Bionics microfluidic mixer | |
| CN108274734B (en) | System and method for manufacturing in-vitro 3D tumor microsphere model with controllable core-shell structure | |
| CN118146949A (en) | Electric field gradient and concentration gradient cell in-vitro culture device and preparation method | |
| Tian et al. | A valve-free 2D concentration gradient generator |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |